Research ArticleTopical Treatment with Xiaozheng Zhitong Paste(XZP) Alleviates Bone Destruction and Bone CancerPain in a Rat Model of Prostate Cancer-InducedBone Pain by Modulating the RANKLRANKOPG Signaling
1Department of Oncology Guangrsquoanmen Hospital China Academy of Chinese Medical Sciences Beixiange 5Xicheng District Beijing 100053 China2Beijing University of Chinese Medicine Beijing North Third Ring Road No 11 Chaoyang District Beijing 100029 China3Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences Nanxiaojie Dongzhimen DistrictBeijing 100700 China4Department of Nephrology Guangrsquoanmen Hospital China Academy of Chinese Medical Sciences Beixiange 5Xicheng District Beijing 100053 China
Correspondence should be addressed to Yanju Bao baoyanju126com and Baojin Hua huabaojin2008126com
Received 7 September 2014 Revised 19 December 2014 Accepted 20 December 2014
Academic Editor Yen-Chin Liu
Copyright copy 2015 Yanju Bao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
To explore the effects and mechanisms of Xiaozheng Zhitong Paste (XZP) on bone cancer pain Wistar rats were inoculated withvehicle or prostate cancer PC-3 into the tibia bone and treated topically with inert paste XZP at 1575 315 or 63 gkg twice perday for 21 daysTheir bone structural damage nociceptive behaviors bone osteoclast and osteoblast activity and the levels of OPGRANL RNAK PTHrP IGF-1 M-CSF IL-8 and TNF-120572were examined In comparison with that in the placebo group significantlyreduced numbers of invaded cancer cells decreased levels of bone damage and mechanical threshold and paw withdrawal latencylower levels of serum TRACP5b ICTP PINP and BAP and less levels of bone osteoblast and osteoclast activity were detected inthe XZP-treated rats (119875 lt 005) Moreover significantly increased levels of bone OPG but significantly decreased levels of RANLRNAK PTHrP IGF-1 M-CSF IL-8 and TNF-120572 were detected in the XZP-treated rats (119875 lt 005 for all) Together XZP treatmentsignificantly mitigated the cancer-induced bone damage and bone osteoclast and osteoblast activity and alleviated prostate cancer-induced bone pain by modulating the RANKLRANKOPG pathway and bone cancer-related inflammation in rats
1 Introduction
Cancer pain is reported to be experienced by 75ndash90 oflate stagemetastatic cancer patients [1] Cancer-induced bonepain (CIBP) is the most common type of cancer pain and isoften debilitating and intractable affecting the quality of lifeand functional status in cancer patients [2 3] Bone cancerpain is not a single entity but is a combination of backgroundand breakthrough pain which is defined as ldquoa transitoryexacerbation of pain experienced by the patient who hasrelatively stable and adequately controlled baseline painrdquo[2] Currently there are many therapeutic options available
for the alleviation of bone cancer pain and they includeexternal beam radiotherapy opioid analgesia nonsteroidalanti-inflammatory drugs (NSAIDs) bisphosphonates localsurgery and anaesthetic techniques [4] However each ofthese treatment options is accompanied by limitations of lesseffectiveness and severe effects [4ndash9] Hence control of bonecancer pain will be of great significance in management ofpatients with bone cancer
According to traditional Chinese medicine (TCM) the-ory cancer pain is the result of tumor-caused stagnation andinsufficiency of Qi and blood flow in the body Thus themain therapeutic goals are to promote Qi and activate blood
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 215892 14 pageshttpdxdoiorg1011552015215892
2 Evidence-Based Complementary and Alternative Medicine
Table 1 Composition of XZP
Pharmaceuticalname
Botanicalsourcefamily Part used Traditional actionsuses Amount (g)
Anticancer clearing awayheat and toxic material 10
by softening hard lumps dispelling nodes and warmingthe channels [10] Our previous study has demonstratedthat herbal analgesic paste Xiaozheng Zhitong Paste (XZP)containing six herbs of Xuejie (Dragonrsquos blood) Yanhusuo(Corydalis rhizoma) Ruxiang (Olibanum) Moyao (Myrrha)Qingdai (Natural Indigo) and Bingpian (Borneolum Syn-theticum) significantly alleviated cancer pain including bonecancer pain in patients with middlelate stage cancer [11]However the mechanisms underlying action of XZP in alle-viating bone cancer pain have not been systemically exploredIn advanced medicine the development of bone cancerpain has been attributed to cancer-related bone destructionreactive muscle spasm cancer-related inflammation andrelease of inflammatory mediators as well as increased con-centrations of calcium [2] During the process of bone cancerpain aberrant activation of osteoclasts and compensativelyincreased osteoblast activity contribute to the bone structuraldamage and are positively regulated by the receptor activatorof nuclear factor-120581B (RANK) ligand (RANKL) [12] butnegatively regulated by osteoprotegerin (OPG) Furthermorethe bone formation and resorption are also regulated bythe parathyroid hormone related proteins (PTHrP) andinsulin-like growth factor 1- (IGF-1-) related signaling [1314] Accordingly the levels of serum tartrate-resistant acidphosphatase 5b (TRACP5b) carboxy-terminal pyridinolinecross-linked telopeptide of type I collagen (ICTP) procol-lagen type I amino-terminal propeptide (PINP) and bonealkaline phosphates (BAP) have been used to estimate bonedestruction in patients with bone metastatic cancers such asprostate cancer [15ndash17] In addition bone cancer can causeinflammation and produce inflammatory cytokines such as
interleukin 8 (IL-8) tumor necrosis factor- (TNF-) 120572 andmacrophage colony-stimulating factor (M-CSF) which feed-back induce bone damage and pain [12 18 19] It is unclearwhether and how XZP treatment can inhibit cancer invasionin the bone and related bone damage as well as osteoclastand osteoblast activity Furthermore it is still unknown onwhether and howXZP treatment canmodulate the activationof the RANKLRANKOPG signaling and the expression ofbone metabolic regulators and inflammatory mediators
The aim of this study was to evaluate the effects ofXZP and to explore the mechanisms underlying its actionin alleviating bone cancer pain We employed a rat modelof bone prostate cancer pain to test the hypothesis thatXZP can alleviate bone cancer pain by modulating theRANKLRANKOPG signaling and the expression of bonemetabolic regulators of PTHrP and IGF-1 and inflammatorymediators of IL-8 M-CSF and TNF-120572 in the bone of rats
2 Materials and Methods
21 Composition andPreparation of XZP XZPwas composedof six traditional Chinese medicines which were purchasedfrom Tong-Ren-Tang (Beijing China) and authenticated byProfessor Hu Shilin Institute of Chinese Materia MedicaChina Academy of Chinese Medical Sciences The basicpharmacological functions of these Chinese medicines areillustrated in Table 1 All the components of XZP were driedand homogenized to fine powders To prepare the XZPindividual powdered medicine (950 g) was extracted twicewith 70 ethanol (1 5 wv) for 1ndash15 h eachThe extracts werefiltrated and concentrated to 1 g per 359 g (crude herbs)
Evidence-Based Complementary and Alternative Medicine 3
followed by being stored at minus20∘C The extracts were mixedwith cataplasm matrix to generate a proper concentration ofXZP before use in vivo
22 High Performance Liquid Chromatography To deter-mine the potential components the XZP was preparedand analyzed by high performance liquid chromatography(HPLC) using gt98 purity of the standards of tetrahy-dropalmatine imperatorin isoimperatorin coptisine andpalmatine chloride (in methanol) that were purchased fromthe National Institute for Food and Drug Control (BeijingChina) Methanol and acetonitrile (HPLC grade FisherScientific New Jersey USA) and pure water (Wahaha GroupChina) were used for HPLC analysis All other chemicalswere of analytical grade The solutions were filtered through045 120583m membranes and subjected to analysis at 280 nmusing an Agilent Zorbax Eclips XDB C18 column (250 times46mm 45 120583m) on a Waters HPLC system (Waters Corpo-ration USA) equipped with 2487 pump an UV detector anEmpower 2 system controller and a waters 717 plus autosam-pler at 30∘C Samples at 10 120583L were injected and acetonitrile-methanol-water-triethylamine (24 32 44 05) at a flow-rateof 10mL per min was used at the mobile phase Accordingto the chromatographic characteristics of these standardcompounds the peak areas of individual compounds in thesamples were used for determining the contents of thesecompounds and expressed as the percent
23 Animals Female Wistar rats weighing 150ndash180 g werefrom the Department of Experimental Animal SciencesPeking University Health Science Center (Beijing China)Individual rats were housed in specific pathogen free (SPF)facility at 24 plusmn 1∘C on a cycle of 1212 h lightdark andfree access to food and water All experiments were carriedout in accordance with the guidelines of the InternationalAssociation for the Study of Pain [20] and approved bythe Animal Care and Use Committee of China Academy ofChinese Medical Sciences
24 Cell Preparation and Bone Cancer Pain Model Prostatecancer PC-3 cells were prepared and bone cancer pain modelwere established as our previous report [21] Briefly individ-ual rats were anesthetized intraperitoneally (ip) with sodiumpentobarbital (45mgkg) and the left tibia of individual ratswas exposedThe left tibia of individual rats was injected with105 PC-3 cells in 10 120583L Hankrsquos solution and the injection sitewas covered by bone wax followed by closing the wound Agroup of rats (119899 = 10) received the same surgery and the samevolume of vehicle injection and served as the Sham controlsAll rats were subjected to the same postoperational cares
25 Treatment and Groups The Sham control rats weretreated topically with the inert paste as the control (119899 = 10per group)The bone cancer-bearing rats were randomly andtreated topically with the inert paste as the placebo the XZPat 1575 gkg (as the low dose) 315 gkg (medium dose) or63 gkg (high dose) evenly applied on the skin of tumor-bearing tibias covered with gauze and a layer of plastic filmsealed and fixed with desensitized adhesive plaster twice
a day at 800 AM and 2000 PM for consecutive 21 daysbeginning one day after Walker 256 cell inoculation [11]
26 Radiological Analysis The cancer-related osteolyticlesions in the tibia of individual rats were examined by X-rayradiology at 21 days after the inoculation The rats wereanaesthetized and exposed to X-ray (Emerald 125) at 40 KVPfor 120 s followed by the development of X-ray film (HenrySchein blue sensitive film) using a film developer (KonicaSRX-101) The radiological score was calculated according toStewartrsquos radiological score [22] 0 normal bone structurewith no sign of deterioration 1 minor loss of medullarybone 2 substantial loss of medullary bone with erosion ofcortical bone and 3 substantial loss of medullary bone withmajor cortical destruction of the proximal epiphysis
The tibias were scanned by micro-CT and reconstructedwith 8 120583m isotropic voxel size on amicro-CT system (eXploreLocus SP GE Medical Systems) The reconstructed 3D imag-ines of femurs were analyzed usingMicroviewer (GEMedicalSystems) as described previously [23] Micro-CT is used tomeasure several histomorphometric variables including bonevolume (BV) total volume (TV) and bone volume fraction(BVTV) [24]
27 Mechanical Threshold and Paw Withdrawal Latency Theeffects of treatment with XZP on the spontaneous nocifensivebehaviors of the different groups of rats were tested for themechanical threshold and paw withdrawal latency (PWL)in a blinded manner as our previous description [21 2526] In brief individual rats were placed in an invertedplastic chamber on the glass surface of the Paw ThermalStimulator System (UCSD San Diego) for 30min beforethe test Mechanical hyperalgesia was measured using asingle rigid filament attached to a handheld transducer(automatic plantar analgesia tester Institute of BiomedicalEngineering Chinese Academy of Medical Science TianjinChina) Animals were acclimated to their surroundings for10min daily for three consecutive days in a plexiglass box ona metal grid surface prior to testing On the testing days ratswere allowed to acclimate for 5ndash10min A rigid filament waspressed perpendicularly against the medial plantar surface ofthe hind paw with an increasing force Brisk paw withdrawalor pawflinching accompanied by head turning biting andorlicking upon application of an increasing force was consid-ered as a positive response The paw withdrawal threshold(PWT)was defined as theminimal force (g) required to evokethe cited positive responses Each hind paw of rats was testedthree times and the data were averagedThe interval betweenconsecutive tests of the same paw was 5min The sameprocedure was performed on days 3 6 9 12 15 18 and 21after tumor inoculation Each hind paw of rats was stimulatedwith a focused beam of radiant heat using an analgesiometer(37360 Ugo Basile Italy) underneath the glass surfaceWhenthe paw was withdrawn from the stimulus the PWL wasautomatically recorded to the nearest 01 s The intensity ofstimuli was adjusted to generate an average baseline PWLof approximately 100 s in naive animals The maximumstimulation was controlled lt20 s to prevent potential tissuedamage Paws were alternated randomly to preclude ldquoorderrdquo
4 Evidence-Based Complementary and Alternative Medicine
effects Individual rats were subjected to four tests with a5min interval before surgery and 2 5 8 11 14 17 and 20 daysafter inoculation in a blinded manner The mean PWL wascalculated for each time point in individual rats
28 Histological Evaluation The rats were deeply anes-thetized by pentobarbital and transcardially perfused withsaline on day 21 after inoculation (119899 = 10 per group) The lefttibia from each animal was dissected fixed in 10 formalinovernight decalcified in 15 EDTA-PBS for 7 days andparaffin-embedded The tissue sections (6 120583m) were stainedwith hematoxylin and eosin (HE) Furthermore the numbersof osteoclasts and osteoblasts in the regions were identi-fied by the tartrate-resistant acid phosphatase (TRAP) oralkaline phosphatase (AP) staining using the TRAP or AKPstaining kits (Nanjing Jiancheng Bioengineering InstituteNanjing China) respectivelyThe numbers of osteoclasts andosteoblasts in 10 sections from each rat were counted under alight microscope (Leica DM 2500) in a blinded manner [25]
29 Enzyme-Linked Immunosorbent Assay (ELISA) Periph-eral blood samples were collected from individual rats at0 7 14 or 21 days after inoculation and the concentrationsof serum tartrate-resistant acid phosphatase 5b (TRACP5b)carboxy-terminal pyridinoline cross-linked telopeptides oftype I collagen (ICTP) procollagen type I amino-terminalpropeptide (PINP) and bone alkaline phosphatase (BAP) inindividual rats were determined by ELISA using the specifickits according to the manufacturerrsquos instruction (ThermoScientific Hudson NH USA) The limitation of detectionfor TRACP5b ICTP PINP and BAP was 0078mIUmL25 ngmL 1875 pgmL and 25UL respectively
210 Quantitative RT-PCR Total RNA was extracted fromindividual rat tibias using the TRIzol reagent and reverselytranscribed to cDNA using the first-strand cDNA synthesiskit (Invitrogen)The relative levels of target genemRNA tran-scripts were determined by quantitative RT-PCR using theSYBR Green system and specific primers on the LightCyclersystem (Roche) The sequences of primers are shown inTable 2The PCR amplification was performed in triplicate at95∘C for 10 minutes and was subjected to 40 cycles of 95∘Cfor 15 s and 60∘C for 30 s The relative levels of each gene toGAPDHmRNA transcripts were calculated
211 Western Blot Assay Total tibia proteins were extractedfrom individual rats by sonication in RIPA buffer containingprotease inhibitors (Roche) After quantification of proteinconcentrations using the BCA protein assay kit (ThermoScientific Rockford IL) the protein samples (20120583glane)were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinyli-dene difluoride (PVDF) membranes (Millipore) After beingblocked with 5 fat-free dry milk the membranes wereincubated with anti-OPG (ab73400) anti-GAPDH (6C5ab8245) anti-IL-8 (ab7747) anti-PTHrP (ab85205) anti-M-CSF (ab99109) anti-IGF1 antibody (ab36532) anti-TNF-120572(ab66579) anti-RANKL (EPR4999 ab124797 Abcam Cam-bridge MA USA) and anti-RANK (H-300 sc-9072 SantaCruz Biotechnology) respectively The bound antibodieswere detected using a horseradish peroxidase- (HRP-) con-jugated secondary antibody and visualized by an enhancedchemiluminescence detection system followed by quantify-ing using the Image Quant LAS 4000 (GE Healthcare)
Evidence-Based Complementary and Alternative Medicine 5
0010
0005
000 1000 2000 3000(min)
(AU
)
4000 5000 6000
0000
AB
C DE
(a)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
A
B C DE
minus004
minus002
(b)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
minus004
minus002
(c)
Tetrahydropalmatine
N
O
O
O
O
Imperatorin
OO O
O
H3C
H3C
Isoimperatorin
O
O
OO
CoptisineO
O
O CH3
O
CH3
H3C N+
Palmatine chloride
O
O
O
ON+
Clminus
(d)
Figure 1 HPLC analysis of the components of XZP XZP were extracted with ethanol and the representative active components in the XZPextracts were characterized by HPLC using the standard components of (A) tetrahydropalmatine (B) imperatorin (C) isoimperatorin (D)coptisine and (E) palmatine chloride Data are representative chromatographic histograms at 280 nm of XZP (a) characterized profile (UVchromatograms at 280 nm) of the standard compounds (a) XZP ethanol-extracts (b) and blank (c) from three independent experiments (d)The structures of standard compounds
6 Evidence-Based Complementary and Alternative Medicine
Control
Low dose Medium dose High dose
Placebo 40
30
20
10
00
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
Radi
olog
ical
scor
e
lowastlowast lowastlowastlowastlowast
(a)
Control
Low dose Medium dose High dose
Placebo
100Bo
ne v
olum
e fra
ctio
n BV
TV
(100
)
80
60
40
20
0
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
lowastlowastlowastlowast
(b)
Control
Low dose Medium dose High dose
Placebo
(c)
Figure 2 XZP treatment reduces the cancer-induced bone damage in rats At 21 days after inoculation the tibial bone structure and damagein the individual rats were characterized by X-rad and micro-CT and quantitatively analyzed Subsequently their tibial bone sections werestained with HE Data are representative images of individual groups of rats (119899 = 10 per group) (a) Radiographs of the bone structure andtumor invasion (b) 3D micro-CT images of the tibias (c) HE analysis of the cancer bone tissues (magnification times400) Low dose mediumdose and high dose The rats were treated with low medium or high dose of XZP (lowastlowast119875 lt 001 versus the control group 119875 lt 005 versus theplacebo group lowastlowast119875 lt 001 versus placebo group)
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Anticancer clearing awayheat and toxic material 10
by softening hard lumps dispelling nodes and warmingthe channels [10] Our previous study has demonstratedthat herbal analgesic paste Xiaozheng Zhitong Paste (XZP)containing six herbs of Xuejie (Dragonrsquos blood) Yanhusuo(Corydalis rhizoma) Ruxiang (Olibanum) Moyao (Myrrha)Qingdai (Natural Indigo) and Bingpian (Borneolum Syn-theticum) significantly alleviated cancer pain including bonecancer pain in patients with middlelate stage cancer [11]However the mechanisms underlying action of XZP in alle-viating bone cancer pain have not been systemically exploredIn advanced medicine the development of bone cancerpain has been attributed to cancer-related bone destructionreactive muscle spasm cancer-related inflammation andrelease of inflammatory mediators as well as increased con-centrations of calcium [2] During the process of bone cancerpain aberrant activation of osteoclasts and compensativelyincreased osteoblast activity contribute to the bone structuraldamage and are positively regulated by the receptor activatorof nuclear factor-120581B (RANK) ligand (RANKL) [12] butnegatively regulated by osteoprotegerin (OPG) Furthermorethe bone formation and resorption are also regulated bythe parathyroid hormone related proteins (PTHrP) andinsulin-like growth factor 1- (IGF-1-) related signaling [1314] Accordingly the levels of serum tartrate-resistant acidphosphatase 5b (TRACP5b) carboxy-terminal pyridinolinecross-linked telopeptide of type I collagen (ICTP) procol-lagen type I amino-terminal propeptide (PINP) and bonealkaline phosphates (BAP) have been used to estimate bonedestruction in patients with bone metastatic cancers such asprostate cancer [15ndash17] In addition bone cancer can causeinflammation and produce inflammatory cytokines such as
interleukin 8 (IL-8) tumor necrosis factor- (TNF-) 120572 andmacrophage colony-stimulating factor (M-CSF) which feed-back induce bone damage and pain [12 18 19] It is unclearwhether and how XZP treatment can inhibit cancer invasionin the bone and related bone damage as well as osteoclastand osteoblast activity Furthermore it is still unknown onwhether and howXZP treatment canmodulate the activationof the RANKLRANKOPG signaling and the expression ofbone metabolic regulators and inflammatory mediators
The aim of this study was to evaluate the effects ofXZP and to explore the mechanisms underlying its actionin alleviating bone cancer pain We employed a rat modelof bone prostate cancer pain to test the hypothesis thatXZP can alleviate bone cancer pain by modulating theRANKLRANKOPG signaling and the expression of bonemetabolic regulators of PTHrP and IGF-1 and inflammatorymediators of IL-8 M-CSF and TNF-120572 in the bone of rats
2 Materials and Methods
21 Composition andPreparation of XZP XZPwas composedof six traditional Chinese medicines which were purchasedfrom Tong-Ren-Tang (Beijing China) and authenticated byProfessor Hu Shilin Institute of Chinese Materia MedicaChina Academy of Chinese Medical Sciences The basicpharmacological functions of these Chinese medicines areillustrated in Table 1 All the components of XZP were driedand homogenized to fine powders To prepare the XZPindividual powdered medicine (950 g) was extracted twicewith 70 ethanol (1 5 wv) for 1ndash15 h eachThe extracts werefiltrated and concentrated to 1 g per 359 g (crude herbs)
Evidence-Based Complementary and Alternative Medicine 3
followed by being stored at minus20∘C The extracts were mixedwith cataplasm matrix to generate a proper concentration ofXZP before use in vivo
22 High Performance Liquid Chromatography To deter-mine the potential components the XZP was preparedand analyzed by high performance liquid chromatography(HPLC) using gt98 purity of the standards of tetrahy-dropalmatine imperatorin isoimperatorin coptisine andpalmatine chloride (in methanol) that were purchased fromthe National Institute for Food and Drug Control (BeijingChina) Methanol and acetonitrile (HPLC grade FisherScientific New Jersey USA) and pure water (Wahaha GroupChina) were used for HPLC analysis All other chemicalswere of analytical grade The solutions were filtered through045 120583m membranes and subjected to analysis at 280 nmusing an Agilent Zorbax Eclips XDB C18 column (250 times46mm 45 120583m) on a Waters HPLC system (Waters Corpo-ration USA) equipped with 2487 pump an UV detector anEmpower 2 system controller and a waters 717 plus autosam-pler at 30∘C Samples at 10 120583L were injected and acetonitrile-methanol-water-triethylamine (24 32 44 05) at a flow-rateof 10mL per min was used at the mobile phase Accordingto the chromatographic characteristics of these standardcompounds the peak areas of individual compounds in thesamples were used for determining the contents of thesecompounds and expressed as the percent
23 Animals Female Wistar rats weighing 150ndash180 g werefrom the Department of Experimental Animal SciencesPeking University Health Science Center (Beijing China)Individual rats were housed in specific pathogen free (SPF)facility at 24 plusmn 1∘C on a cycle of 1212 h lightdark andfree access to food and water All experiments were carriedout in accordance with the guidelines of the InternationalAssociation for the Study of Pain [20] and approved bythe Animal Care and Use Committee of China Academy ofChinese Medical Sciences
24 Cell Preparation and Bone Cancer Pain Model Prostatecancer PC-3 cells were prepared and bone cancer pain modelwere established as our previous report [21] Briefly individ-ual rats were anesthetized intraperitoneally (ip) with sodiumpentobarbital (45mgkg) and the left tibia of individual ratswas exposedThe left tibia of individual rats was injected with105 PC-3 cells in 10 120583L Hankrsquos solution and the injection sitewas covered by bone wax followed by closing the wound Agroup of rats (119899 = 10) received the same surgery and the samevolume of vehicle injection and served as the Sham controlsAll rats were subjected to the same postoperational cares
25 Treatment and Groups The Sham control rats weretreated topically with the inert paste as the control (119899 = 10per group)The bone cancer-bearing rats were randomly andtreated topically with the inert paste as the placebo the XZPat 1575 gkg (as the low dose) 315 gkg (medium dose) or63 gkg (high dose) evenly applied on the skin of tumor-bearing tibias covered with gauze and a layer of plastic filmsealed and fixed with desensitized adhesive plaster twice
a day at 800 AM and 2000 PM for consecutive 21 daysbeginning one day after Walker 256 cell inoculation [11]
26 Radiological Analysis The cancer-related osteolyticlesions in the tibia of individual rats were examined by X-rayradiology at 21 days after the inoculation The rats wereanaesthetized and exposed to X-ray (Emerald 125) at 40 KVPfor 120 s followed by the development of X-ray film (HenrySchein blue sensitive film) using a film developer (KonicaSRX-101) The radiological score was calculated according toStewartrsquos radiological score [22] 0 normal bone structurewith no sign of deterioration 1 minor loss of medullarybone 2 substantial loss of medullary bone with erosion ofcortical bone and 3 substantial loss of medullary bone withmajor cortical destruction of the proximal epiphysis
The tibias were scanned by micro-CT and reconstructedwith 8 120583m isotropic voxel size on amicro-CT system (eXploreLocus SP GE Medical Systems) The reconstructed 3D imag-ines of femurs were analyzed usingMicroviewer (GEMedicalSystems) as described previously [23] Micro-CT is used tomeasure several histomorphometric variables including bonevolume (BV) total volume (TV) and bone volume fraction(BVTV) [24]
27 Mechanical Threshold and Paw Withdrawal Latency Theeffects of treatment with XZP on the spontaneous nocifensivebehaviors of the different groups of rats were tested for themechanical threshold and paw withdrawal latency (PWL)in a blinded manner as our previous description [21 2526] In brief individual rats were placed in an invertedplastic chamber on the glass surface of the Paw ThermalStimulator System (UCSD San Diego) for 30min beforethe test Mechanical hyperalgesia was measured using asingle rigid filament attached to a handheld transducer(automatic plantar analgesia tester Institute of BiomedicalEngineering Chinese Academy of Medical Science TianjinChina) Animals were acclimated to their surroundings for10min daily for three consecutive days in a plexiglass box ona metal grid surface prior to testing On the testing days ratswere allowed to acclimate for 5ndash10min A rigid filament waspressed perpendicularly against the medial plantar surface ofthe hind paw with an increasing force Brisk paw withdrawalor pawflinching accompanied by head turning biting andorlicking upon application of an increasing force was consid-ered as a positive response The paw withdrawal threshold(PWT)was defined as theminimal force (g) required to evokethe cited positive responses Each hind paw of rats was testedthree times and the data were averagedThe interval betweenconsecutive tests of the same paw was 5min The sameprocedure was performed on days 3 6 9 12 15 18 and 21after tumor inoculation Each hind paw of rats was stimulatedwith a focused beam of radiant heat using an analgesiometer(37360 Ugo Basile Italy) underneath the glass surfaceWhenthe paw was withdrawn from the stimulus the PWL wasautomatically recorded to the nearest 01 s The intensity ofstimuli was adjusted to generate an average baseline PWLof approximately 100 s in naive animals The maximumstimulation was controlled lt20 s to prevent potential tissuedamage Paws were alternated randomly to preclude ldquoorderrdquo
4 Evidence-Based Complementary and Alternative Medicine
effects Individual rats were subjected to four tests with a5min interval before surgery and 2 5 8 11 14 17 and 20 daysafter inoculation in a blinded manner The mean PWL wascalculated for each time point in individual rats
28 Histological Evaluation The rats were deeply anes-thetized by pentobarbital and transcardially perfused withsaline on day 21 after inoculation (119899 = 10 per group) The lefttibia from each animal was dissected fixed in 10 formalinovernight decalcified in 15 EDTA-PBS for 7 days andparaffin-embedded The tissue sections (6 120583m) were stainedwith hematoxylin and eosin (HE) Furthermore the numbersof osteoclasts and osteoblasts in the regions were identi-fied by the tartrate-resistant acid phosphatase (TRAP) oralkaline phosphatase (AP) staining using the TRAP or AKPstaining kits (Nanjing Jiancheng Bioengineering InstituteNanjing China) respectivelyThe numbers of osteoclasts andosteoblasts in 10 sections from each rat were counted under alight microscope (Leica DM 2500) in a blinded manner [25]
29 Enzyme-Linked Immunosorbent Assay (ELISA) Periph-eral blood samples were collected from individual rats at0 7 14 or 21 days after inoculation and the concentrationsof serum tartrate-resistant acid phosphatase 5b (TRACP5b)carboxy-terminal pyridinoline cross-linked telopeptides oftype I collagen (ICTP) procollagen type I amino-terminalpropeptide (PINP) and bone alkaline phosphatase (BAP) inindividual rats were determined by ELISA using the specifickits according to the manufacturerrsquos instruction (ThermoScientific Hudson NH USA) The limitation of detectionfor TRACP5b ICTP PINP and BAP was 0078mIUmL25 ngmL 1875 pgmL and 25UL respectively
210 Quantitative RT-PCR Total RNA was extracted fromindividual rat tibias using the TRIzol reagent and reverselytranscribed to cDNA using the first-strand cDNA synthesiskit (Invitrogen)The relative levels of target genemRNA tran-scripts were determined by quantitative RT-PCR using theSYBR Green system and specific primers on the LightCyclersystem (Roche) The sequences of primers are shown inTable 2The PCR amplification was performed in triplicate at95∘C for 10 minutes and was subjected to 40 cycles of 95∘Cfor 15 s and 60∘C for 30 s The relative levels of each gene toGAPDHmRNA transcripts were calculated
211 Western Blot Assay Total tibia proteins were extractedfrom individual rats by sonication in RIPA buffer containingprotease inhibitors (Roche) After quantification of proteinconcentrations using the BCA protein assay kit (ThermoScientific Rockford IL) the protein samples (20120583glane)were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinyli-dene difluoride (PVDF) membranes (Millipore) After beingblocked with 5 fat-free dry milk the membranes wereincubated with anti-OPG (ab73400) anti-GAPDH (6C5ab8245) anti-IL-8 (ab7747) anti-PTHrP (ab85205) anti-M-CSF (ab99109) anti-IGF1 antibody (ab36532) anti-TNF-120572(ab66579) anti-RANKL (EPR4999 ab124797 Abcam Cam-bridge MA USA) and anti-RANK (H-300 sc-9072 SantaCruz Biotechnology) respectively The bound antibodieswere detected using a horseradish peroxidase- (HRP-) con-jugated secondary antibody and visualized by an enhancedchemiluminescence detection system followed by quantify-ing using the Image Quant LAS 4000 (GE Healthcare)
Evidence-Based Complementary and Alternative Medicine 5
0010
0005
000 1000 2000 3000(min)
(AU
)
4000 5000 6000
0000
AB
C DE
(a)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
A
B C DE
minus004
minus002
(b)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
minus004
minus002
(c)
Tetrahydropalmatine
N
O
O
O
O
Imperatorin
OO O
O
H3C
H3C
Isoimperatorin
O
O
OO
CoptisineO
O
O CH3
O
CH3
H3C N+
Palmatine chloride
O
O
O
ON+
Clminus
(d)
Figure 1 HPLC analysis of the components of XZP XZP were extracted with ethanol and the representative active components in the XZPextracts were characterized by HPLC using the standard components of (A) tetrahydropalmatine (B) imperatorin (C) isoimperatorin (D)coptisine and (E) palmatine chloride Data are representative chromatographic histograms at 280 nm of XZP (a) characterized profile (UVchromatograms at 280 nm) of the standard compounds (a) XZP ethanol-extracts (b) and blank (c) from three independent experiments (d)The structures of standard compounds
6 Evidence-Based Complementary and Alternative Medicine
Control
Low dose Medium dose High dose
Placebo 40
30
20
10
00
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
Radi
olog
ical
scor
e
lowastlowast lowastlowastlowastlowast
(a)
Control
Low dose Medium dose High dose
Placebo
100Bo
ne v
olum
e fra
ctio
n BV
TV
(100
)
80
60
40
20
0
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
lowastlowastlowastlowast
(b)
Control
Low dose Medium dose High dose
Placebo
(c)
Figure 2 XZP treatment reduces the cancer-induced bone damage in rats At 21 days after inoculation the tibial bone structure and damagein the individual rats were characterized by X-rad and micro-CT and quantitatively analyzed Subsequently their tibial bone sections werestained with HE Data are representative images of individual groups of rats (119899 = 10 per group) (a) Radiographs of the bone structure andtumor invasion (b) 3D micro-CT images of the tibias (c) HE analysis of the cancer bone tissues (magnification times400) Low dose mediumdose and high dose The rats were treated with low medium or high dose of XZP (lowastlowast119875 lt 001 versus the control group 119875 lt 005 versus theplacebo group lowastlowast119875 lt 001 versus placebo group)
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Evidence-Based Complementary and Alternative Medicine 3
followed by being stored at minus20∘C The extracts were mixedwith cataplasm matrix to generate a proper concentration ofXZP before use in vivo
22 High Performance Liquid Chromatography To deter-mine the potential components the XZP was preparedand analyzed by high performance liquid chromatography(HPLC) using gt98 purity of the standards of tetrahy-dropalmatine imperatorin isoimperatorin coptisine andpalmatine chloride (in methanol) that were purchased fromthe National Institute for Food and Drug Control (BeijingChina) Methanol and acetonitrile (HPLC grade FisherScientific New Jersey USA) and pure water (Wahaha GroupChina) were used for HPLC analysis All other chemicalswere of analytical grade The solutions were filtered through045 120583m membranes and subjected to analysis at 280 nmusing an Agilent Zorbax Eclips XDB C18 column (250 times46mm 45 120583m) on a Waters HPLC system (Waters Corpo-ration USA) equipped with 2487 pump an UV detector anEmpower 2 system controller and a waters 717 plus autosam-pler at 30∘C Samples at 10 120583L were injected and acetonitrile-methanol-water-triethylamine (24 32 44 05) at a flow-rateof 10mL per min was used at the mobile phase Accordingto the chromatographic characteristics of these standardcompounds the peak areas of individual compounds in thesamples were used for determining the contents of thesecompounds and expressed as the percent
23 Animals Female Wistar rats weighing 150ndash180 g werefrom the Department of Experimental Animal SciencesPeking University Health Science Center (Beijing China)Individual rats were housed in specific pathogen free (SPF)facility at 24 plusmn 1∘C on a cycle of 1212 h lightdark andfree access to food and water All experiments were carriedout in accordance with the guidelines of the InternationalAssociation for the Study of Pain [20] and approved bythe Animal Care and Use Committee of China Academy ofChinese Medical Sciences
24 Cell Preparation and Bone Cancer Pain Model Prostatecancer PC-3 cells were prepared and bone cancer pain modelwere established as our previous report [21] Briefly individ-ual rats were anesthetized intraperitoneally (ip) with sodiumpentobarbital (45mgkg) and the left tibia of individual ratswas exposedThe left tibia of individual rats was injected with105 PC-3 cells in 10 120583L Hankrsquos solution and the injection sitewas covered by bone wax followed by closing the wound Agroup of rats (119899 = 10) received the same surgery and the samevolume of vehicle injection and served as the Sham controlsAll rats were subjected to the same postoperational cares
25 Treatment and Groups The Sham control rats weretreated topically with the inert paste as the control (119899 = 10per group)The bone cancer-bearing rats were randomly andtreated topically with the inert paste as the placebo the XZPat 1575 gkg (as the low dose) 315 gkg (medium dose) or63 gkg (high dose) evenly applied on the skin of tumor-bearing tibias covered with gauze and a layer of plastic filmsealed and fixed with desensitized adhesive plaster twice
a day at 800 AM and 2000 PM for consecutive 21 daysbeginning one day after Walker 256 cell inoculation [11]
26 Radiological Analysis The cancer-related osteolyticlesions in the tibia of individual rats were examined by X-rayradiology at 21 days after the inoculation The rats wereanaesthetized and exposed to X-ray (Emerald 125) at 40 KVPfor 120 s followed by the development of X-ray film (HenrySchein blue sensitive film) using a film developer (KonicaSRX-101) The radiological score was calculated according toStewartrsquos radiological score [22] 0 normal bone structurewith no sign of deterioration 1 minor loss of medullarybone 2 substantial loss of medullary bone with erosion ofcortical bone and 3 substantial loss of medullary bone withmajor cortical destruction of the proximal epiphysis
The tibias were scanned by micro-CT and reconstructedwith 8 120583m isotropic voxel size on amicro-CT system (eXploreLocus SP GE Medical Systems) The reconstructed 3D imag-ines of femurs were analyzed usingMicroviewer (GEMedicalSystems) as described previously [23] Micro-CT is used tomeasure several histomorphometric variables including bonevolume (BV) total volume (TV) and bone volume fraction(BVTV) [24]
27 Mechanical Threshold and Paw Withdrawal Latency Theeffects of treatment with XZP on the spontaneous nocifensivebehaviors of the different groups of rats were tested for themechanical threshold and paw withdrawal latency (PWL)in a blinded manner as our previous description [21 2526] In brief individual rats were placed in an invertedplastic chamber on the glass surface of the Paw ThermalStimulator System (UCSD San Diego) for 30min beforethe test Mechanical hyperalgesia was measured using asingle rigid filament attached to a handheld transducer(automatic plantar analgesia tester Institute of BiomedicalEngineering Chinese Academy of Medical Science TianjinChina) Animals were acclimated to their surroundings for10min daily for three consecutive days in a plexiglass box ona metal grid surface prior to testing On the testing days ratswere allowed to acclimate for 5ndash10min A rigid filament waspressed perpendicularly against the medial plantar surface ofthe hind paw with an increasing force Brisk paw withdrawalor pawflinching accompanied by head turning biting andorlicking upon application of an increasing force was consid-ered as a positive response The paw withdrawal threshold(PWT)was defined as theminimal force (g) required to evokethe cited positive responses Each hind paw of rats was testedthree times and the data were averagedThe interval betweenconsecutive tests of the same paw was 5min The sameprocedure was performed on days 3 6 9 12 15 18 and 21after tumor inoculation Each hind paw of rats was stimulatedwith a focused beam of radiant heat using an analgesiometer(37360 Ugo Basile Italy) underneath the glass surfaceWhenthe paw was withdrawn from the stimulus the PWL wasautomatically recorded to the nearest 01 s The intensity ofstimuli was adjusted to generate an average baseline PWLof approximately 100 s in naive animals The maximumstimulation was controlled lt20 s to prevent potential tissuedamage Paws were alternated randomly to preclude ldquoorderrdquo
4 Evidence-Based Complementary and Alternative Medicine
effects Individual rats were subjected to four tests with a5min interval before surgery and 2 5 8 11 14 17 and 20 daysafter inoculation in a blinded manner The mean PWL wascalculated for each time point in individual rats
28 Histological Evaluation The rats were deeply anes-thetized by pentobarbital and transcardially perfused withsaline on day 21 after inoculation (119899 = 10 per group) The lefttibia from each animal was dissected fixed in 10 formalinovernight decalcified in 15 EDTA-PBS for 7 days andparaffin-embedded The tissue sections (6 120583m) were stainedwith hematoxylin and eosin (HE) Furthermore the numbersof osteoclasts and osteoblasts in the regions were identi-fied by the tartrate-resistant acid phosphatase (TRAP) oralkaline phosphatase (AP) staining using the TRAP or AKPstaining kits (Nanjing Jiancheng Bioengineering InstituteNanjing China) respectivelyThe numbers of osteoclasts andosteoblasts in 10 sections from each rat were counted under alight microscope (Leica DM 2500) in a blinded manner [25]
29 Enzyme-Linked Immunosorbent Assay (ELISA) Periph-eral blood samples were collected from individual rats at0 7 14 or 21 days after inoculation and the concentrationsof serum tartrate-resistant acid phosphatase 5b (TRACP5b)carboxy-terminal pyridinoline cross-linked telopeptides oftype I collagen (ICTP) procollagen type I amino-terminalpropeptide (PINP) and bone alkaline phosphatase (BAP) inindividual rats were determined by ELISA using the specifickits according to the manufacturerrsquos instruction (ThermoScientific Hudson NH USA) The limitation of detectionfor TRACP5b ICTP PINP and BAP was 0078mIUmL25 ngmL 1875 pgmL and 25UL respectively
210 Quantitative RT-PCR Total RNA was extracted fromindividual rat tibias using the TRIzol reagent and reverselytranscribed to cDNA using the first-strand cDNA synthesiskit (Invitrogen)The relative levels of target genemRNA tran-scripts were determined by quantitative RT-PCR using theSYBR Green system and specific primers on the LightCyclersystem (Roche) The sequences of primers are shown inTable 2The PCR amplification was performed in triplicate at95∘C for 10 minutes and was subjected to 40 cycles of 95∘Cfor 15 s and 60∘C for 30 s The relative levels of each gene toGAPDHmRNA transcripts were calculated
211 Western Blot Assay Total tibia proteins were extractedfrom individual rats by sonication in RIPA buffer containingprotease inhibitors (Roche) After quantification of proteinconcentrations using the BCA protein assay kit (ThermoScientific Rockford IL) the protein samples (20120583glane)were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinyli-dene difluoride (PVDF) membranes (Millipore) After beingblocked with 5 fat-free dry milk the membranes wereincubated with anti-OPG (ab73400) anti-GAPDH (6C5ab8245) anti-IL-8 (ab7747) anti-PTHrP (ab85205) anti-M-CSF (ab99109) anti-IGF1 antibody (ab36532) anti-TNF-120572(ab66579) anti-RANKL (EPR4999 ab124797 Abcam Cam-bridge MA USA) and anti-RANK (H-300 sc-9072 SantaCruz Biotechnology) respectively The bound antibodieswere detected using a horseradish peroxidase- (HRP-) con-jugated secondary antibody and visualized by an enhancedchemiluminescence detection system followed by quantify-ing using the Image Quant LAS 4000 (GE Healthcare)
Evidence-Based Complementary and Alternative Medicine 5
0010
0005
000 1000 2000 3000(min)
(AU
)
4000 5000 6000
0000
AB
C DE
(a)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
A
B C DE
minus004
minus002
(b)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
minus004
minus002
(c)
Tetrahydropalmatine
N
O
O
O
O
Imperatorin
OO O
O
H3C
H3C
Isoimperatorin
O
O
OO
CoptisineO
O
O CH3
O
CH3
H3C N+
Palmatine chloride
O
O
O
ON+
Clminus
(d)
Figure 1 HPLC analysis of the components of XZP XZP were extracted with ethanol and the representative active components in the XZPextracts were characterized by HPLC using the standard components of (A) tetrahydropalmatine (B) imperatorin (C) isoimperatorin (D)coptisine and (E) palmatine chloride Data are representative chromatographic histograms at 280 nm of XZP (a) characterized profile (UVchromatograms at 280 nm) of the standard compounds (a) XZP ethanol-extracts (b) and blank (c) from three independent experiments (d)The structures of standard compounds
6 Evidence-Based Complementary and Alternative Medicine
Control
Low dose Medium dose High dose
Placebo 40
30
20
10
00
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
Radi
olog
ical
scor
e
lowastlowast lowastlowastlowastlowast
(a)
Control
Low dose Medium dose High dose
Placebo
100Bo
ne v
olum
e fra
ctio
n BV
TV
(100
)
80
60
40
20
0
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
lowastlowastlowastlowast
(b)
Control
Low dose Medium dose High dose
Placebo
(c)
Figure 2 XZP treatment reduces the cancer-induced bone damage in rats At 21 days after inoculation the tibial bone structure and damagein the individual rats were characterized by X-rad and micro-CT and quantitatively analyzed Subsequently their tibial bone sections werestained with HE Data are representative images of individual groups of rats (119899 = 10 per group) (a) Radiographs of the bone structure andtumor invasion (b) 3D micro-CT images of the tibias (c) HE analysis of the cancer bone tissues (magnification times400) Low dose mediumdose and high dose The rats were treated with low medium or high dose of XZP (lowastlowast119875 lt 001 versus the control group 119875 lt 005 versus theplacebo group lowastlowast119875 lt 001 versus placebo group)
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
effects Individual rats were subjected to four tests with a5min interval before surgery and 2 5 8 11 14 17 and 20 daysafter inoculation in a blinded manner The mean PWL wascalculated for each time point in individual rats
28 Histological Evaluation The rats were deeply anes-thetized by pentobarbital and transcardially perfused withsaline on day 21 after inoculation (119899 = 10 per group) The lefttibia from each animal was dissected fixed in 10 formalinovernight decalcified in 15 EDTA-PBS for 7 days andparaffin-embedded The tissue sections (6 120583m) were stainedwith hematoxylin and eosin (HE) Furthermore the numbersof osteoclasts and osteoblasts in the regions were identi-fied by the tartrate-resistant acid phosphatase (TRAP) oralkaline phosphatase (AP) staining using the TRAP or AKPstaining kits (Nanjing Jiancheng Bioengineering InstituteNanjing China) respectivelyThe numbers of osteoclasts andosteoblasts in 10 sections from each rat were counted under alight microscope (Leica DM 2500) in a blinded manner [25]
29 Enzyme-Linked Immunosorbent Assay (ELISA) Periph-eral blood samples were collected from individual rats at0 7 14 or 21 days after inoculation and the concentrationsof serum tartrate-resistant acid phosphatase 5b (TRACP5b)carboxy-terminal pyridinoline cross-linked telopeptides oftype I collagen (ICTP) procollagen type I amino-terminalpropeptide (PINP) and bone alkaline phosphatase (BAP) inindividual rats were determined by ELISA using the specifickits according to the manufacturerrsquos instruction (ThermoScientific Hudson NH USA) The limitation of detectionfor TRACP5b ICTP PINP and BAP was 0078mIUmL25 ngmL 1875 pgmL and 25UL respectively
210 Quantitative RT-PCR Total RNA was extracted fromindividual rat tibias using the TRIzol reagent and reverselytranscribed to cDNA using the first-strand cDNA synthesiskit (Invitrogen)The relative levels of target genemRNA tran-scripts were determined by quantitative RT-PCR using theSYBR Green system and specific primers on the LightCyclersystem (Roche) The sequences of primers are shown inTable 2The PCR amplification was performed in triplicate at95∘C for 10 minutes and was subjected to 40 cycles of 95∘Cfor 15 s and 60∘C for 30 s The relative levels of each gene toGAPDHmRNA transcripts were calculated
211 Western Blot Assay Total tibia proteins were extractedfrom individual rats by sonication in RIPA buffer containingprotease inhibitors (Roche) After quantification of proteinconcentrations using the BCA protein assay kit (ThermoScientific Rockford IL) the protein samples (20120583glane)were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinyli-dene difluoride (PVDF) membranes (Millipore) After beingblocked with 5 fat-free dry milk the membranes wereincubated with anti-OPG (ab73400) anti-GAPDH (6C5ab8245) anti-IL-8 (ab7747) anti-PTHrP (ab85205) anti-M-CSF (ab99109) anti-IGF1 antibody (ab36532) anti-TNF-120572(ab66579) anti-RANKL (EPR4999 ab124797 Abcam Cam-bridge MA USA) and anti-RANK (H-300 sc-9072 SantaCruz Biotechnology) respectively The bound antibodieswere detected using a horseradish peroxidase- (HRP-) con-jugated secondary antibody and visualized by an enhancedchemiluminescence detection system followed by quantify-ing using the Image Quant LAS 4000 (GE Healthcare)
Evidence-Based Complementary and Alternative Medicine 5
0010
0005
000 1000 2000 3000(min)
(AU
)
4000 5000 6000
0000
AB
C DE
(a)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
A
B C DE
minus004
minus002
(b)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
minus004
minus002
(c)
Tetrahydropalmatine
N
O
O
O
O
Imperatorin
OO O
O
H3C
H3C
Isoimperatorin
O
O
OO
CoptisineO
O
O CH3
O
CH3
H3C N+
Palmatine chloride
O
O
O
ON+
Clminus
(d)
Figure 1 HPLC analysis of the components of XZP XZP were extracted with ethanol and the representative active components in the XZPextracts were characterized by HPLC using the standard components of (A) tetrahydropalmatine (B) imperatorin (C) isoimperatorin (D)coptisine and (E) palmatine chloride Data are representative chromatographic histograms at 280 nm of XZP (a) characterized profile (UVchromatograms at 280 nm) of the standard compounds (a) XZP ethanol-extracts (b) and blank (c) from three independent experiments (d)The structures of standard compounds
6 Evidence-Based Complementary and Alternative Medicine
Control
Low dose Medium dose High dose
Placebo 40
30
20
10
00
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
Radi
olog
ical
scor
e
lowastlowast lowastlowastlowastlowast
(a)
Control
Low dose Medium dose High dose
Placebo
100Bo
ne v
olum
e fra
ctio
n BV
TV
(100
)
80
60
40
20
0
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
lowastlowastlowastlowast
(b)
Control
Low dose Medium dose High dose
Placebo
(c)
Figure 2 XZP treatment reduces the cancer-induced bone damage in rats At 21 days after inoculation the tibial bone structure and damagein the individual rats were characterized by X-rad and micro-CT and quantitatively analyzed Subsequently their tibial bone sections werestained with HE Data are representative images of individual groups of rats (119899 = 10 per group) (a) Radiographs of the bone structure andtumor invasion (b) 3D micro-CT images of the tibias (c) HE analysis of the cancer bone tissues (magnification times400) Low dose mediumdose and high dose The rats were treated with low medium or high dose of XZP (lowastlowast119875 lt 001 versus the control group 119875 lt 005 versus theplacebo group lowastlowast119875 lt 001 versus placebo group)
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Evidence-Based Complementary and Alternative Medicine 5
0010
0005
000 1000 2000 3000(min)
(AU
)
4000 5000 6000
0000
AB
C DE
(a)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
A
B C DE
minus004
minus002
(b)
(min)
(AU
)
000
000
002
1000 2000 3000 4000 5000 6000
minus004
minus002
(c)
Tetrahydropalmatine
N
O
O
O
O
Imperatorin
OO O
O
H3C
H3C
Isoimperatorin
O
O
OO
CoptisineO
O
O CH3
O
CH3
H3C N+
Palmatine chloride
O
O
O
ON+
Clminus
(d)
Figure 1 HPLC analysis of the components of XZP XZP were extracted with ethanol and the representative active components in the XZPextracts were characterized by HPLC using the standard components of (A) tetrahydropalmatine (B) imperatorin (C) isoimperatorin (D)coptisine and (E) palmatine chloride Data are representative chromatographic histograms at 280 nm of XZP (a) characterized profile (UVchromatograms at 280 nm) of the standard compounds (a) XZP ethanol-extracts (b) and blank (c) from three independent experiments (d)The structures of standard compounds
6 Evidence-Based Complementary and Alternative Medicine
Control
Low dose Medium dose High dose
Placebo 40
30
20
10
00
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
Radi
olog
ical
scor
e
lowastlowast lowastlowastlowastlowast
(a)
Control
Low dose Medium dose High dose
Placebo
100Bo
ne v
olum
e fra
ctio
n BV
TV
(100
)
80
60
40
20
0
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
lowastlowastlowastlowast
(b)
Control
Low dose Medium dose High dose
Placebo
(c)
Figure 2 XZP treatment reduces the cancer-induced bone damage in rats At 21 days after inoculation the tibial bone structure and damagein the individual rats were characterized by X-rad and micro-CT and quantitatively analyzed Subsequently their tibial bone sections werestained with HE Data are representative images of individual groups of rats (119899 = 10 per group) (a) Radiographs of the bone structure andtumor invasion (b) 3D micro-CT images of the tibias (c) HE analysis of the cancer bone tissues (magnification times400) Low dose mediumdose and high dose The rats were treated with low medium or high dose of XZP (lowastlowast119875 lt 001 versus the control group 119875 lt 005 versus theplacebo group lowastlowast119875 lt 001 versus placebo group)
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
6 Evidence-Based Complementary and Alternative Medicine
Control
Low dose Medium dose High dose
Placebo 40
30
20
10
00
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
Radi
olog
ical
scor
e
lowastlowast lowastlowastlowastlowast
(a)
Control
Low dose Medium dose High dose
Placebo
100Bo
ne v
olum
e fra
ctio
n BV
TV
(100
)
80
60
40
20
0
Con
trol
Plac
ebo
grou
p
Low
dos
e
Med
ium
dos
e
Hig
h do
se
lowastlowastlowastlowast
(b)
Control
Low dose Medium dose High dose
Placebo
(c)
Figure 2 XZP treatment reduces the cancer-induced bone damage in rats At 21 days after inoculation the tibial bone structure and damagein the individual rats were characterized by X-rad and micro-CT and quantitatively analyzed Subsequently their tibial bone sections werestained with HE Data are representative images of individual groups of rats (119899 = 10 per group) (a) Radiographs of the bone structure andtumor invasion (b) 3D micro-CT images of the tibias (c) HE analysis of the cancer bone tissues (magnification times400) Low dose mediumdose and high dose The rats were treated with low medium or high dose of XZP (lowastlowast119875 lt 001 versus the control group 119875 lt 005 versus theplacebo group lowastlowast119875 lt 001 versus placebo group)
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Evidence-Based Complementary and Alternative Medicine 7
Days after tumor cell implantation
Mec
hani
cal t
hres
hold
(g)
00
3
6
9
12
3 6 9 12 15 18 21
lowastlowast
lowast
lowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast
lowastlowastlowast
lowast
lowast
lowastlowastlowast
lowastlowastlowast
ControlPlacebo group
Low doseMedium doseHigh dose
(a)
Days after tumor cell implantation
Paw
with
draw
al la
tenc
y (s
)
0
5
10
15
20
25
0 3 6 9 12 15 2118
ControlPlacebo group
Low doseMedium doseHigh dose
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowastlowast lowastlowast
lowastlowast lowastlowast
lowastlowast
lowastlowastlowast
lowastlowastlowast
lowast lowast lowastlowast
lowastlowastlowast
(b)
Figure 3 XZP treatment mitigates the bone cancer-related nociceptive behaviors in rats The mechanical allodynia and paw withdrawallatency of individual rats before and at the indicated time points after inoculation were measured Data are expressed as the means plusmn SD ofindividual groups of rats (119899 = 10 per group) (a)The changes in themechanical allodynia (b)The changes in thermal hyperalgesia lowast119875 lt 005lowastlowast
119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
212 Statistical Analysis Data are expressed as the mean plusmnstandard deviation (SD) and analyzed by two-way repeatedANOVA or by one-way ANOVA followed by a Newman-Keuls test using SPSS 130 statistical software A 119875 value oflt005 was considered statistically significant
3 Results
31 HPLC Analysis of XZP Our previous study has shownthat XZP prepared from six herbs of Xuejie (Dragonrsquos blood)Yanhusuo (Corydalis rhizoma) Ruxiang (Olibanum) Moyao(Myrrha) Qingdai (Natural Indigo) and Bingpian (Borne-olum Syntheticum) significantly alleviates bone cancer painin patients with middlelate stage of cancer [11] To under-stand the effect of XZP themajor components in the ethanol-extracted XZP were analyzed by HPLC We found that theXZP contained tetrahydropalmatine (00568) imperatorin(00041) isoimperatorin (00122) coptisine (00358)and palmatine chloride (0112 Figure 1)
32 The Effect of XZP on the Implanted Cancer Growthand Bone Destruction At 21 days after inoculation all ratswere sacrificed and their tibial bones were examined by X-rad and micro-CT as well as pathology while there is noradiological change in the tibial bones of the control groupof rats (Figure 2(a)) Less degrees of medullary bone loss andthe cortical bone erosion were apparent in the rats that hadbeen treated with XZP Quantitative analysis revealed thatthe radiological scores in the rats that had been treated withdifferent doses of XZP were significantly less than that ofthe placebo group (119875 lt 005 or 119875 lt 001) although theywere still significantly higher than that of the healthy controlsMicro-CT examinations indicated that the percentages ofbone volume in total volume in the XZP-treated rats were
significantly greater than that of the placebo group of rats(119875 lt 005 or 119875 lt 001 Figure 2(b)) and the effect ofXZP on preserving the bone structure appeared to be dose-dependent In addition histological examination revealedthat while many cancer cells are invaded in the bone tissuesof the placebo group of rats the numbers of invaded cancercells in the bone of the XZP-treated rats were obviouslyreduced particularly from the rats that had been treated witha higher dose of XZP (Figure 2(c)) Collectively treatmentwith XZP mitigated the cancer invasion into the bone andcancer-related bone destruction in rats
33 The Effect of XZP Treatment on the Nociceptive Behaviorsin Rats Bone cancer usually causes severe pain in humansand nociceptive responses in animals The effects of XZPon bone cancer-related nociceptive behaviors of the PATand PWL in the different groups of rats were measuredlongitudinally after inoculation While similar levels of PWTwere observed in the control rats throughout the obser-vation period the levels of PWT were gradually reduced(Figure 3(a)) In contrast treatment with different dosesof XZP significantly mitigated the mechanical allodyniain a dose and time-dependent manner A similar patternof the PWL was observed in the different groups of rats(Figure 3(b)) Hence treatment with XZP reduced themechanical and thermal nociceptive behaviors in rats withbone cancer
34 The Effect of XZP Treatment on the Levels of Osteoclastand Osteoblast Activity in Bone Cancer Rats To understandthe effect of XZP treatment on the levels of osteoclast andosteoblast activity the levels of serumTRACP5b ICTP PINPand BAP in individual rats were measured longitudinally byELISA First there was no significant difference in the levels
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
8 Evidence-Based Complementary and Alternative Medicine
Tart
rate
-res
istan
t aci
d ph
osph
atas
e 5b
(UL
)
ControlPlacebo groupLow dose
Medium doseHigh dose
00
20
40
60
lowastlowast
lowast lowastlowast
lowast
lowast
lowastlowast
7 14 21
(d)
(a)
0
5
10
15
20
0 7 14 21
(d)
lowast
lowast
lowastlowast
lowast lowast
lowastlowast
lowast
Carb
oxy-
term
inal
telo
pept
ide
of ty
pe I
colla
gen
(120583g
L)
ControlPlacebo groupLow dose
Medium doseHigh dose
(b)
0
0
140
210
280
350
420
PIN
P (120583
gL)
lowastlowast
lowastlowast
lowast
lowast
lowast
lowast
0 7 14 21
(d)
ControlPlacebo groupLow dose
Medium doseHigh dose
(c)
0
2
4
6
8BA
LP (120583
gL) lowast lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
Figure 4 XZP treatment modulates the levels of serum TRACP5b ICTP PINP and BALP in ratsThe levels of serumTRACP5b ICTP PINPand BALP in individual rats were analyzed at the indicated time points before and after prostate cancer cell inoculation Data are expressedas the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of serum TRACP5b (b)The levels of serum ICTP (c)The levels of serum PINP (d)The levels of serum BAP lowast119875 lt 005 versus the control group 119875 lt 005 versus theplacebo group
of serum TRACP5b ICTP PINP and BAP among thedifferent groups of rats before inoculation (Figure 4) Secondthe levels of serum TRACP5b ICTP PINP and BAP in theplacebo group of rats were significantly higher than that ofthe control throughout the observation period (119875 lt 001)In contrast the levels of serum TRACP5b ICTP PINP andBAP in the XZP-treated rats particularly for the rats thatreceived a high dose of XZP were significantly lower thanthat of the placebo group (119875 lt 005) although they remainedsignificantly higher than that of the controls (119875 lt 005)
In parallel the activity of osteoclasts and osteoblastsin the bone tissues from the different groups of rats was
further evaluated by AP and TRAP staining respectivelyThere were a few AP+ osteoblasts in the bone sections ofthe control rats and increased numbers of AP+ osteoblastswere detected in the bone sections of the placebo group ofrats but not obviously in the bone sections from the XZP-treated rats (Figure 5(a)) Quantitative analysis indicated thatthe numbers of AP+ osteoblasts in the bone sections fromthe XZP-treated rats were significantly less than that in theplacebo rats at 14 and 21 days after inoculation (119875 lt 005)but remained significantly greater than that in the controls(119875 lt 005) Furthermore the numbers of TRAP+ osteoclastsin the XZP-treated rats were significantly greater than that
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Evidence-Based Complementary and Alternative Medicine 9
Control Placebo
Low dose Medium dose High dose
0
100
200
300
400
500
NO
bT
A (
mm
2)
lowast
lowast
lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(a)
Control Placebo
Low dose Medium dose High dose
0
20
40
60
80
100
0 7 14 21
NO
cT
A (
mm
2)
lowastlowast
lowastlowast
lowast
lowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
(d)
(b)
Figure 5 Histological evaluation of osteoblast and osteoclast activity andmacrophage infiltrationThe numbers of osteoblasts and osteoclastsin the cancer-invaded tibial bones of individual groups of rats were characterized by AP and TRAP-enzymatic staining respectively Theyellow-brown staining represents AP+ osteoblasts or TRAP+ osteoclasts respectively Data are representative images (magnification times400)and are expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Characterizationof osteoblasts (b) characterization of osteoclasts lowast119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
in the controls (119875 lt 005) but were significantly less thanin the placebo group of rats at 7 14 and 21 days afterinoculation (119875 lt 005 Figure 5(b)) Together treatmentwith XZP mitigated the cancer-stimulated osteoclast andosteoblast activity in vivo
35 XZP Treatment Modulates the OPGRANKLRANKSignaling and Other Bone Regulator Expressions in theBone of Rats The bone metabolism is regulated by theOPGRANKLRANK signaling inflammatory mediatorsand hormone and IGF-1 [12ndash14] To understand molecular
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
10 Evidence-Based Complementary and Alternative Medicine
00
20
40
60
80
100
Relat
ive O
PG m
RNA
leve
ls(O
PGG
APD
H)
lowast
lowastlowast
lowast
lowastlowast
lowastlowast
lowast
ControlPlacebo group
Medium dose High dose
Low dose
0 7 14 21
(d)
(a)
00
50
100
150
Relat
ive R
AN
KL m
RNA
leve
ls(R
AN
KLG
APD
H)
lowast lowast lowast lowast lowast lowast
lowast
lowastlowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(b)
00
40
80
120
Relat
ive R
AN
K m
RNA
leve
ls(R
AN
KG
APD
H)
lowast lowast lowast lowast
lowastlowastlowast lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(c)
00
30
60
90
120Re
lativ
e IL8
mRN
A le
vels
(IL8
GA
PDH
)
lowast
lowastlowast
lowast lowast lowast lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(d)
00
20
40
60
80
Relat
ive P
THrP
mRN
A le
vels
(PTH
rPG
APD
H)
lowastlowast
lowast lowastlowast
lowastlowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(e)
00
30
60
90
Relat
ive M
-CSF
mRN
A le
vels
(M-C
SFG
APD
H)
lowastlowastlowast
lowast lowastlowast
lowast
lowast
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(f)
Figure 6 Continued
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Evidence-Based Complementary and Alternative Medicine 11
120
00
30
60
90
Relat
ive I
GF-
1 m
RNA
leve
ls(IG
F-1
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(g)
00
30
60
90
lowast
lowast lowastlowastlowast
Relat
ive T
NF-120572
mRN
A le
vels
(TN
F-120572
GA
PDH
)
ControlPlacebo groupLow dose
Medium doseHigh dose
0 7 14 21
(d)
(h)
Figure 6 Quantitative RT-PCR analysis of the relative levels of target gene mRNA transcripts in the tibial bones of individual groups ofrats The relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 mRNA transcripts to the control GAPDH in thetibial bones of individual rats at the indicated time points were determined by quantitative RT-PCR Data are expressed as the means plusmn SDof individual groups of rats (119899 = 10 per group) from three separated experiments (a) The levels of OPG mRNA transcripts (b) the levels ofRANKLmRNA transcripts (c) the levels of RNAKmRNA transcripts (d) the levels of IL-8mRNA transcripts (e) the levels of PTHrPmRNAtranscripts (f) the levels of M-CSF mRNA transcripts (g) the levels of IGF-1 mRNA transcripts (h) the levels of TNF-120572mRNA transcriptslowast
119875 lt 005 versus the control group 119875 lt 005 versus the placebo group
mechanisms by which XZP inhibited the cancer-related bonedestruction the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones from thedifferent groups of rats were analyzed by quantitative RT-PCRThe relative levels of OPGmRNA transcripts to controlGAPDH in the XZP-treated rats at different time points postinoculation were significantly higher than that in the placebogroup (119875 lt 005 or 119875 lt 001) but were significantlyhigher than that in the control (119875 lt 005 Figure 6(a))In contrast the relative levels of RANKL IL-8 PTHrP M-CSF IGF-1 and TNF-120572 but not RANK mRNA transcripts inthe placebo group at different time points after inoculationwere significantly higher than that in the controls (119875 lt 005or 119875 lt 001 Figures 6(b)ndash6(h)) except that there was nosignificant difference in the relative levels of IGF-1 M-CSFand TNF-120572 mRNA transcripts between these two groups ofrats at earlier time points after inoculationThe relative levelsof RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572mRNA transcripts in the XZP-treated rats were significantlylower than that in the placebo group of rats (119875 lt 005 or119875 lt 001) except that the relative levels of IL-8 PTHrP M-CSF and TNF-120572mRNA were higher than that in the placebogroup at earlier time points after inoculation
FurtherWestern blot analysis revealed that in comparisonwith that in the placebo group of rats significantly higherlevels of OPG and lower levels of RANKL RANK IL-8PTHrPM-CSF IGF-1 and TNF-120572were detected in the bonesof the control group of rats (Figure 7) In contrast the relativelevels of OPG in the bones from the XZP-treated rats signif-icantly increased while the relative levels of RANKL RANK
IL-8 PTHrP M-CSF IGF-1 and TNF-120572 in the bones fromthe XZP-treated rats were significantly reduced (119875 lt 005119875 lt 001 or 119875 lt 0001) The modulatory effects of differentdoses of XZP were dose-dependent in rats Collectively thesedata indicated that XZP treatment modulated the expressionof OPGRANKLRANK pathway events and other bonemetabolic regulators as well as inflammatorymediators in thecancer bones of rats
4 Discussion
In this study we examined the effects of XZP treatment onthe bone prostate cancer nociceptive behaviors and poten-tial mechanisms underlying the action of XZP treatmentin a rat model of prostate cancer bone pain We foundthat XZP treatment significantly mitigated cancer invasionand bone damage as well as nociceptive behaviors in ratsconsistent with our previous findings [11] Furthermore wefound that XZP treatment significantly reduced the levelsof serum TRACP5b CIPT PINP and BAP and the AP+osteoblasts and TRAP+ osteoclasts in rats related to that ofplacebo group demonstrating that XZP treatment mitigatedthe bone cancer-induced aberrant activation of osteoclastsand osteoblasts In addition we found that XZP treatmentupregulated OPG expression but downregulated RANKLRANK Il-8 M-CSF TNF-120572 PTHrP and IGF-1 expression inthe tibial bones of rats as compared with that in the placebogroup These data indicated that XZP treatment inhibitedthe activation of bone cancer-related RANKLRANKOPGpathway and inflammation leading to a reduction in
12 Evidence-Based Complementary and Alternative Medicine
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Figure 7Western blot analysis of the relative levels of OPG RANKL RANK IL-8 PTHrP M-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of ratsThe relative levels of OPG RANKL RANK IL-8 PTHrPM-CSF IGF-1 and TNF-120572 expression in the tibialbones of individual groups of rats at the indicated time points were determined by Western blot assays Data are representative images andare expressed as the means plusmn SD of individual groups of rats (119899 = 10 per group) from three separated experiments (a) Western blot analysis(b) quantitative analysis lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus the control group 119875 lt 005 versus the placebo group
the cancer-related bone destruction and alleviation of bonecancer pain in rats To the best of our knowledge this was thefirst study on the mechanisms underlying the action of XZPOur findings may provide new insights into understandingthe molecular pathogenesis of bone cancer pain and aid indesign of new therapies for intervention of bone cancer pain
Bone cancer pain is associated with bone destructionwhich is attributed to aberrant activation of osteoclasts [3 2728] The functional development of osteoclasts is positivelyregulated by the RANKLRANK-related signaling but is neg-atively regulated by OPG [12 29] Furthermore the activityof osteoclasts is also regulated by the IGF-1-related signalingand PTHrP and other hormones [13ndash18] In this study wefound that in comparison with that in the placebo groupXZP treatment significantly mitigated the bone cancer-upregulated RANKL RANK PTHrP IGF-1 IL-8 M-CSFand TNF-120572 but increased the levels of OPG expression in the
tibial bones of rats accompanied by decreased numbers ofosteoclasts and osteoblasts These data were consistent withprevious findings [30ndash33] and support the notion that theRANKLRANKOPG signaling is crucial for the develop-ment and progression of bone cancer pain [12 34ndash36] It islikely that bone metastasis of cancer cells recruits inflamma-tory infiltrates which secrete inflammatory mediators suchas IL-8 TNF-120572 andM-CSF andpromotes osteoclastogenesisleading to the bone destruction and bone cancer pain Hencethe RANKLRANKOPG signaling may be a target for thedesign of new therapies for intervention of bone cancer pain
Aberrant activation of osteoclasts also compensativelypromotes the activity of osteoblasts to balance the boneabsorption and formation [12 35 37 38] We detected highlevels of serumPINP andBAP and increased numbers of AP+osteoblasts in the tibial bones of the placebo group of ratsIndeed the levels of serum PINP and BAP have been used
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
Evidence-Based Complementary and Alternative Medicine 13
as biomarkers for evaluating the bone turnover in patientswith bonemetastatic cancer [15ndash17]However XZP treatmentsignificantly reduced the values of serum TRACP5b andCIPT but also decreased the levels of serumPINP and BAP inrats further supporting that aberrant activation of osteoclastsenhanced the activity of osteoblasts in the bone cancerrats Therefore aberrant activation of osteoclasts should betherapeutic targets for treatment of bone cancer pain [39 40]
In TCM tumor-caused stagnation and insufficiency ofQi and blood flow in the body are responsible for thedevelopment of bone cancer pain while increase in the Qiand blood flow such as softening hard lumps dispellingnodes and warming the channels is crucial for alleviationof bone cancer pain [10] We found that XZP treatmentsignificantly alleviated the bone cancer-related nociceptivebehaviors Indeed the herbs in the XZP formula have beenused for the control of pain in TCM for many yearsDragonrsquos blood contains cochinchinenin A cochinchineninB and loureirin B and can inhibit the expression of Cox2and substance P and reduce the levels of intracellular cal-cium leading to potent analgesic activity [41 42] Yanhusuo(Corydalis rhizome) can also significantly inhibit formalin-evoked spontaneous nociceptive responses in rodents [43]and combination of Yanhusuo (Corydalis rhizome) andBaizhi(Angelicae dahuricae) alleviates pain in a clinical trial [44]Ruxiang (Olibanum) and Moyao (Myrrha) can also haveanti-inflammatory and analgesic activities respectively [45]It is possible that these drugs in the XZP synergisticallyinhibited inflammation and promoted the Qi and blood flowalleviating bone cancer nociception in rats
In conclusion our data demonstrated that XZP treatmentsignificantly reduced cancer invasion related osteoclast activ-ity and bone destruction as well as nociceptive behaviorsin rats by modulating the RANKLRANKOPG signalingand the expression of inflammatory mediators and bonemetabolic regulators Therefore our findings may provide anew basis for the design of therapies for intervention of bonecancer pain
Disclosure
Yanju Bao and Yebo Gao are co-first authors
Conflict of Interests
All authors declare no conflict of interests
Acknowledgment
The current work was partially supported by the grants ofNational Natural Science Foundation Project of China (no81302961 no 81273718 and no 81202931)
References
[1] M A C Sabino and P W Mantyh ldquoPathophysiology of bonecancer painrdquo Journal of Supportive Oncology vol 3 no 1 pp15ndash24 2005
[2] S Mercadante ldquoMalignant bone pain pathophysiology andtreatmentrdquo Pain vol 69 no 1-2 pp 1ndash18 1997
[3] R E Coleman ldquoClinical features of metastatic bone disease andrisk of skeletal morbidityrdquo Clinical Cancer Research vol 12 no20 part 2 pp 6243sndash6249s 2006
[4] E Chow L Zeng N Salvo K Dennis M Tsao and S LutzldquoUpdate on the systematic review of palliative radiotherapytrials for bone metastasesrdquo Clinical Oncology vol 24 no 2 pp112ndash124 2012
[5] R K Portenoy D Payne and P Jacobsen ldquoBreakthrough paincharacteristics and impact in patients with cancer painrdquo Painvol 81 no 1-2 pp 129ndash134 1999
[6] H J McQuay S L Collins D Carroll and R A MooreldquoRadiotherapy for the palliation of painful bone metastasesrdquoCochrane Database of Systematic Reviews no 2 Article IDCD001793 2000
[7] M D Michaelson andM R Smith ldquoBisphosphonates for treat-ment and prevention of bone metastasesrdquo Journal of ClinicalOncology vol 23 no 32 pp 8219ndash8224 2005
[8] F K L Chan ldquoPrimer managing NSAID-induced ulcercomplicationsmdashbalancing gastrointestinal and cardiovascularrisksrdquoNature Clinical Practice Gastroenterology andHepatologyvol 3 no 10 pp 563ndash573 2006
[9] M Lapeyre-Mestre AM RuedaDe CastroM-P Bareille et alldquoNon-steroidal anti-inflammatory drug-related hepatic damagein France and Spain analysis fromnational spontaneous report-ing systemsrdquo Fundamental and Clinical Pharmacology vol 20no 4 pp 391ndash395 2006
[10] C Lin X Lin and J Yang ldquoAn observation on combined useof chemotherapy and traditional Chinese medicine to relievecancer painrdquo Journal of Traditional ChineseMedicine vol 16 no4 pp 267ndash269 1996
[11] Y-J Bao B-J Hua W Hou H-S Lin X-B Zhang and G-X Yang ldquoAlleviation of cancerous pain by external compresswith Xiaozheng Zhitong Pasterdquo Chinese Journal of IntegrativeMedicine vol 16 no 4 pp 309ndash314 2010
[12] N M Luger P Honore M A C Sabino et al ldquoOsteoprotegerindiminishes advanced bone cancer painrdquo Cancer Research vol61 no 10 pp 4038ndash4047 2001
[13] S Adami A Zivelonghi V Braga et al ldquoInsulin-like growthfactor-1 is associated with bone formation markers PTH andbone mineral density in healthy premenopausal womenrdquo Bonevol 46 no 1 pp 244ndash247 2010
[14] T Kubota H Z Elalieh N Saless et al ldquoInsulin-like growthfactor-1 receptor in mature osteoblasts is required for periostealbone formation induced by reloadingrdquo Acta Astronautica vol92 no 1 pp 73ndash78 2013
[15] N Abildgaard K Brixen J E Kristensen E F Eriksen J LNielsen and L Heickendorff ldquoComparison of five biochemicalmarkers of bone resorption in multiple myeloma elevated pre-treatment levels of S-ICTP and U-Ntx are predictive for earlyprogression of the bone disease during standard chemotherapyrdquoBritish Journal of Haematology vol 120 no 2 pp 235ndash2422003
[16] D J Leeming M Koizumi I Byrjalsen B Li P Qvist andL B Tanko ldquoThe relative use of eight collagenous and nonco-llagenous markers for diagnosis of skeletal metastases inbreast prostate or lung cancer patientsrdquo Cancer EpidemiologyBiomarkers and Prevention vol 15 no 1 pp 32ndash38 2006
[17] A G Zafeirakis G A Papatheodorou and G S LimourisldquoClinical and imaging correlations of bone turnover markers
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
14 Evidence-Based Complementary and Alternative Medicine
in prostate cancer patients with bone only metastasesrdquo NuclearMedicine Communications vol 31 no 3 pp 249ndash253 2010
[18] P Mantyh ldquoBone cancer pain causes consequences and thera-peutic opportunitiesrdquo Pain vol 154 supplement 1 pp S54ndashS622013
[19] T Yoneda K Hata M Nakanishi et al ldquoInvolvement of acidicmicroenvironment in the pathophysiology of cancer-associatedbone painrdquo Bone vol 48 no 1 pp 100ndash105 2011
[20] M Zimmermann ldquoEthical guidelines for investigations ofexperimental pain in conscious animalsrdquo Pain vol 16 no 2 pp109ndash110 1983
[21] Y Bao BHuaWHou et al ldquoInvolvement of protease-activatedreceptor 2 in nociceptive behavior in a rat model of bonecancerrdquo Journal of Molecular Neuroscience vol 52 no 4 pp566ndash576 2014
[22] S M Sweitzer R W Colburn M Rutkowski and J ADeLeo ldquoAcute peripheral inflammation induces moderate glialactivation and spinal IL-1120573 expression that correlates with painbehavior in the ratrdquo Brain Research vol 829 no 1-2 pp 209ndash221 1999
[23] F Lamoureux M Baudrsquohuin L Rodriguez Calleja et al ldquoSelec-tive inhibition of BETbromodomain epigenetic signalling inter-feres with the bone-associated tumour vicious cyclerdquo NatureCommunications vol 5 p 3511 2014
[24] A Parsa N Ibrahim B Hassan P van der Stelt and DWismeijer ldquoBone quality evaluation at dental implant site usingmultislice CT micro-CT and cone beam CTrdquo Clinical OralImplants Research vol 26 no 1 pp e1ndashe7 2015
[25] JWang R Zhang CDong et al ldquoTopical treatmentwith Tong-Luo-San-Jie gel alleviates bone cancer pain in ratsrdquo Journal ofEthnopharmacology vol 143 no 3 pp 905ndash913 2012
[26] Y Bao W Hou R Liu et al ldquoPAR2-mediated upregulation ofBDNF contributes to central sensitization in bone cancer painrdquoMolecular Pain vol 10 no 1 article 28 2014
[27] R E Coleman ldquoMetastatic bone disease clinical featurespathophysiology and treatment strategiesrdquo Cancer TreatmentReviews vol 27 no 3 pp 165ndash176 2001
[28] V DePuy K J Anstrom L D Castel K A Schulman KP Weinfurt and F Saad ldquoEffects of skeletal morbidities onlongitudinal patient-reported outcomes and survival in patientswith metastatic prostate cancerrdquo Supportive Care in Cancer vol15 no 7 pp 869ndash876 2007
[29] J R Canon M Roudier R Bryant et al ldquoInhibition of RANKLblocks skeletal tumor progression and improves survival ina mouse model of breast cancer bone metastasisrdquo Clinical ampExperimental Metastasis vol 25 no 2 pp 119ndash129 2008
[30] D BMach S D Rogers M C Sabino et al ldquoOrigins of skeletalpain sensory and sympathetic innervation of themouse femurrdquoNeuroscience vol 113 no 1 pp 155ndash166 2002
[31] S J Medhurst K Walker M Bowes et al ldquoA rat model of bonecancer painrdquo Pain vol 96 no 1-2 pp 129ndash140 2002
[32] P W Wacnik L J Kehl T M Trempe M L RamnaraineA J Beitz and G L Wilcox ldquoTumor implantation in mousehumerus evokes movement-related hyperalgesia exceeding thatevoked by intramuscular carrageenanrdquo Pain vol 101 no 1-2 pp175ndash186 2003
[33] M El Mouedden and T F Meert ldquoEvaluation of pain-relatedbehavior bone destruction and effectiveness of fentanyl sufen-tanil and morphine in a murine model of cancer painrdquoPharmacology Biochemistry and Behavior vol 82 no 1 pp 109ndash119 2005
[34] D R Clohisy and P W Mantyh ldquoBone cancer pain and therole of RANKLOPGrdquo Journal of Musculoskeletal NeuronalInteractions vol 4 no 3 pp 293ndash300 2004
[35] M P Roudier S D Bain and W C Dougall ldquoEffects of theRANKL inhibitor osteoprotegerin on the pain andhistopathol-ogy of bone cancer in ratsrdquo Clinical and Experimental Metasta-sis vol 23 no 3-4 pp 167ndash175 2006
[36] S Fili M Karalaki and B Schaller ldquoMechanism of bonemetastasis the role of osteoprotegerin and of the host-tissuemicroenvironment-related survival factorsrdquoCancer Letters vol283 no 1 pp 10ndash19 2009
[37] M C Bezerra J F Carvalho A S Prokopowitsch and R M RPereira ldquoRANK RANKL and osteoprotegerin in arthritic bonelossrdquo Brazilian Journal of Medical and Biological Research vol38 no 2 pp 161ndash170 2005
[38] Y-D Bai F-S Yang K Xuan Y-X Bai and B-L WuldquoInhibition of RANKRANKL signal transduction pathwaya promising approach for osteoporosis treatmentrdquo MedicalHypotheses vol 71 no 2 pp 256ndash258 2008
[39] M El Mouedden and T F Meert ldquoThe impact of the opioidsfentanyl and morphine on nociception and bone destruction ina murine model of bone cancer painrdquo Pharmacology Biochem-istry and Behavior vol 87 no 1 pp 30ndash40 2007
[40] P Zwolak A Z Dudek V D Bodempudi et al ldquoLocal irra-diation in combination with bevacizumab enhances radiationcontrol of bone destruction and cancer-induced pain in amodelof bonemetastasesrdquo International Journal of Cancer vol 122 no3 pp 681ndash688 2008
[41] Y S Li J X Wang M M Jia M Liu X-J Li and H-B Tang ldquoDragonrsquos blood inhibits chronic inflammatory andneuropathic pain responses by blocking the synthesis andrelease of substance P in ratsrdquo Journal of PharmacologicalSciences vol 118 no 1 pp 43ndash54 2012
[42] L-S Wei S Chen X-J Huang J Yao and X-M Liu ldquoMaterialbasis for inhibition of dragonrsquos blood on capsaicin-inducedTRPV1 receptor currents in rat dorsal root ganglion neuronsrdquoEuropean Journal of Pharmacology vol 702 no 1ndash3 pp 275ndash284 2013
[43] C Wang S Wang G Fan and H Zou ldquoScreening of antinoci-ceptive components in Corydalis yanhusuoWTWang by com-prehensive two-dimensional liquid chromatographytandemmass spectrometryrdquoAnalytical andBioanalytical Chemistry vol396 no 5 pp 1731ndash1740 2010
[44] C-S Yuan S R Mehendale C-Z Wang et al ldquoEffects ofCorydalis yanhusuo and Angelicae dahuricae on cold pressor-induced pain in humans a controlled trialrdquo The Journal ofClinical Pharmacology vol 44 no 11 pp 1323ndash1327 2004
[45] S Su YHua YWang et al ldquoEvaluation of the anti-inflammato-ry and analgesic properties of individual and combined extractsfrom Commiphora myrrha and Boswellia carteriirdquo Journal ofEthnopharmacology vol 139 no 2 pp 649ndash656 2012
![[XLS]workroom.homestead.comworkroom.homestead.com/Scholarships.xlsx · Web viewAll Submissions Must be Topically Focused Upon the Social and/or Experimental Orgone-Biophysical Aspects](https://static.documents.pub/doc/80x56/5ae523537f8b9a3d3b8b6f68/xls-viewall-submissions-must-be-topically-focused-upon-the-social-andor-experimental.jpg)
