1Guangzhou Hospital of Traditional Chinese Medicine Guangzhou 510130 China2Nanfang Hospital Southern Medical University Guangzhou 510515 China
Correspondence should be addressed to Zhuoming Lu 739835365qqcom
Received 27 April 2016 Revised 1 September 2016 Accepted 10 October 2016
Academic Editor Monica Borgatti
Copyright copy 2016 Zhuoming Lu 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
A previous study conducted by our group demonstrated that Radix Astragali compounded with Codonopsis pilosula and Plastrumtestudinis was effective in treating pediatric 120573-thalassemia in a randomized controlled clinical trial However the mechanism ofaction that underpins this treatment remains to be elucidated Blood was collected from patients participating in this clinical trialand nucleated red blood cell-enriched mononuclear cells were isolated to facilitate the extraction of RNA and protein RT-PCR wasused to monitor the expression of globin genes and p38 MAPK and total and phosphorylated p38 MAPK expression was assessedusing Western blot analysis Expression of 120572- 120573- and A120574-globin mRNAs was not significantly affected following treatment withR Astragali or the compounded formulation However G120574-globin mRNA levels increased significantly in both treatment groups(when compared with pretreatment levels) following 12 weeks of treatment Moreover posttreatment G120574-globin expression wassignificantly higher in both treatment groups compared with the control group Although neither p38 MAPK mRNA nor proteinlevels were affected by the treatments posttreatment phosphorylation of p38 MAPK was significantly increased in the R Astragaliand compounded formulation groups compared with the control group These data suggest that the molecular mechanisms thatunderpin the efficacious use of R Astragali (and its compounded formulation) in pediatric 120573-thalassemia treatment facilitate theinduction of G120574-globin expression following activation of p38 MAPK
1 Introduction
Hemoglobin (Hb) disorders particularly 120573-thalassemia arethe most common single gene disorders caused by mutationsin the 120573-globin locus Resultant disorders cause abnormalor reduced adult hemoglobin (HbA) production and excessunmatched 120572-chains in developing erythroblasts Clinicalsymptoms include bone marrow expansion splenomegalyand a severe anemia that requires regular blood transfusionsClinical management of 120573-thalassemia patients includeslifelong blood transfusions and chelation therapy to removeexcess transfused iron [1] and in some cases bone marrowtransplantation [2 3]120573-Thalassemia syndromes are classifiedaccording to severity based on steady-state Hb and trans-fusion dependency 120573-Thalassemia major and 120573-thalassemiaintermedia are both caused by the inheritance of two120573-globingene mutations [4] Furthermore 120573-thalassemia results inmoderate anemia in childhood which often progresses to
transfusion dependency over time iron loading and uniquecomplications related to expanded erythropoiesis and hemol-ysis
Although allogeneic hematopoietic stem cell transplan-tation (HSCT) and gene transfer therapy can be used forpatients with 120573-thalassemia modern medicines and therequisite financial resources for these procedures are notavailable to most 120573-hemoglobinopathy patients [5ndash7] Thepharmacological induction of fetal hemoglobin (HbF) maybe a promising alternative therapeutic option for treatment ofthese patients HbF is comprised of two 120572-globin and two 120574-globin chains (120572
21205742) and is highly expressed during fetal and
early postnatal periods however HbF levels subsequentlydecline following concurrent increases inHbA (120572
21205732) expres-
sion An increase in HbF levels can functionally compensatefor the shortfall in HbA synthesis in 120573-thalassemia patientsand synthesized 120574-chains can neutralize excess unbalanced120572-chains to reduce erythrocyte damage and ameliorate anemia
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 7468979 8 pageshttpdxdoiorg10115520167468979
2 Evidence-Based Complementary and Alternative Medicine
andmany of the complications associated with 120573-thalassemia[5] Pharmacological induction of HbF also represents apotentially less expensive and more widely applicable meansto treat 120573-hemoglobinopathies worldwide Drugs that facili-tate the induction of 120574-globin include hydroxyurea (HU) 5-azacytidine butyrate and erythropoietin among which HUis the only candidate that has been proven to improve theclinical course of 120573-thalassemia [8ndash11] However these agentsare unsuitable in relation to efficacy safety and ease of useFor example responses to HU are variable and weakenedresponses are often observed following long-term adminis-tration In addition 120574-globin induction agents may causemyelotoxicity and are potential carcinogens Furthermorethe cost of these treatment regimens is a significant limita-tion [7] Thus effective and safe alternative gene inductionreagents are highly desirable [12ndash14]
Traditional Chinese medicine (TCM) alone or in com-bination with other treatment regimens has been utilizedto treat anemia [2 15ndash17] It has been suggested that Yisuishengxue granules [18] and Plastrum testudinis (tortoiseplastron) [19] prolong erythrocyte lifespans via antilipidperoxidation and membrane stabilization Astragalus mem-branaceus (Radix Astragali [AMW] ldquoHuang Qirdquo in Chinese)roots have previously been used in TCM to treat anemia[16 20] Our previous in vitro experiments indicated thatPlastrum testudinis AMW and Codonopsis pilosula couldenhance 120574-globin mRNA expression and HbF productionin both human erythroid K562 cell cultures and erythroidprogenitors from patients with 120573-thalassemia [19 21] Sub-sequently we conducted a randomized controlled double-blind clinical trial with AMW and its compounded formu-lation (AMW + Codonopsis pilosula + Plastrum testudinis)to treat 120573-thalassemia [14] The resultant data indicated thatfollowing 12 weeks of treatment AMW and the compoundedformulation increased the level of HbF erythrocytes meancorpuscular Hb and reticulocytes in 120573-thalassemia withno reported side effects [14] Thus AMW and the com-pounded formulation can ameliorate 120573-thalassemia symp-toms by inducing HbF gene expression Furthermore anadditional study performed by our group demonstratedthat Radix Astragali and the compounded formulation canindependently induce p38 phosphorylation in K562 cellsand cultured human erythroid progenitor cells from theperipheral blood of 120573-thalassemia patients [22] Howeverthese results require further validation Thus we aim toelucidate the mechanism of action pertaining to AMW andits compounded formulation in 120573-thalassemia patients withstudies focusing on globin gene expression and downstreamsignal pathways
2 Materials and Methods
Research in this study was performed in accordance withthe Declaration of Helsinki and the study was approved bythe Ethics Committee of Guangzhou Hospital of TraditionalChinese Medicine All participating patients or their legalguardians signed informed consent forms before initiatingthe study
21 Samples The blood samples that were utilized in thisstudy were from participants of a previous randomized con-trolled double-blinded clinical trial [14] The trial included35 patients who were between two and 18 years of age withconfirmed 120573-thalassemia based on the Criteria for Diagnosisand Treatment of Hematopathy [23] The subjects had notreceived a blood transfusion or treatment for anemia inthe previous 12 weeks prior to enrollment and associatedhemoglobin levels ranged from 45 to 10 gdL at the timeof enrollment Thirteen of the subjects were treated with RAstragali (AMW) 11 patients were treated with the com-pounded formulation (AMW + Codonopsis pilosula granule[CPG] + Plastrum testudinis granule [PTG]) and 11 patientswere treated with a placebo Pilot experiments performedin our laboratory (data not shown) indicated that a samplesize greater than 10 subjects per group was required for thisanalysis
Peripheral blood was drawn from the patients beforetreatment and 12 weeks after treatment as designated in theclinical trial [14] All of the patients (or their legal guardians)permitted us to use the blood samples that were extracted inthe present study to study the molecular mechanisms under-lying the treatment effects Blood samples were collected intowhole-blood tubes containing Na-EDTA and aliquots fromthese samples were used for this study
22 Treatment The treatment procedure and informationpertaining to the effectiveness of treatments with AMW andthe compounded formulation were previously reported byLu et al (2012) [14] Briefly AMW and the compoundedformulation (AMW + CPG + PTG) were produced byGuangdong Yifang Pharmaceutical Co Ltd (GuangdongProvince China) Herbs were extracted and granulated with10 g of R Astragali per g of AMW 10 g of Codonopsis pilosulain 3 g of CPG and 10 g of Plastrum testudinis in 07 g of PTGThe placebo was prepared using dextrin and an identicalprotocol to that used for herb granulation All granules wereadministered orally with warm water in the fasted state andtreatment was administered for 12 weeks
23 Peripheral BloodMononuclear Cell Isolation Amononu-clear cell population that consists mostly of nucleatedred blood cells was isolated from peripheral blood usingdiscontinuous Percoll density gradient centrifugation Twomilliliters of peripheral blood from each of the pedi-atric patients was placed in a centrifuge tube Each bloodaliquot was subsequently diluted with 2mL of PBS (pH74) A double-density Percoll column was prepared withone milliliter each of 1090 gmL and 1075 gmL dilutedPercoll cell isolation media (Pharmacia) The 1090 gmLPercoll solution was layered at the bottom of a centrifugetube and the 1075 gmL Percoll solution was slowly andgently dispensed from a syringe over the denser solutionwithout disturbing the interface Four milliliters of dilutedperipheral blood sample was slowly added to the top ofthe double-density Percoll column and centrifuged at roomtemperature at 400 rpm for 30minThe cell layer between the1090 gmL and 1075 gmL Percoll media was isolated Thiscell population contained mostly nucleated red blood cells
Evidence-Based Complementary and Alternative Medicine 3
and a small number of lymphocytes and granulocytes [24]These cells were washed three times with two volumes of PBSThis was followed by a centrifugation step at 1250 rpm for10min to discard the supernatant and keep the pellet Thesecells were used for RNA and protein extraction
24 Measurement of Globin mRNA RNA was isolated fromnucleated red blood cell-enriched mononuclear cells usingTRIzol reagent (Sangon Biotech Shanghai China) accord-ing to the manufacturerrsquos instructions One hundred unitsof M-MLV (Invitrogen Carlsbad CA) were used to syn-thesize cDNA from 4 120583L of total RNA Quantitative real-time PCR was then performed using SYBR Green PCRMaster Mix (Applied Biosystems Norwalk CT) and an ABI3900 real-time system (Applied Biosystems) The followingprimer and probe sequences were used 120572-globin forwardprimer (51015840-GAGGCCCTGGAGAGGATGTT-31015840) and 120572-globin reverse primer (51015840-CGTGGCTCAGGTCGAAGTG-31015840) 120573-globin forward primer (51015840-GGCAACCCTAAGGTG-AAGGC-31015840) and 120573-globin reverse primer (51015840-GCAGCT-CACTCAGTGTGGCA-31015840) A120574-globin forward primer (51015840-GAGCTCACTGCCCATGAAT-31015840) and A120574-globin reverseprimer (51015840-CTCTCAGCAGAATAGATTTATTATTTCT-31015840) G120574-globin forward primer (51015840-AGCTCACTGCCCATG-AAG-31015840) and G120574-globin reverse primer (51015840-CTCTTAGCA-GAATAGATTTATTATTTCA-31015840) and p38 MAPK forwardprimer (51015840-ACGTTCTACCGGCAGGAGCT-31015840) and p38MAPK reverse primer (51015840-AAGCAGCACACACAGAGC-CA-31015840) Human 120573-actin was used as an endogenous controlthe forward primer sequence used for the amplification of thislocus was 51015840-GCGCGGCTACAGCTT CA-31015840 and the reverseprimer sequence was 51015840-TCTCCTTAATGTCACGCACGA-T-31015840 All data were analyzed after normalization to 120573-actinexpression and each reactionwas performed in triplicate RT-PCR experiments were performed in triplicate
25 Preparation of Standard cDNA and Standard CurveThree cDNA samples were randomly selected from eachgroup and amplified using the corresponding primer pair toprepare standard control DNA PCR products were separatedand extracted from agarose gels DNA concentration wasmeasured using spectrophotometry (260 nm) DNA wasconsidered to be of high quality if the OD260 nmOD280 nmratio was between 18 and 20
mRNA was measured following 10-fold serial dilution ofstandard DNA (108 to 104 copies in ultrapure water) Thisdetermination was performed in triplicate following the useof a SYBR Green real-time RT-PCR assay Associated Ctvalues were plotted against DNA copy number to constructa standard curve
26 SYBR Green Real-Time RT-PCR Assay A real-time RT-PCR assay was carried out using the SYBRGreenMaster Mix(Invitrogen) Kit A concentration of 02 120583M of each primerwas used in each reaction RT-PCR was performed in a finalvolume of 25 120583L with each reaction containing 5 120583L of 5 timesSYBR Green Master Mix 05 120583L of each of 10 120583M forwardand reverse primers 05 120583L of dNTPs (10mmolL) 2 120583L of
cDNA 05 120583L of Taq (3U120583L) and 165 120583L of ultrapure waterThe optimized thermal cycling conditions were as follows 40cycles of 93∘C for 3min 93∘C for 15 s 55∘C for 25 s and 72∘Cfor 25 s Fluorescence was measured at the end of each cycle
27 Western Blot and Antibodies For whole-cell extractpreparation nucleated red blood cell-enriched mononuclearcells were lysed in radioimmunoprecipitation assay (RIPA)buffer containing a complete protease inhibitor cocktail(Roche Penzberg Upper Bavaria Germany) for 10minat 4∘C and the extract (50 120583g) was separated using SDS-PAGE and transferred onto polyvinyl difluoride (PVDF)membranes Membranes were incubated with rabbit anti-p38MAPK antibody (1 500) goat anti-mouse 120573-actin antibody(Santa Cruz Biotechnology Santa Cruz CA) or rabbit anti-phospho-p38 MAPK antibody (1 500 Bioworld TechnologySt Louis ParkMN) HRP-conjugated anti-goat or anti-rabbitsecondary antibodies (both 1 5000) were purchased fromSigma Aldrich (St Louis MO) and Santa Cruz Biotechnol-ogy respectively The sample sizes pertaining to the Westernblot analyses for the three groups were similar to thosedescribed for previous experiments that is 13 patients treatedwith R Astragali 11 patients treated with the compoundedformulation and 11 patients treated with the placebo drugwere analyzed during the Western blot analyses
28 Statistical Methods Statistical analysis was conductedusing SPSS software (Version 130) Results are presentedas means plusmn standard deviation (SD) Greenhouse-Geissercorrections for nonsphericity were taken into accountwhen determining significance Studentrsquos t-test or one-wayANOVA was used to assess statistical significance amongmean values for groups Following ANOVA analysis FisherrsquosLeast Significant Difference (LSD) test was performed toconfirm data and compare means (119901 le 005 was consideredstatistically significant)
3 Results and Discussion
31 Standard Curves and Assay Reliability A mononuclearcell population that consists mostly of nucleated red bloodcells was harvested from patients and RNA was subsequentlyextracted For each sample 120574- and 120573-globin mRNA levelswere measured using a fluorescence-based RT-PCR assay toanalyze the molecular mechanisms associated with 12 weeksof AMW and compounded formulation treatment Ct valueswere plotted against diluted standard DNA copy numberand the associated standard curve displayed a linear rangeacross 3-4 log units of DNA copy number (108ndash104 copies120583Lfor G120574-globin 109ndash105 copies120583L for 120572- 120573- and A120574-globinand 109 to 106 copies120583L for p38 MAPK) Linear regressionanalysis was used to establish standard curves for 120572-globin(119910 = minus3047119909+36290 1198772 = 0999) 120573-globin (119910 = minus3008119909+36606 1198772 = 0999) G120574-globin (119910 = minus3128119909 + 357961198772 = 0999) A120574-globin (119910 = minus3438119909 + 39598 1198772 = 0990)p38MAPR (119910 = minus3082119909 + 36240 1198772 = 0999) and 120573-actin(119910 = minus3394119909 + 39996 1198772 = 1000)
4 Evidence-Based Complementary and Alternative Medicine
32 G120574-Globin mRNA before and after Treatment Cellsexpress 120572- 120573- and 120574-globin genes during fetal and adultdevelopment in order to synthesize hemoglobin Adulthemoglobin A is a tetramer containing two 120572-globin sub-units and two 120573-globin subunits (120572
21205732) Conversely fetal
hemoglobin is composed of two 120572-globin and two 120574-globinsubunits Evaluation of globin gene expression in nucleatedred blood cell-enriched mononuclear cells collected frompatients before and after treatment using standard RT-qPCRand primer pairs specific to human 120572- 120573- and 120574-globinrevealed expression of mRNA transcripts specific to theseglobin genes RT-PCR data indicate that G120574-globin mRNAdid not differ significantly between groups before treatment(119901 = 0923) but was significantly different between groupsafter treatment (119901 = 0025) Further multiple comparisonsshowed that G120574-globin mRNAs from the compounded for-mulation group (119901 = 0010) and the Radix Astragali treat-ment group (119901 = 0036) were significantly higher than thecontrol group no significant difference was found betweenthe compounded formulation group and the Radix Astragalitreatment group (119901 = 0516) Significant differences occurredbetween the placebo and treatment groups however the dif-ference between theAMWand the compounded formulationgroup was not significant (Figure 1) The G120574-globin mRNAlevels in the compounded formulation group were 165-fold higher following treatment compared with levels beforetreatment (119901 = 0004) Similarly the G120574-globin mRNA levelsin the Radix Astragali treatment group were 153-fold higherfollowing treatment compared with levels before treatment(119901 = 0002) These differences were deemed significantIn contrast no significant differences were observed in G120574-globin levels in patients from the control group before andafter placebo drug treatment (119901 = 0293) (Figure 1) sug-gesting that AMW and its compounded formulation signif-icantly increased G120574-globin mRNA transcription The latterphenomenon might explain the increased HbF levels thatwere observed in the clinical trial However neither AMWnor its compounded formulation affected120572-120573- orA120574-globinmRNA levels after 12 weeks of treatment (Figure 2)
33 p38 MAPK Signaling Pathway Changes before and afterTreatment p38 MAPK was initially identified as a proteinkinase that is activated by stress This kinase has also beenshown to coordinate cellular responses during erythropoiesis[25ndash27] p38 MAPK is essential for hemoglobin synthesisand p38 mice exhibit severe anemia and die in utero dueto defective angiogenesis and placental insufficiency [28] Inresponse to sodium butyrate and trichostatin A treatment oferythroid cells p38 MAPK mediates HbF induction by acti-vating cAMP response element binding protein 1 (CREB1)p38 signaling regulates G120574-globin transcription during ery-throid maturation via a downstream effector CREB1 whichbinds to the G120574-globin cAMP response element (G-CRE)Moreover gain of p38 or CREB1 function augments 120574-globintranscription and these regulatory effects were observed to beconserved under tested physiological conditions in primaryerythroid cells [29]
0
02
04
06
08
1
12
14
Control AMW Compoundedformulation
BeforeAfter
Rela
tive G
120574-g
lobi
n m
RNA
leve
l lowast
lowast
Figure 1 Relative G120574-globin gene expression before and aftertreatment of pediatric 120573-thalassemia lowast119901 lt 005 compared with thecorresponding control 119901 lt 001 compared with before treatmentof the same group
Given that the p38 MAPK signaling pathway is the keymediator pertaining to stress induction of 120574-globin mRNA[30] we proposed that the induction of G120574-globin geneexpression following AMW and compounded formulationtreatment may increase p38 MAPK expression or activa-tion Indeed a previous study performed by our groupdemonstrated that one of the compounded agents Plastrumtestudinis facilitated 120574-globin mRNA accumulation and HbFsynthesis in K562 cells following activation of the p38MAPKsignaling pathway These results were validated followingthe observation that the latter effects were suppressed afterpretreatment of the K562 cells with the p38 MAPK inhibitorSB203580 [30] Furthermore a separate study conducted byour group demonstrated that aqueous extracts ofRadixAstra-gali aqueous extracts of the compounded formulation and05mmolL sodium butyrate all independently induced p38phosphorylation in K562 cells and cultured human erythroidprogenitor cells from the peripheral blood of 120573-thalassemiaAll of these reactions demonstrated a similar time-effect pro-file phosphorylated p38 levels began to rise at 6ndash8 h peakedat 48ndash60 h and later began to decline Once more SB203580inhibited the increases in p38 phosphorylation that wereinitially induced by the aqueous extracts [22]
Thus we measured total p38 MAPK (t-p38) and phos-phorylated p38 MAPK (p-p38) levels in nucleated redblood cell-enriched mononuclear cells collected in AMW-compounded formulation- or placebo-treated patients Weobserved that t-p38 mRNA (Figure 3(a) 119901 = 0999 and 0965before and after treatment) and protein (Figures 3(b) and3(c) 119901 = 0554 and 0476 before and after treatment) levelswere not significantly different between the groups Howeverp-p38 protein levels were comparable in all three groups
Evidence-Based Complementary and Alternative Medicine 5
0
50
100
150
200
250
300
Control AMW
Rela
tive120572
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(a)
0
1
2
3
4
5
6
7
Control AMW
Rela
tive120573
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(b)
0
01
02
03
04
05
06
07
Control AMW
Rela
tive A
120574-g
lobi
n m
RNA
leve
l
Compoundedformulation
BeforeAfter
(c)
Figure 2 Relative globin gene expression before and after treatment of pediatric 120573-thalassemia (a) 120572-globin (b) 120573-globin and (c) A120574-globin
(Figure 3(d) 119901 = 0813) prior to treatment and proteinlevels were significantly different after treatment (Figure 3(d)119901 = 0013) ANOVA analysis and subsequent LSD analysisindicated that p-p38 levels were significantly higher afterAMW and compounded formulation treatments comparedto placebo treatment Compared with p-p38 protein levelsthat were measured before treatment the compounded for-mulation and AMW groups expressed 23- and 22-fold moreprotein respectively after treatment (Figure 3) Howeverdifferences between these treatment groups were not signifi-cantly different
We previously reported clinical outcomes pertainingto AMW and compounded formulation treatments [14]Following 12 weeks of treatment pediatric patients with
120573-thalassemia had increased fetal Hb erythrocytes andmean corpuscular Hb and reticulocytes Furthermore noadverse effects were observed with respect to leucocytesneutrophils platelets or hepatic and renal function Thetreatment was effective in 64 and 62 of the children with120573-thalassemia in the compounded formulation and AMWgroups respectively These outcomes suggest that AMWand the compounded formulation are effective and safeMoreover increases in Hb and HbF levels were observedin AMW- and compounded formulation-treated patientsindicating that both treatments may be effective and safe inrelation to HbF induction in patients
Traditional Chinese herbal medicine (TCM) is believedto cause relatively few side effects However current reports
6 Evidence-Based Complementary and Alternative Medicine
0
5
10
15
20
25
30
35
40
45
50
Control AMW
Rela
tive p
38
MA
PK m
RNA
leve
l
Compoundedformulation
BeforeAfter
(a)
1 2 3 4 5 6
p38 MAPK
p-p38 MAPK
120573-Actin
(b)
0
02
04
06
08
1
12
14
16
18
2
Control AMW
Relat
ive p
38 M
APK
pro
tein
leve
l
Compoundedformulation
BeforeAfter
(c)
0
02
04
06
08
1
12
Control AMW
lowastlowast
lowast
Rela
tive p
-p38
MA
PK p
rote
in le
vel
Compoundedformulation
BeforeAfter
(d)
Figure 3 Changes in p38MAPKexpression and activation before and after treatment of pediatric120573-thalassemia (a) Changes inmRNA levels(b) Representative images ofWestern blot analyses Graphs of blot quantification data pertaining to total p38MAPK and phosphorylated p38MAPK (p-p38 MAPK) levels are shown in (c) and (d) respectively lowast119901 lt 005 and lowastlowast119901 lt 001 compared with the corresponding control119901 lt 001 compared with before treatment of the same group
pertaining to the use of TCM in relation to 120573-thalassemiaare complicated and the identification of a single 120574-globininducer from TCM preparations is difficult We previouslyreported that AMW and an associated compounded formu-lation for pediatric 120573-thalassemia can significantly improvehematological parameters [14 16] resulting in elevated HbFlevels in patients treated for twelve weeks with AMW or thecompounded formulation Compared with butyrate admin-istration induction was lasting longer and did not inhibiterythroid cells Thalassemia is a hematological disorder andseveral TCM preparations have been reported to improve
hematological parameters in anemic patients [17] Morespecifically Plastrum testudinis has been shown to induce 120574-globin expression in erythroid cells (unpublished data)
Based on TCM theory and effects observed in vitro andin vivo we developed an AMW formulation and a com-bined AMW CPG and TPG formulation for 120573-thalassemiatreatment As part of this study we report that treatmentwith AMW or the compounded formulation for 12 weekssignificantly affected G120574-globin mRNA levels and increasedphosphorylated p38 MAPK levels with no concomitantchanges to total p38 MAPK mRNA and protein levels These
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
2 Evidence-Based Complementary and Alternative Medicine
andmany of the complications associated with 120573-thalassemia[5] Pharmacological induction of HbF also represents apotentially less expensive and more widely applicable meansto treat 120573-hemoglobinopathies worldwide Drugs that facili-tate the induction of 120574-globin include hydroxyurea (HU) 5-azacytidine butyrate and erythropoietin among which HUis the only candidate that has been proven to improve theclinical course of 120573-thalassemia [8ndash11] However these agentsare unsuitable in relation to efficacy safety and ease of useFor example responses to HU are variable and weakenedresponses are often observed following long-term adminis-tration In addition 120574-globin induction agents may causemyelotoxicity and are potential carcinogens Furthermorethe cost of these treatment regimens is a significant limita-tion [7] Thus effective and safe alternative gene inductionreagents are highly desirable [12ndash14]
Traditional Chinese medicine (TCM) alone or in com-bination with other treatment regimens has been utilizedto treat anemia [2 15ndash17] It has been suggested that Yisuishengxue granules [18] and Plastrum testudinis (tortoiseplastron) [19] prolong erythrocyte lifespans via antilipidperoxidation and membrane stabilization Astragalus mem-branaceus (Radix Astragali [AMW] ldquoHuang Qirdquo in Chinese)roots have previously been used in TCM to treat anemia[16 20] Our previous in vitro experiments indicated thatPlastrum testudinis AMW and Codonopsis pilosula couldenhance 120574-globin mRNA expression and HbF productionin both human erythroid K562 cell cultures and erythroidprogenitors from patients with 120573-thalassemia [19 21] Sub-sequently we conducted a randomized controlled double-blind clinical trial with AMW and its compounded formu-lation (AMW + Codonopsis pilosula + Plastrum testudinis)to treat 120573-thalassemia [14] The resultant data indicated thatfollowing 12 weeks of treatment AMW and the compoundedformulation increased the level of HbF erythrocytes meancorpuscular Hb and reticulocytes in 120573-thalassemia withno reported side effects [14] Thus AMW and the com-pounded formulation can ameliorate 120573-thalassemia symp-toms by inducing HbF gene expression Furthermore anadditional study performed by our group demonstratedthat Radix Astragali and the compounded formulation canindependently induce p38 phosphorylation in K562 cellsand cultured human erythroid progenitor cells from theperipheral blood of 120573-thalassemia patients [22] Howeverthese results require further validation Thus we aim toelucidate the mechanism of action pertaining to AMW andits compounded formulation in 120573-thalassemia patients withstudies focusing on globin gene expression and downstreamsignal pathways
2 Materials and Methods
Research in this study was performed in accordance withthe Declaration of Helsinki and the study was approved bythe Ethics Committee of Guangzhou Hospital of TraditionalChinese Medicine All participating patients or their legalguardians signed informed consent forms before initiatingthe study
21 Samples The blood samples that were utilized in thisstudy were from participants of a previous randomized con-trolled double-blinded clinical trial [14] The trial included35 patients who were between two and 18 years of age withconfirmed 120573-thalassemia based on the Criteria for Diagnosisand Treatment of Hematopathy [23] The subjects had notreceived a blood transfusion or treatment for anemia inthe previous 12 weeks prior to enrollment and associatedhemoglobin levels ranged from 45 to 10 gdL at the timeof enrollment Thirteen of the subjects were treated with RAstragali (AMW) 11 patients were treated with the com-pounded formulation (AMW + Codonopsis pilosula granule[CPG] + Plastrum testudinis granule [PTG]) and 11 patientswere treated with a placebo Pilot experiments performedin our laboratory (data not shown) indicated that a samplesize greater than 10 subjects per group was required for thisanalysis
Peripheral blood was drawn from the patients beforetreatment and 12 weeks after treatment as designated in theclinical trial [14] All of the patients (or their legal guardians)permitted us to use the blood samples that were extracted inthe present study to study the molecular mechanisms under-lying the treatment effects Blood samples were collected intowhole-blood tubes containing Na-EDTA and aliquots fromthese samples were used for this study
22 Treatment The treatment procedure and informationpertaining to the effectiveness of treatments with AMW andthe compounded formulation were previously reported byLu et al (2012) [14] Briefly AMW and the compoundedformulation (AMW + CPG + PTG) were produced byGuangdong Yifang Pharmaceutical Co Ltd (GuangdongProvince China) Herbs were extracted and granulated with10 g of R Astragali per g of AMW 10 g of Codonopsis pilosulain 3 g of CPG and 10 g of Plastrum testudinis in 07 g of PTGThe placebo was prepared using dextrin and an identicalprotocol to that used for herb granulation All granules wereadministered orally with warm water in the fasted state andtreatment was administered for 12 weeks
23 Peripheral BloodMononuclear Cell Isolation Amononu-clear cell population that consists mostly of nucleatedred blood cells was isolated from peripheral blood usingdiscontinuous Percoll density gradient centrifugation Twomilliliters of peripheral blood from each of the pedi-atric patients was placed in a centrifuge tube Each bloodaliquot was subsequently diluted with 2mL of PBS (pH74) A double-density Percoll column was prepared withone milliliter each of 1090 gmL and 1075 gmL dilutedPercoll cell isolation media (Pharmacia) The 1090 gmLPercoll solution was layered at the bottom of a centrifugetube and the 1075 gmL Percoll solution was slowly andgently dispensed from a syringe over the denser solutionwithout disturbing the interface Four milliliters of dilutedperipheral blood sample was slowly added to the top ofthe double-density Percoll column and centrifuged at roomtemperature at 400 rpm for 30minThe cell layer between the1090 gmL and 1075 gmL Percoll media was isolated Thiscell population contained mostly nucleated red blood cells
Evidence-Based Complementary and Alternative Medicine 3
and a small number of lymphocytes and granulocytes [24]These cells were washed three times with two volumes of PBSThis was followed by a centrifugation step at 1250 rpm for10min to discard the supernatant and keep the pellet Thesecells were used for RNA and protein extraction
24 Measurement of Globin mRNA RNA was isolated fromnucleated red blood cell-enriched mononuclear cells usingTRIzol reagent (Sangon Biotech Shanghai China) accord-ing to the manufacturerrsquos instructions One hundred unitsof M-MLV (Invitrogen Carlsbad CA) were used to syn-thesize cDNA from 4 120583L of total RNA Quantitative real-time PCR was then performed using SYBR Green PCRMaster Mix (Applied Biosystems Norwalk CT) and an ABI3900 real-time system (Applied Biosystems) The followingprimer and probe sequences were used 120572-globin forwardprimer (51015840-GAGGCCCTGGAGAGGATGTT-31015840) and 120572-globin reverse primer (51015840-CGTGGCTCAGGTCGAAGTG-31015840) 120573-globin forward primer (51015840-GGCAACCCTAAGGTG-AAGGC-31015840) and 120573-globin reverse primer (51015840-GCAGCT-CACTCAGTGTGGCA-31015840) A120574-globin forward primer (51015840-GAGCTCACTGCCCATGAAT-31015840) and A120574-globin reverseprimer (51015840-CTCTCAGCAGAATAGATTTATTATTTCT-31015840) G120574-globin forward primer (51015840-AGCTCACTGCCCATG-AAG-31015840) and G120574-globin reverse primer (51015840-CTCTTAGCA-GAATAGATTTATTATTTCA-31015840) and p38 MAPK forwardprimer (51015840-ACGTTCTACCGGCAGGAGCT-31015840) and p38MAPK reverse primer (51015840-AAGCAGCACACACAGAGC-CA-31015840) Human 120573-actin was used as an endogenous controlthe forward primer sequence used for the amplification of thislocus was 51015840-GCGCGGCTACAGCTT CA-31015840 and the reverseprimer sequence was 51015840-TCTCCTTAATGTCACGCACGA-T-31015840 All data were analyzed after normalization to 120573-actinexpression and each reactionwas performed in triplicate RT-PCR experiments were performed in triplicate
25 Preparation of Standard cDNA and Standard CurveThree cDNA samples were randomly selected from eachgroup and amplified using the corresponding primer pair toprepare standard control DNA PCR products were separatedand extracted from agarose gels DNA concentration wasmeasured using spectrophotometry (260 nm) DNA wasconsidered to be of high quality if the OD260 nmOD280 nmratio was between 18 and 20
mRNA was measured following 10-fold serial dilution ofstandard DNA (108 to 104 copies in ultrapure water) Thisdetermination was performed in triplicate following the useof a SYBR Green real-time RT-PCR assay Associated Ctvalues were plotted against DNA copy number to constructa standard curve
26 SYBR Green Real-Time RT-PCR Assay A real-time RT-PCR assay was carried out using the SYBRGreenMaster Mix(Invitrogen) Kit A concentration of 02 120583M of each primerwas used in each reaction RT-PCR was performed in a finalvolume of 25 120583L with each reaction containing 5 120583L of 5 timesSYBR Green Master Mix 05 120583L of each of 10 120583M forwardand reverse primers 05 120583L of dNTPs (10mmolL) 2 120583L of
cDNA 05 120583L of Taq (3U120583L) and 165 120583L of ultrapure waterThe optimized thermal cycling conditions were as follows 40cycles of 93∘C for 3min 93∘C for 15 s 55∘C for 25 s and 72∘Cfor 25 s Fluorescence was measured at the end of each cycle
27 Western Blot and Antibodies For whole-cell extractpreparation nucleated red blood cell-enriched mononuclearcells were lysed in radioimmunoprecipitation assay (RIPA)buffer containing a complete protease inhibitor cocktail(Roche Penzberg Upper Bavaria Germany) for 10minat 4∘C and the extract (50 120583g) was separated using SDS-PAGE and transferred onto polyvinyl difluoride (PVDF)membranes Membranes were incubated with rabbit anti-p38MAPK antibody (1 500) goat anti-mouse 120573-actin antibody(Santa Cruz Biotechnology Santa Cruz CA) or rabbit anti-phospho-p38 MAPK antibody (1 500 Bioworld TechnologySt Louis ParkMN) HRP-conjugated anti-goat or anti-rabbitsecondary antibodies (both 1 5000) were purchased fromSigma Aldrich (St Louis MO) and Santa Cruz Biotechnol-ogy respectively The sample sizes pertaining to the Westernblot analyses for the three groups were similar to thosedescribed for previous experiments that is 13 patients treatedwith R Astragali 11 patients treated with the compoundedformulation and 11 patients treated with the placebo drugwere analyzed during the Western blot analyses
28 Statistical Methods Statistical analysis was conductedusing SPSS software (Version 130) Results are presentedas means plusmn standard deviation (SD) Greenhouse-Geissercorrections for nonsphericity were taken into accountwhen determining significance Studentrsquos t-test or one-wayANOVA was used to assess statistical significance amongmean values for groups Following ANOVA analysis FisherrsquosLeast Significant Difference (LSD) test was performed toconfirm data and compare means (119901 le 005 was consideredstatistically significant)
3 Results and Discussion
31 Standard Curves and Assay Reliability A mononuclearcell population that consists mostly of nucleated red bloodcells was harvested from patients and RNA was subsequentlyextracted For each sample 120574- and 120573-globin mRNA levelswere measured using a fluorescence-based RT-PCR assay toanalyze the molecular mechanisms associated with 12 weeksof AMW and compounded formulation treatment Ct valueswere plotted against diluted standard DNA copy numberand the associated standard curve displayed a linear rangeacross 3-4 log units of DNA copy number (108ndash104 copies120583Lfor G120574-globin 109ndash105 copies120583L for 120572- 120573- and A120574-globinand 109 to 106 copies120583L for p38 MAPK) Linear regressionanalysis was used to establish standard curves for 120572-globin(119910 = minus3047119909+36290 1198772 = 0999) 120573-globin (119910 = minus3008119909+36606 1198772 = 0999) G120574-globin (119910 = minus3128119909 + 357961198772 = 0999) A120574-globin (119910 = minus3438119909 + 39598 1198772 = 0990)p38MAPR (119910 = minus3082119909 + 36240 1198772 = 0999) and 120573-actin(119910 = minus3394119909 + 39996 1198772 = 1000)
4 Evidence-Based Complementary and Alternative Medicine
32 G120574-Globin mRNA before and after Treatment Cellsexpress 120572- 120573- and 120574-globin genes during fetal and adultdevelopment in order to synthesize hemoglobin Adulthemoglobin A is a tetramer containing two 120572-globin sub-units and two 120573-globin subunits (120572
21205732) Conversely fetal
hemoglobin is composed of two 120572-globin and two 120574-globinsubunits Evaluation of globin gene expression in nucleatedred blood cell-enriched mononuclear cells collected frompatients before and after treatment using standard RT-qPCRand primer pairs specific to human 120572- 120573- and 120574-globinrevealed expression of mRNA transcripts specific to theseglobin genes RT-PCR data indicate that G120574-globin mRNAdid not differ significantly between groups before treatment(119901 = 0923) but was significantly different between groupsafter treatment (119901 = 0025) Further multiple comparisonsshowed that G120574-globin mRNAs from the compounded for-mulation group (119901 = 0010) and the Radix Astragali treat-ment group (119901 = 0036) were significantly higher than thecontrol group no significant difference was found betweenthe compounded formulation group and the Radix Astragalitreatment group (119901 = 0516) Significant differences occurredbetween the placebo and treatment groups however the dif-ference between theAMWand the compounded formulationgroup was not significant (Figure 1) The G120574-globin mRNAlevels in the compounded formulation group were 165-fold higher following treatment compared with levels beforetreatment (119901 = 0004) Similarly the G120574-globin mRNA levelsin the Radix Astragali treatment group were 153-fold higherfollowing treatment compared with levels before treatment(119901 = 0002) These differences were deemed significantIn contrast no significant differences were observed in G120574-globin levels in patients from the control group before andafter placebo drug treatment (119901 = 0293) (Figure 1) sug-gesting that AMW and its compounded formulation signif-icantly increased G120574-globin mRNA transcription The latterphenomenon might explain the increased HbF levels thatwere observed in the clinical trial However neither AMWnor its compounded formulation affected120572-120573- orA120574-globinmRNA levels after 12 weeks of treatment (Figure 2)
33 p38 MAPK Signaling Pathway Changes before and afterTreatment p38 MAPK was initially identified as a proteinkinase that is activated by stress This kinase has also beenshown to coordinate cellular responses during erythropoiesis[25ndash27] p38 MAPK is essential for hemoglobin synthesisand p38 mice exhibit severe anemia and die in utero dueto defective angiogenesis and placental insufficiency [28] Inresponse to sodium butyrate and trichostatin A treatment oferythroid cells p38 MAPK mediates HbF induction by acti-vating cAMP response element binding protein 1 (CREB1)p38 signaling regulates G120574-globin transcription during ery-throid maturation via a downstream effector CREB1 whichbinds to the G120574-globin cAMP response element (G-CRE)Moreover gain of p38 or CREB1 function augments 120574-globintranscription and these regulatory effects were observed to beconserved under tested physiological conditions in primaryerythroid cells [29]
0
02
04
06
08
1
12
14
Control AMW Compoundedformulation
BeforeAfter
Rela
tive G
120574-g
lobi
n m
RNA
leve
l lowast
lowast
Figure 1 Relative G120574-globin gene expression before and aftertreatment of pediatric 120573-thalassemia lowast119901 lt 005 compared with thecorresponding control 119901 lt 001 compared with before treatmentof the same group
Given that the p38 MAPK signaling pathway is the keymediator pertaining to stress induction of 120574-globin mRNA[30] we proposed that the induction of G120574-globin geneexpression following AMW and compounded formulationtreatment may increase p38 MAPK expression or activa-tion Indeed a previous study performed by our groupdemonstrated that one of the compounded agents Plastrumtestudinis facilitated 120574-globin mRNA accumulation and HbFsynthesis in K562 cells following activation of the p38MAPKsignaling pathway These results were validated followingthe observation that the latter effects were suppressed afterpretreatment of the K562 cells with the p38 MAPK inhibitorSB203580 [30] Furthermore a separate study conducted byour group demonstrated that aqueous extracts ofRadixAstra-gali aqueous extracts of the compounded formulation and05mmolL sodium butyrate all independently induced p38phosphorylation in K562 cells and cultured human erythroidprogenitor cells from the peripheral blood of 120573-thalassemiaAll of these reactions demonstrated a similar time-effect pro-file phosphorylated p38 levels began to rise at 6ndash8 h peakedat 48ndash60 h and later began to decline Once more SB203580inhibited the increases in p38 phosphorylation that wereinitially induced by the aqueous extracts [22]
Thus we measured total p38 MAPK (t-p38) and phos-phorylated p38 MAPK (p-p38) levels in nucleated redblood cell-enriched mononuclear cells collected in AMW-compounded formulation- or placebo-treated patients Weobserved that t-p38 mRNA (Figure 3(a) 119901 = 0999 and 0965before and after treatment) and protein (Figures 3(b) and3(c) 119901 = 0554 and 0476 before and after treatment) levelswere not significantly different between the groups Howeverp-p38 protein levels were comparable in all three groups
Evidence-Based Complementary and Alternative Medicine 5
0
50
100
150
200
250
300
Control AMW
Rela
tive120572
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(a)
0
1
2
3
4
5
6
7
Control AMW
Rela
tive120573
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(b)
0
01
02
03
04
05
06
07
Control AMW
Rela
tive A
120574-g
lobi
n m
RNA
leve
l
Compoundedformulation
BeforeAfter
(c)
Figure 2 Relative globin gene expression before and after treatment of pediatric 120573-thalassemia (a) 120572-globin (b) 120573-globin and (c) A120574-globin
(Figure 3(d) 119901 = 0813) prior to treatment and proteinlevels were significantly different after treatment (Figure 3(d)119901 = 0013) ANOVA analysis and subsequent LSD analysisindicated that p-p38 levels were significantly higher afterAMW and compounded formulation treatments comparedto placebo treatment Compared with p-p38 protein levelsthat were measured before treatment the compounded for-mulation and AMW groups expressed 23- and 22-fold moreprotein respectively after treatment (Figure 3) Howeverdifferences between these treatment groups were not signifi-cantly different
We previously reported clinical outcomes pertainingto AMW and compounded formulation treatments [14]Following 12 weeks of treatment pediatric patients with
120573-thalassemia had increased fetal Hb erythrocytes andmean corpuscular Hb and reticulocytes Furthermore noadverse effects were observed with respect to leucocytesneutrophils platelets or hepatic and renal function Thetreatment was effective in 64 and 62 of the children with120573-thalassemia in the compounded formulation and AMWgroups respectively These outcomes suggest that AMWand the compounded formulation are effective and safeMoreover increases in Hb and HbF levels were observedin AMW- and compounded formulation-treated patientsindicating that both treatments may be effective and safe inrelation to HbF induction in patients
Traditional Chinese herbal medicine (TCM) is believedto cause relatively few side effects However current reports
6 Evidence-Based Complementary and Alternative Medicine
0
5
10
15
20
25
30
35
40
45
50
Control AMW
Rela
tive p
38
MA
PK m
RNA
leve
l
Compoundedformulation
BeforeAfter
(a)
1 2 3 4 5 6
p38 MAPK
p-p38 MAPK
120573-Actin
(b)
0
02
04
06
08
1
12
14
16
18
2
Control AMW
Relat
ive p
38 M
APK
pro
tein
leve
l
Compoundedformulation
BeforeAfter
(c)
0
02
04
06
08
1
12
Control AMW
lowastlowast
lowast
Rela
tive p
-p38
MA
PK p
rote
in le
vel
Compoundedformulation
BeforeAfter
(d)
Figure 3 Changes in p38MAPKexpression and activation before and after treatment of pediatric120573-thalassemia (a) Changes inmRNA levels(b) Representative images ofWestern blot analyses Graphs of blot quantification data pertaining to total p38MAPK and phosphorylated p38MAPK (p-p38 MAPK) levels are shown in (c) and (d) respectively lowast119901 lt 005 and lowastlowast119901 lt 001 compared with the corresponding control119901 lt 001 compared with before treatment of the same group
pertaining to the use of TCM in relation to 120573-thalassemiaare complicated and the identification of a single 120574-globininducer from TCM preparations is difficult We previouslyreported that AMW and an associated compounded formu-lation for pediatric 120573-thalassemia can significantly improvehematological parameters [14 16] resulting in elevated HbFlevels in patients treated for twelve weeks with AMW or thecompounded formulation Compared with butyrate admin-istration induction was lasting longer and did not inhibiterythroid cells Thalassemia is a hematological disorder andseveral TCM preparations have been reported to improve
hematological parameters in anemic patients [17] Morespecifically Plastrum testudinis has been shown to induce 120574-globin expression in erythroid cells (unpublished data)
Based on TCM theory and effects observed in vitro andin vivo we developed an AMW formulation and a com-bined AMW CPG and TPG formulation for 120573-thalassemiatreatment As part of this study we report that treatmentwith AMW or the compounded formulation for 12 weekssignificantly affected G120574-globin mRNA levels and increasedphosphorylated p38 MAPK levels with no concomitantchanges to total p38 MAPK mRNA and protein levels These
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
Evidence-Based Complementary and Alternative Medicine 3
and a small number of lymphocytes and granulocytes [24]These cells were washed three times with two volumes of PBSThis was followed by a centrifugation step at 1250 rpm for10min to discard the supernatant and keep the pellet Thesecells were used for RNA and protein extraction
24 Measurement of Globin mRNA RNA was isolated fromnucleated red blood cell-enriched mononuclear cells usingTRIzol reagent (Sangon Biotech Shanghai China) accord-ing to the manufacturerrsquos instructions One hundred unitsof M-MLV (Invitrogen Carlsbad CA) were used to syn-thesize cDNA from 4 120583L of total RNA Quantitative real-time PCR was then performed using SYBR Green PCRMaster Mix (Applied Biosystems Norwalk CT) and an ABI3900 real-time system (Applied Biosystems) The followingprimer and probe sequences were used 120572-globin forwardprimer (51015840-GAGGCCCTGGAGAGGATGTT-31015840) and 120572-globin reverse primer (51015840-CGTGGCTCAGGTCGAAGTG-31015840) 120573-globin forward primer (51015840-GGCAACCCTAAGGTG-AAGGC-31015840) and 120573-globin reverse primer (51015840-GCAGCT-CACTCAGTGTGGCA-31015840) A120574-globin forward primer (51015840-GAGCTCACTGCCCATGAAT-31015840) and A120574-globin reverseprimer (51015840-CTCTCAGCAGAATAGATTTATTATTTCT-31015840) G120574-globin forward primer (51015840-AGCTCACTGCCCATG-AAG-31015840) and G120574-globin reverse primer (51015840-CTCTTAGCA-GAATAGATTTATTATTTCA-31015840) and p38 MAPK forwardprimer (51015840-ACGTTCTACCGGCAGGAGCT-31015840) and p38MAPK reverse primer (51015840-AAGCAGCACACACAGAGC-CA-31015840) Human 120573-actin was used as an endogenous controlthe forward primer sequence used for the amplification of thislocus was 51015840-GCGCGGCTACAGCTT CA-31015840 and the reverseprimer sequence was 51015840-TCTCCTTAATGTCACGCACGA-T-31015840 All data were analyzed after normalization to 120573-actinexpression and each reactionwas performed in triplicate RT-PCR experiments were performed in triplicate
25 Preparation of Standard cDNA and Standard CurveThree cDNA samples were randomly selected from eachgroup and amplified using the corresponding primer pair toprepare standard control DNA PCR products were separatedand extracted from agarose gels DNA concentration wasmeasured using spectrophotometry (260 nm) DNA wasconsidered to be of high quality if the OD260 nmOD280 nmratio was between 18 and 20
mRNA was measured following 10-fold serial dilution ofstandard DNA (108 to 104 copies in ultrapure water) Thisdetermination was performed in triplicate following the useof a SYBR Green real-time RT-PCR assay Associated Ctvalues were plotted against DNA copy number to constructa standard curve
26 SYBR Green Real-Time RT-PCR Assay A real-time RT-PCR assay was carried out using the SYBRGreenMaster Mix(Invitrogen) Kit A concentration of 02 120583M of each primerwas used in each reaction RT-PCR was performed in a finalvolume of 25 120583L with each reaction containing 5 120583L of 5 timesSYBR Green Master Mix 05 120583L of each of 10 120583M forwardand reverse primers 05 120583L of dNTPs (10mmolL) 2 120583L of
cDNA 05 120583L of Taq (3U120583L) and 165 120583L of ultrapure waterThe optimized thermal cycling conditions were as follows 40cycles of 93∘C for 3min 93∘C for 15 s 55∘C for 25 s and 72∘Cfor 25 s Fluorescence was measured at the end of each cycle
27 Western Blot and Antibodies For whole-cell extractpreparation nucleated red blood cell-enriched mononuclearcells were lysed in radioimmunoprecipitation assay (RIPA)buffer containing a complete protease inhibitor cocktail(Roche Penzberg Upper Bavaria Germany) for 10minat 4∘C and the extract (50 120583g) was separated using SDS-PAGE and transferred onto polyvinyl difluoride (PVDF)membranes Membranes were incubated with rabbit anti-p38MAPK antibody (1 500) goat anti-mouse 120573-actin antibody(Santa Cruz Biotechnology Santa Cruz CA) or rabbit anti-phospho-p38 MAPK antibody (1 500 Bioworld TechnologySt Louis ParkMN) HRP-conjugated anti-goat or anti-rabbitsecondary antibodies (both 1 5000) were purchased fromSigma Aldrich (St Louis MO) and Santa Cruz Biotechnol-ogy respectively The sample sizes pertaining to the Westernblot analyses for the three groups were similar to thosedescribed for previous experiments that is 13 patients treatedwith R Astragali 11 patients treated with the compoundedformulation and 11 patients treated with the placebo drugwere analyzed during the Western blot analyses
28 Statistical Methods Statistical analysis was conductedusing SPSS software (Version 130) Results are presentedas means plusmn standard deviation (SD) Greenhouse-Geissercorrections for nonsphericity were taken into accountwhen determining significance Studentrsquos t-test or one-wayANOVA was used to assess statistical significance amongmean values for groups Following ANOVA analysis FisherrsquosLeast Significant Difference (LSD) test was performed toconfirm data and compare means (119901 le 005 was consideredstatistically significant)
3 Results and Discussion
31 Standard Curves and Assay Reliability A mononuclearcell population that consists mostly of nucleated red bloodcells was harvested from patients and RNA was subsequentlyextracted For each sample 120574- and 120573-globin mRNA levelswere measured using a fluorescence-based RT-PCR assay toanalyze the molecular mechanisms associated with 12 weeksof AMW and compounded formulation treatment Ct valueswere plotted against diluted standard DNA copy numberand the associated standard curve displayed a linear rangeacross 3-4 log units of DNA copy number (108ndash104 copies120583Lfor G120574-globin 109ndash105 copies120583L for 120572- 120573- and A120574-globinand 109 to 106 copies120583L for p38 MAPK) Linear regressionanalysis was used to establish standard curves for 120572-globin(119910 = minus3047119909+36290 1198772 = 0999) 120573-globin (119910 = minus3008119909+36606 1198772 = 0999) G120574-globin (119910 = minus3128119909 + 357961198772 = 0999) A120574-globin (119910 = minus3438119909 + 39598 1198772 = 0990)p38MAPR (119910 = minus3082119909 + 36240 1198772 = 0999) and 120573-actin(119910 = minus3394119909 + 39996 1198772 = 1000)
4 Evidence-Based Complementary and Alternative Medicine
32 G120574-Globin mRNA before and after Treatment Cellsexpress 120572- 120573- and 120574-globin genes during fetal and adultdevelopment in order to synthesize hemoglobin Adulthemoglobin A is a tetramer containing two 120572-globin sub-units and two 120573-globin subunits (120572
21205732) Conversely fetal
hemoglobin is composed of two 120572-globin and two 120574-globinsubunits Evaluation of globin gene expression in nucleatedred blood cell-enriched mononuclear cells collected frompatients before and after treatment using standard RT-qPCRand primer pairs specific to human 120572- 120573- and 120574-globinrevealed expression of mRNA transcripts specific to theseglobin genes RT-PCR data indicate that G120574-globin mRNAdid not differ significantly between groups before treatment(119901 = 0923) but was significantly different between groupsafter treatment (119901 = 0025) Further multiple comparisonsshowed that G120574-globin mRNAs from the compounded for-mulation group (119901 = 0010) and the Radix Astragali treat-ment group (119901 = 0036) were significantly higher than thecontrol group no significant difference was found betweenthe compounded formulation group and the Radix Astragalitreatment group (119901 = 0516) Significant differences occurredbetween the placebo and treatment groups however the dif-ference between theAMWand the compounded formulationgroup was not significant (Figure 1) The G120574-globin mRNAlevels in the compounded formulation group were 165-fold higher following treatment compared with levels beforetreatment (119901 = 0004) Similarly the G120574-globin mRNA levelsin the Radix Astragali treatment group were 153-fold higherfollowing treatment compared with levels before treatment(119901 = 0002) These differences were deemed significantIn contrast no significant differences were observed in G120574-globin levels in patients from the control group before andafter placebo drug treatment (119901 = 0293) (Figure 1) sug-gesting that AMW and its compounded formulation signif-icantly increased G120574-globin mRNA transcription The latterphenomenon might explain the increased HbF levels thatwere observed in the clinical trial However neither AMWnor its compounded formulation affected120572-120573- orA120574-globinmRNA levels after 12 weeks of treatment (Figure 2)
33 p38 MAPK Signaling Pathway Changes before and afterTreatment p38 MAPK was initially identified as a proteinkinase that is activated by stress This kinase has also beenshown to coordinate cellular responses during erythropoiesis[25ndash27] p38 MAPK is essential for hemoglobin synthesisand p38 mice exhibit severe anemia and die in utero dueto defective angiogenesis and placental insufficiency [28] Inresponse to sodium butyrate and trichostatin A treatment oferythroid cells p38 MAPK mediates HbF induction by acti-vating cAMP response element binding protein 1 (CREB1)p38 signaling regulates G120574-globin transcription during ery-throid maturation via a downstream effector CREB1 whichbinds to the G120574-globin cAMP response element (G-CRE)Moreover gain of p38 or CREB1 function augments 120574-globintranscription and these regulatory effects were observed to beconserved under tested physiological conditions in primaryerythroid cells [29]
0
02
04
06
08
1
12
14
Control AMW Compoundedformulation
BeforeAfter
Rela
tive G
120574-g
lobi
n m
RNA
leve
l lowast
lowast
Figure 1 Relative G120574-globin gene expression before and aftertreatment of pediatric 120573-thalassemia lowast119901 lt 005 compared with thecorresponding control 119901 lt 001 compared with before treatmentof the same group
Given that the p38 MAPK signaling pathway is the keymediator pertaining to stress induction of 120574-globin mRNA[30] we proposed that the induction of G120574-globin geneexpression following AMW and compounded formulationtreatment may increase p38 MAPK expression or activa-tion Indeed a previous study performed by our groupdemonstrated that one of the compounded agents Plastrumtestudinis facilitated 120574-globin mRNA accumulation and HbFsynthesis in K562 cells following activation of the p38MAPKsignaling pathway These results were validated followingthe observation that the latter effects were suppressed afterpretreatment of the K562 cells with the p38 MAPK inhibitorSB203580 [30] Furthermore a separate study conducted byour group demonstrated that aqueous extracts ofRadixAstra-gali aqueous extracts of the compounded formulation and05mmolL sodium butyrate all independently induced p38phosphorylation in K562 cells and cultured human erythroidprogenitor cells from the peripheral blood of 120573-thalassemiaAll of these reactions demonstrated a similar time-effect pro-file phosphorylated p38 levels began to rise at 6ndash8 h peakedat 48ndash60 h and later began to decline Once more SB203580inhibited the increases in p38 phosphorylation that wereinitially induced by the aqueous extracts [22]
Thus we measured total p38 MAPK (t-p38) and phos-phorylated p38 MAPK (p-p38) levels in nucleated redblood cell-enriched mononuclear cells collected in AMW-compounded formulation- or placebo-treated patients Weobserved that t-p38 mRNA (Figure 3(a) 119901 = 0999 and 0965before and after treatment) and protein (Figures 3(b) and3(c) 119901 = 0554 and 0476 before and after treatment) levelswere not significantly different between the groups Howeverp-p38 protein levels were comparable in all three groups
Evidence-Based Complementary and Alternative Medicine 5
0
50
100
150
200
250
300
Control AMW
Rela
tive120572
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(a)
0
1
2
3
4
5
6
7
Control AMW
Rela
tive120573
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(b)
0
01
02
03
04
05
06
07
Control AMW
Rela
tive A
120574-g
lobi
n m
RNA
leve
l
Compoundedformulation
BeforeAfter
(c)
Figure 2 Relative globin gene expression before and after treatment of pediatric 120573-thalassemia (a) 120572-globin (b) 120573-globin and (c) A120574-globin
(Figure 3(d) 119901 = 0813) prior to treatment and proteinlevels were significantly different after treatment (Figure 3(d)119901 = 0013) ANOVA analysis and subsequent LSD analysisindicated that p-p38 levels were significantly higher afterAMW and compounded formulation treatments comparedto placebo treatment Compared with p-p38 protein levelsthat were measured before treatment the compounded for-mulation and AMW groups expressed 23- and 22-fold moreprotein respectively after treatment (Figure 3) Howeverdifferences between these treatment groups were not signifi-cantly different
We previously reported clinical outcomes pertainingto AMW and compounded formulation treatments [14]Following 12 weeks of treatment pediatric patients with
120573-thalassemia had increased fetal Hb erythrocytes andmean corpuscular Hb and reticulocytes Furthermore noadverse effects were observed with respect to leucocytesneutrophils platelets or hepatic and renal function Thetreatment was effective in 64 and 62 of the children with120573-thalassemia in the compounded formulation and AMWgroups respectively These outcomes suggest that AMWand the compounded formulation are effective and safeMoreover increases in Hb and HbF levels were observedin AMW- and compounded formulation-treated patientsindicating that both treatments may be effective and safe inrelation to HbF induction in patients
Traditional Chinese herbal medicine (TCM) is believedto cause relatively few side effects However current reports
6 Evidence-Based Complementary and Alternative Medicine
0
5
10
15
20
25
30
35
40
45
50
Control AMW
Rela
tive p
38
MA
PK m
RNA
leve
l
Compoundedformulation
BeforeAfter
(a)
1 2 3 4 5 6
p38 MAPK
p-p38 MAPK
120573-Actin
(b)
0
02
04
06
08
1
12
14
16
18
2
Control AMW
Relat
ive p
38 M
APK
pro
tein
leve
l
Compoundedformulation
BeforeAfter
(c)
0
02
04
06
08
1
12
Control AMW
lowastlowast
lowast
Rela
tive p
-p38
MA
PK p
rote
in le
vel
Compoundedformulation
BeforeAfter
(d)
Figure 3 Changes in p38MAPKexpression and activation before and after treatment of pediatric120573-thalassemia (a) Changes inmRNA levels(b) Representative images ofWestern blot analyses Graphs of blot quantification data pertaining to total p38MAPK and phosphorylated p38MAPK (p-p38 MAPK) levels are shown in (c) and (d) respectively lowast119901 lt 005 and lowastlowast119901 lt 001 compared with the corresponding control119901 lt 001 compared with before treatment of the same group
pertaining to the use of TCM in relation to 120573-thalassemiaare complicated and the identification of a single 120574-globininducer from TCM preparations is difficult We previouslyreported that AMW and an associated compounded formu-lation for pediatric 120573-thalassemia can significantly improvehematological parameters [14 16] resulting in elevated HbFlevels in patients treated for twelve weeks with AMW or thecompounded formulation Compared with butyrate admin-istration induction was lasting longer and did not inhibiterythroid cells Thalassemia is a hematological disorder andseveral TCM preparations have been reported to improve
hematological parameters in anemic patients [17] Morespecifically Plastrum testudinis has been shown to induce 120574-globin expression in erythroid cells (unpublished data)
Based on TCM theory and effects observed in vitro andin vivo we developed an AMW formulation and a com-bined AMW CPG and TPG formulation for 120573-thalassemiatreatment As part of this study we report that treatmentwith AMW or the compounded formulation for 12 weekssignificantly affected G120574-globin mRNA levels and increasedphosphorylated p38 MAPK levels with no concomitantchanges to total p38 MAPK mRNA and protein levels These
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
4 Evidence-Based Complementary and Alternative Medicine
32 G120574-Globin mRNA before and after Treatment Cellsexpress 120572- 120573- and 120574-globin genes during fetal and adultdevelopment in order to synthesize hemoglobin Adulthemoglobin A is a tetramer containing two 120572-globin sub-units and two 120573-globin subunits (120572
21205732) Conversely fetal
hemoglobin is composed of two 120572-globin and two 120574-globinsubunits Evaluation of globin gene expression in nucleatedred blood cell-enriched mononuclear cells collected frompatients before and after treatment using standard RT-qPCRand primer pairs specific to human 120572- 120573- and 120574-globinrevealed expression of mRNA transcripts specific to theseglobin genes RT-PCR data indicate that G120574-globin mRNAdid not differ significantly between groups before treatment(119901 = 0923) but was significantly different between groupsafter treatment (119901 = 0025) Further multiple comparisonsshowed that G120574-globin mRNAs from the compounded for-mulation group (119901 = 0010) and the Radix Astragali treat-ment group (119901 = 0036) were significantly higher than thecontrol group no significant difference was found betweenthe compounded formulation group and the Radix Astragalitreatment group (119901 = 0516) Significant differences occurredbetween the placebo and treatment groups however the dif-ference between theAMWand the compounded formulationgroup was not significant (Figure 1) The G120574-globin mRNAlevels in the compounded formulation group were 165-fold higher following treatment compared with levels beforetreatment (119901 = 0004) Similarly the G120574-globin mRNA levelsin the Radix Astragali treatment group were 153-fold higherfollowing treatment compared with levels before treatment(119901 = 0002) These differences were deemed significantIn contrast no significant differences were observed in G120574-globin levels in patients from the control group before andafter placebo drug treatment (119901 = 0293) (Figure 1) sug-gesting that AMW and its compounded formulation signif-icantly increased G120574-globin mRNA transcription The latterphenomenon might explain the increased HbF levels thatwere observed in the clinical trial However neither AMWnor its compounded formulation affected120572-120573- orA120574-globinmRNA levels after 12 weeks of treatment (Figure 2)
33 p38 MAPK Signaling Pathway Changes before and afterTreatment p38 MAPK was initially identified as a proteinkinase that is activated by stress This kinase has also beenshown to coordinate cellular responses during erythropoiesis[25ndash27] p38 MAPK is essential for hemoglobin synthesisand p38 mice exhibit severe anemia and die in utero dueto defective angiogenesis and placental insufficiency [28] Inresponse to sodium butyrate and trichostatin A treatment oferythroid cells p38 MAPK mediates HbF induction by acti-vating cAMP response element binding protein 1 (CREB1)p38 signaling regulates G120574-globin transcription during ery-throid maturation via a downstream effector CREB1 whichbinds to the G120574-globin cAMP response element (G-CRE)Moreover gain of p38 or CREB1 function augments 120574-globintranscription and these regulatory effects were observed to beconserved under tested physiological conditions in primaryerythroid cells [29]
0
02
04
06
08
1
12
14
Control AMW Compoundedformulation
BeforeAfter
Rela
tive G
120574-g
lobi
n m
RNA
leve
l lowast
lowast
Figure 1 Relative G120574-globin gene expression before and aftertreatment of pediatric 120573-thalassemia lowast119901 lt 005 compared with thecorresponding control 119901 lt 001 compared with before treatmentof the same group
Given that the p38 MAPK signaling pathway is the keymediator pertaining to stress induction of 120574-globin mRNA[30] we proposed that the induction of G120574-globin geneexpression following AMW and compounded formulationtreatment may increase p38 MAPK expression or activa-tion Indeed a previous study performed by our groupdemonstrated that one of the compounded agents Plastrumtestudinis facilitated 120574-globin mRNA accumulation and HbFsynthesis in K562 cells following activation of the p38MAPKsignaling pathway These results were validated followingthe observation that the latter effects were suppressed afterpretreatment of the K562 cells with the p38 MAPK inhibitorSB203580 [30] Furthermore a separate study conducted byour group demonstrated that aqueous extracts ofRadixAstra-gali aqueous extracts of the compounded formulation and05mmolL sodium butyrate all independently induced p38phosphorylation in K562 cells and cultured human erythroidprogenitor cells from the peripheral blood of 120573-thalassemiaAll of these reactions demonstrated a similar time-effect pro-file phosphorylated p38 levels began to rise at 6ndash8 h peakedat 48ndash60 h and later began to decline Once more SB203580inhibited the increases in p38 phosphorylation that wereinitially induced by the aqueous extracts [22]
Thus we measured total p38 MAPK (t-p38) and phos-phorylated p38 MAPK (p-p38) levels in nucleated redblood cell-enriched mononuclear cells collected in AMW-compounded formulation- or placebo-treated patients Weobserved that t-p38 mRNA (Figure 3(a) 119901 = 0999 and 0965before and after treatment) and protein (Figures 3(b) and3(c) 119901 = 0554 and 0476 before and after treatment) levelswere not significantly different between the groups Howeverp-p38 protein levels were comparable in all three groups
Evidence-Based Complementary and Alternative Medicine 5
0
50
100
150
200
250
300
Control AMW
Rela
tive120572
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(a)
0
1
2
3
4
5
6
7
Control AMW
Rela
tive120573
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(b)
0
01
02
03
04
05
06
07
Control AMW
Rela
tive A
120574-g
lobi
n m
RNA
leve
l
Compoundedformulation
BeforeAfter
(c)
Figure 2 Relative globin gene expression before and after treatment of pediatric 120573-thalassemia (a) 120572-globin (b) 120573-globin and (c) A120574-globin
(Figure 3(d) 119901 = 0813) prior to treatment and proteinlevels were significantly different after treatment (Figure 3(d)119901 = 0013) ANOVA analysis and subsequent LSD analysisindicated that p-p38 levels were significantly higher afterAMW and compounded formulation treatments comparedto placebo treatment Compared with p-p38 protein levelsthat were measured before treatment the compounded for-mulation and AMW groups expressed 23- and 22-fold moreprotein respectively after treatment (Figure 3) Howeverdifferences between these treatment groups were not signifi-cantly different
We previously reported clinical outcomes pertainingto AMW and compounded formulation treatments [14]Following 12 weeks of treatment pediatric patients with
120573-thalassemia had increased fetal Hb erythrocytes andmean corpuscular Hb and reticulocytes Furthermore noadverse effects were observed with respect to leucocytesneutrophils platelets or hepatic and renal function Thetreatment was effective in 64 and 62 of the children with120573-thalassemia in the compounded formulation and AMWgroups respectively These outcomes suggest that AMWand the compounded formulation are effective and safeMoreover increases in Hb and HbF levels were observedin AMW- and compounded formulation-treated patientsindicating that both treatments may be effective and safe inrelation to HbF induction in patients
Traditional Chinese herbal medicine (TCM) is believedto cause relatively few side effects However current reports
6 Evidence-Based Complementary and Alternative Medicine
0
5
10
15
20
25
30
35
40
45
50
Control AMW
Rela
tive p
38
MA
PK m
RNA
leve
l
Compoundedformulation
BeforeAfter
(a)
1 2 3 4 5 6
p38 MAPK
p-p38 MAPK
120573-Actin
(b)
0
02
04
06
08
1
12
14
16
18
2
Control AMW
Relat
ive p
38 M
APK
pro
tein
leve
l
Compoundedformulation
BeforeAfter
(c)
0
02
04
06
08
1
12
Control AMW
lowastlowast
lowast
Rela
tive p
-p38
MA
PK p
rote
in le
vel
Compoundedformulation
BeforeAfter
(d)
Figure 3 Changes in p38MAPKexpression and activation before and after treatment of pediatric120573-thalassemia (a) Changes inmRNA levels(b) Representative images ofWestern blot analyses Graphs of blot quantification data pertaining to total p38MAPK and phosphorylated p38MAPK (p-p38 MAPK) levels are shown in (c) and (d) respectively lowast119901 lt 005 and lowastlowast119901 lt 001 compared with the corresponding control119901 lt 001 compared with before treatment of the same group
pertaining to the use of TCM in relation to 120573-thalassemiaare complicated and the identification of a single 120574-globininducer from TCM preparations is difficult We previouslyreported that AMW and an associated compounded formu-lation for pediatric 120573-thalassemia can significantly improvehematological parameters [14 16] resulting in elevated HbFlevels in patients treated for twelve weeks with AMW or thecompounded formulation Compared with butyrate admin-istration induction was lasting longer and did not inhibiterythroid cells Thalassemia is a hematological disorder andseveral TCM preparations have been reported to improve
hematological parameters in anemic patients [17] Morespecifically Plastrum testudinis has been shown to induce 120574-globin expression in erythroid cells (unpublished data)
Based on TCM theory and effects observed in vitro andin vivo we developed an AMW formulation and a com-bined AMW CPG and TPG formulation for 120573-thalassemiatreatment As part of this study we report that treatmentwith AMW or the compounded formulation for 12 weekssignificantly affected G120574-globin mRNA levels and increasedphosphorylated p38 MAPK levels with no concomitantchanges to total p38 MAPK mRNA and protein levels These
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
Evidence-Based Complementary and Alternative Medicine 5
0
50
100
150
200
250
300
Control AMW
Rela
tive120572
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(a)
0
1
2
3
4
5
6
7
Control AMW
Rela
tive120573
-glo
bin
mRN
A le
vel
Compoundedformulation
BeforeAfter
(b)
0
01
02
03
04
05
06
07
Control AMW
Rela
tive A
120574-g
lobi
n m
RNA
leve
l
Compoundedformulation
BeforeAfter
(c)
Figure 2 Relative globin gene expression before and after treatment of pediatric 120573-thalassemia (a) 120572-globin (b) 120573-globin and (c) A120574-globin
(Figure 3(d) 119901 = 0813) prior to treatment and proteinlevels were significantly different after treatment (Figure 3(d)119901 = 0013) ANOVA analysis and subsequent LSD analysisindicated that p-p38 levels were significantly higher afterAMW and compounded formulation treatments comparedto placebo treatment Compared with p-p38 protein levelsthat were measured before treatment the compounded for-mulation and AMW groups expressed 23- and 22-fold moreprotein respectively after treatment (Figure 3) Howeverdifferences between these treatment groups were not signifi-cantly different
We previously reported clinical outcomes pertainingto AMW and compounded formulation treatments [14]Following 12 weeks of treatment pediatric patients with
120573-thalassemia had increased fetal Hb erythrocytes andmean corpuscular Hb and reticulocytes Furthermore noadverse effects were observed with respect to leucocytesneutrophils platelets or hepatic and renal function Thetreatment was effective in 64 and 62 of the children with120573-thalassemia in the compounded formulation and AMWgroups respectively These outcomes suggest that AMWand the compounded formulation are effective and safeMoreover increases in Hb and HbF levels were observedin AMW- and compounded formulation-treated patientsindicating that both treatments may be effective and safe inrelation to HbF induction in patients
Traditional Chinese herbal medicine (TCM) is believedto cause relatively few side effects However current reports
6 Evidence-Based Complementary and Alternative Medicine
0
5
10
15
20
25
30
35
40
45
50
Control AMW
Rela
tive p
38
MA
PK m
RNA
leve
l
Compoundedformulation
BeforeAfter
(a)
1 2 3 4 5 6
p38 MAPK
p-p38 MAPK
120573-Actin
(b)
0
02
04
06
08
1
12
14
16
18
2
Control AMW
Relat
ive p
38 M
APK
pro
tein
leve
l
Compoundedformulation
BeforeAfter
(c)
0
02
04
06
08
1
12
Control AMW
lowastlowast
lowast
Rela
tive p
-p38
MA
PK p
rote
in le
vel
Compoundedformulation
BeforeAfter
(d)
Figure 3 Changes in p38MAPKexpression and activation before and after treatment of pediatric120573-thalassemia (a) Changes inmRNA levels(b) Representative images ofWestern blot analyses Graphs of blot quantification data pertaining to total p38MAPK and phosphorylated p38MAPK (p-p38 MAPK) levels are shown in (c) and (d) respectively lowast119901 lt 005 and lowastlowast119901 lt 001 compared with the corresponding control119901 lt 001 compared with before treatment of the same group
pertaining to the use of TCM in relation to 120573-thalassemiaare complicated and the identification of a single 120574-globininducer from TCM preparations is difficult We previouslyreported that AMW and an associated compounded formu-lation for pediatric 120573-thalassemia can significantly improvehematological parameters [14 16] resulting in elevated HbFlevels in patients treated for twelve weeks with AMW or thecompounded formulation Compared with butyrate admin-istration induction was lasting longer and did not inhibiterythroid cells Thalassemia is a hematological disorder andseveral TCM preparations have been reported to improve
hematological parameters in anemic patients [17] Morespecifically Plastrum testudinis has been shown to induce 120574-globin expression in erythroid cells (unpublished data)
Based on TCM theory and effects observed in vitro andin vivo we developed an AMW formulation and a com-bined AMW CPG and TPG formulation for 120573-thalassemiatreatment As part of this study we report that treatmentwith AMW or the compounded formulation for 12 weekssignificantly affected G120574-globin mRNA levels and increasedphosphorylated p38 MAPK levels with no concomitantchanges to total p38 MAPK mRNA and protein levels These
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
6 Evidence-Based Complementary and Alternative Medicine
0
5
10
15
20
25
30
35
40
45
50
Control AMW
Rela
tive p
38
MA
PK m
RNA
leve
l
Compoundedformulation
BeforeAfter
(a)
1 2 3 4 5 6
p38 MAPK
p-p38 MAPK
120573-Actin
(b)
0
02
04
06
08
1
12
14
16
18
2
Control AMW
Relat
ive p
38 M
APK
pro
tein
leve
l
Compoundedformulation
BeforeAfter
(c)
0
02
04
06
08
1
12
Control AMW
lowastlowast
lowast
Rela
tive p
-p38
MA
PK p
rote
in le
vel
Compoundedformulation
BeforeAfter
(d)
Figure 3 Changes in p38MAPKexpression and activation before and after treatment of pediatric120573-thalassemia (a) Changes inmRNA levels(b) Representative images ofWestern blot analyses Graphs of blot quantification data pertaining to total p38MAPK and phosphorylated p38MAPK (p-p38 MAPK) levels are shown in (c) and (d) respectively lowast119901 lt 005 and lowastlowast119901 lt 001 compared with the corresponding control119901 lt 001 compared with before treatment of the same group
pertaining to the use of TCM in relation to 120573-thalassemiaare complicated and the identification of a single 120574-globininducer from TCM preparations is difficult We previouslyreported that AMW and an associated compounded formu-lation for pediatric 120573-thalassemia can significantly improvehematological parameters [14 16] resulting in elevated HbFlevels in patients treated for twelve weeks with AMW or thecompounded formulation Compared with butyrate admin-istration induction was lasting longer and did not inhibiterythroid cells Thalassemia is a hematological disorder andseveral TCM preparations have been reported to improve
hematological parameters in anemic patients [17] Morespecifically Plastrum testudinis has been shown to induce 120574-globin expression in erythroid cells (unpublished data)
Based on TCM theory and effects observed in vitro andin vivo we developed an AMW formulation and a com-bined AMW CPG and TPG formulation for 120573-thalassemiatreatment As part of this study we report that treatmentwith AMW or the compounded formulation for 12 weekssignificantly affected G120574-globin mRNA levels and increasedphosphorylated p38 MAPK levels with no concomitantchanges to total p38 MAPK mRNA and protein levels These
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
Evidence-Based Complementary and Alternative Medicine 7
results suggest that AMW and the compounded formulationare 120574-globin gene-inducing reagents However because TCMtargets multiple receptors definitive mechanisms of actionare often difficult to identify Larger studies are requiredto confirm our preliminary results regarding 120574-globin geneinduction and improvements in relation to the occurrenceof anemia In addition we believe that additional factorsincluding iron load 120573-thalassemia complications such ashepatosplenomegaly heart failure delayed growth and devel-opment and long-term safety dose optimization and treat-ment regimens pertaining to TCM are also worth exploringThus treatment with AMW and the compounded formu-lation can induce G120574-globin gene expression in vivo Thisinduction is likely to stimulate HbF synthesis and enhancetotal hemoglobin levels in patients with 120573-thalassemia Fur-thermore independent studies performed by our groupand another group demonstrated that the agents used inthe treatment regimens used in the current study do notelicit these HbF stimulation phenomena through cytotoxicmechanisms [21 31] Thus we believe that HbF synthesisstimulation may occur via the p38 MAPK signaling pathway
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contributions
Zhuoming Lu designed the study recruited and treatedthe patients performed the molecular biology experimentsanalyzed the data and authored the manuscript XinhuaQian assisted with study design patient recruitment andtreatment and molecular biology experiments ChunhongZhang Zhiwen Chen and Guangliang Du assisted with therecruitment and treatment of patients and the executionof molecular biology experiments All authors read andapproved the final manuscript
Acknowledgments
This study was supported by grants fromGuangdongAdmin-istration Bureau of Chinese Medicine Guangdong China(nos 2010285 and 20141208) and the Natural Science Foun-dation of Guangdong Province (no 2015A030310311) SpecialCollaborative Grants from Guangdong Provincial Depart-ment of Science and Technology (no 2011B032200002)helped to fund this study The authors thank Mr WeijieXu (from Guangzhou Qenequick Biotechnology) for hisassistance with molecular biology experiments
References
[1] H L Muncie Jr and J S Campbell ldquoAlpha and beta tha-lassemiardquoAmerican Family Physician vol 80 no 4 pp 339ndash3712009
[2] Y Xiang J-W Yu Y-B Cheng et al ldquoStudy on composing pre-scription laws of treating aplastic anemia by Chinese medicine
using applying data mining techniquerdquo Zhongguo Zhong Xi YiJie He Za Zhi vol 33 no 7 pp 906ndash910 2013
[3] D Rund and E Rachmilewitz ldquo120573-thalassemiardquo The New Eng-land Journal of Medicine vol 353 no 11 pp 1135ndash1146 2005
[4] S L Schrier ldquoPathobiology of thalassemic erythrocytesrdquo Cur-rent Opinion in Hematology vol 4 no 2 pp 75ndash78 1997
[5] B Modell and M Darlison ldquoGlobal epidemiology ofhaemoglobin disorders and derived service indicatorsrdquoBulletin of the World Health Organization vol 86 no 6 pp480ndash487 2008
[6] M CWalters ldquoGene therapy and bonemarrow transplantationfor thalassemia changing of the guardrdquoMolecularTherapy vol18 no 9 p 1577 2010
[7] D J Weatherall ldquoThe challenge of thalassemia for the develop-ing countriesrdquo Annals of the New York Academy of Sciences vol1054 pp 11ndash17 2005
[8] M Banan ldquoHydroxyurea treatment in 120573-thalassemia patientsto respond or not to respondrdquo Annals of Hematology vol 92no 3 pp 289ndash299 2013
[9] V V Bohara S Ray P Chakrabarti S S Ray U K Nath andU Chaudhuri ldquoOptimizing the dose of hydroxyurea therapy forpatients with 120573-thalassemia intermedia (Hb E-120573-thalassemia)a single center study from eastern Indiardquo Hemoglobin vol 38no 1 pp 44ndash48 2014
[10] M Kosaryan M Zafari A Alipur and A Hedayatizadeh-Omran ldquoThe effect and side effect of hydroxyurea therapy onpatients with 120573-thalassemia a systematic review to December2012rdquo Hemoglobin vol 38 no 4 pp 262ndash271 2014
[11] N Bianchi C Zuccato I Lampronti M Borgatti and RGambari ldquoFetal hemoglobin inducers from the natural worlda novel approach for identification of drugs for the treatmentof 120573-thalassemia and sickle-cell anemiardquo Evidence-Based Com-plementary and Alternative Medicine vol 6 no 2 pp 141ndash1512009
[12] C H Lowrey and AW Nienhuis ldquoBrief report treatment withazacitidine of patients with end-stage 120573- thalassemiardquoThe NewEngland Journal of Medicine vol 329 no 12 pp 845ndash848 1993
[13] X Zhu and B Zhu ldquoEffect of Astragalus membranaceusinjection on megakaryocyte hematopoiesis in anemic micerdquoHua Xi Yi Ke Da Xue Xue Bao vol 32 no 4 pp 590ndash592 2001
[14] Z-M Lu X-H Qian Z-W Chen C-H Zhang L-S Guoand J Chen ldquoProspective clinical study of radix astragali andits compound prescription for treatment of beta-thalassemia inchildrenrdquo Zhongguo Dang Dai Er Ke Za Zhi vol 14 no 5 pp344ndash349 2012
[15] Y Zhang B-D Ye L-L Qian et al ldquoTreatment of myelodys-plastic syndrome by hematopoietic stem cell transplantationcombined with Chinese medical syndrome typing a clinicalstudyrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol 35 no 1 pp53ndash56 2015
[16] M Yang X-H Qian D-H Zhao and S-Z Fu ldquoEffectsof Astragalus polysaccharide on the erythroid lineage andmicroarray analysis in K562 cellsrdquo Journal of Ethnopharmacol-ogy vol 127 no 2 pp 242ndash250 2010
[17] Y-C Linn J Lu L-C Lim H Sun J Sun and Y ZhouldquoTraditional Chinese herbal medicine in the supportive man-agement of patients with chronic cytopaenicmarrow diseasesmdasha phase III clinical studyrdquo ComplementaryTherapies in ClinicalPractice vol 17 no 3 pp 152ndash156 2011
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
8 Evidence-Based Complementary and Alternative Medicine
[18] Y-M Liu Z-K Wu L-M Chai et al ldquoEffect on expression ofmice alpha-hemoglobin stabilizing protein in different develop-mental stages treated with Yisui Shengxue granulesrdquo ZhongguoZhong Yao Za Zhi vol 32 no 7 pp 609ndash612 2007
[19] X Qian J Chen D Zhao L Guo and X Qian ldquoPlastrumtestudinis induces 120574-globin gene expression through epigenetichistone modifications within the 120574-globin gene promoter viaactivation of the p38 MAPK signaling pathwayrdquo InternationalJournal of MolecularMedicine vol 31 no 6 pp 1418ndash1428 2013
[20] M S Chang D R Kim E B Ko et al ldquoTreatment withastragali radix and angelicae radix enhances erythropoietingene expression in the cyclophosphamide-induced anemic ratrdquoJournal of Medicinal Food vol 12 no 3 pp 637ndash642 2009
[21] Z-M Guo H-J Li and X-H Qian ldquo120574-globin synthesis inK562 cells induced with Tortois plastron Astragali Salviaemiltiorrhizae and Codonopsis pilosulaerdquo Zhongguo Shi Yan XueYe Xue Za Zhi vol 16 no 3 pp 520ndash524 2008
[22] D Zhao Role of p38 MAP kinase activation in radix astragali-mediated 120574-globin gene expression [MS thesis] Southern Med-ical University Guangzhou China 2008
[24] K H Kwon Y J Jeon H S Hwang et al ldquoA high yield of fetalnucleated red blood cells isolated using optimal osmolality anda double-density gradient systemrdquo Prenatal Diagnosis vol 27no 13 pp 1245ndash1250 2007
[25] EMartın-Blanco ldquop38MAPK signalling cascades ancient rolesnew functionsrdquo BioEssays vol 22 no 7 pp 637ndash645 2000
[26] V P Cokic R D Smith A Biancotto C T Noguchi R K Puriand A N Schechter ldquoGlobin gene expression in correlationwith G protein-related genes during erythroid differentiationrdquoBMC Genomics vol 14 no 1 article 116 2013
[27] Y-C Chou R-L Chen Z-S Lai J-S Song Y-S Chao and C-K J Shen ldquoPharmacological induction of human fetal globingene in hydroxyurea-resistant primary adult erythroid cellsrdquoMolecular and Cellular Biology vol 35 no 14 pp 2541ndash25532015
[28] D S Steinbrech B JMehrara P B Saadeh et al ldquoVEGF expres-sion in an osteoblast-like cell line is regulated by a hypoxiaresponse mechanismrdquo American Journal of PhysiologymdashCellPhysiology vol 278 no 4 pp C853ndashC860 2000
[29] V Ramakrishnan and B S Pace ldquoRegulation of 120574-globin geneexpression involves signaling through the p38 MAPKCREB1pathwayrdquo Blood Cells Molecules amp Diseases vol 47 no 1 pp12ndash22 2011
[30] B S Pace X-H Qian J Sangerman et al ldquop38 MAP kinaseactivation mediates 120574-globin gene induction in erythroid pro-genitorsrdquo Experimental Hematology vol 31 no 11 pp 1089ndash1096 2003
[31] M Yang D H Zhao and X H Qian ldquoEffects of the mainextracts of Astragalus membranaceus on inducing the erythroiddifferentiation of K562 cellsrdquoMedical Journal of Chinese PeoplersquosLiberation Army vol 33 no 3 pp 293ndash295 2008
