Research ArticleProtective Effect and Mechanism of TotalFlavones from Rhododendron simsii Planch Flower on CulturedRat Cardiomyocytes with Anoxia and Reoxygenation
Yi Jiao1 Yi-Fei Fan1 Yu-Ling Wang1 Jun-Yan Zhang1 Shuo Chen2 and Zhi-Wu Chen1
1Department of Pharmacology Anhui Medical University Hefei Anhui 230032 China2Xinglin College Liaoning University of Traditional Chinese Medicine Shenyang Liaoning 110167 China
Correspondence should be addressed to Zhi-Wu Chen chpharmzw163com
Received 14 August 2014 Revised 25 December 2014 Accepted 31 December 2014
Academic Editor Hyunsu Bae
Copyright copy 2015 Yi Jiao et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Many flavonoids have cardioprotection against myocardial ischemiareperfusion (IR) injury Total flavones from Rhododendronsimsii Planch flower (TFR) can protect myocardial ischemic injuries However its protective mechanism is still unknown Thepresent study was designed to investigate the mechanism of TFR on myocardial IR and anoxiareoxygenation (AR) injuries Ratmodel of myocardial IR injury was made and myocardial infarction was determined AR injury was induced in cultured ratcardiomyocytes cellular damage was evaluated by measuring cell viability LDH and cTnT releases andMDA content Expressionsof ROCK
1and ROCK
2protein were examined by Western blot analysis and K+ currents were recorded by using whole-cell
patch clamp technique TFR 20sim80mgkg markedly reduced IR-induced myocardial infarction TFR 37sim300mgL significantlyinhibited AR-induced reduction of cell viability LDH and cTnT releases and MDA production Exposure to AR significantlyincreased ROCK
1and ROCK
2expressions in rat cardiomyocytes but TFR 333sim300mgL obviously inhibited this increase
300mgL TFR significantly augmented inward rectifier K+ current and other K+ currents in rat cardiomyocytes These resultsindicate that TFR has a protective effect on rat cardiomyocytes AR damage and the protective mechanism may be engaged withthe inhibition of ROCK
1and ROCK
2and activation of K+ channels
1 Introduction
Ischemic cardiovascular disease the most common heartdisease is the main cause of mortality and morbidity world-wide Coronary arteries and their branches supply oxygen-rich blood to myocardium Stopping of blood supply to theheart muscle leads to myocardial ischemic injury Restora-tion of blood supply is essential to prevent irreversibleinjury However sudden blood flow returning to ischemicmyocardium may paradoxically augment myocardial injuryThis is referred to as myocardial ischemiareperfusion(IR) injury [1] characterized by myocardial inflammatoryresponses metabolic disorder cardiac dysfunction and sub-sequent myocardial cell death Alike oxygen deprivationfollowed by reoxygenation causes anoxia and reoxygenation(AR) injury
The RhoARho-kinase signaling pathway has an impor-tant role in many pathological processes The Rho-kinase(Rho-associated coiled-coil forming protein kinase ROCK)is a serinethreonine kinase belonging to the AGC (PKAPKGPKC) family ROCK was identified as the first effectorsof RhoA it mediates some essential cellular functions includ-ing cell shape motility cellular contractility proliferationcoronary vasospasm and inflammation [2] There are twoisoforms of ROCK ROCK
1and ROCK
2 Recent studies
demonstrate that ROCKs play a major role in the pathogen-esis of myocardial infarction [3] ROCK activation occurs inrat reperfused myocardium specifically and this activation isdeleterious Moreover inhibition of ROCKs has a protectiveeffect against myocardial IR injury in rat [4] Y-27632 aROCK inhibitor significantly reduced IR injury-inducedinfarct size and cardiomyocyte apoptosis by attenuating
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 863531 10 pageshttpdxdoiorg1011552015863531
2 Evidence-Based Complementary and Alternative Medicine
inflammatory responses [5]These studies suggest that ROCKinhibitionmay be a novel therapeutic target for the treatmentof ischemic cardiovascular diseases
Multiple types of K+ channels are present in the cardio-vascular system and play crucial roles It is a known factthat K+ channel blocker can block the infarct-limiting effectsof ischemic preconditioning and K+ channel openers mimicthe protective effect [6] The opening of ATP-sensitive K+channel (KATP channel) could protect cardiac myocytesagainst ischemic injuries The cardiac KATP channel consistsof two distinct proteins an inwardly rectifying potassiumchannel pore subunit (Kir62) and the sulfonylurea receptor(SUR2A) 51015840-AMP-activated protein kinase (AMPK) is anenergy sensor protein kinase that takes part in regulatingcellular energy it interacted with Kir62 subsequently trig-gers and promotesKir62KATP channel opening and inducescardioprotection However Kir62 knockout could eliminatethe cardioprotective effect of AMPK [4 7] Adenosine-mediated protection against IR injury was found to beabolished by KATP channel inhibition in humanmyocardium[8] Inward rectifierK+ channel is distinct fromKATP channelwhich plays an important role in the repolarization of cardiacmyocytes Inhibition of inward rectifier K+ channels (Kir21or Kir22) abolished protection of ischemic preconditioningin rabbit cardiomyocytes [9]
Flavonoid compounds are widely distributed in manyChinese herbs and natural plants and have various bio-logical activities and pharmacological functions includingvasorelaxing as well as cardioprotection against myocardialIR injury [10ndash12] Therefore Chinese herbs are importantresources to develop valid and safe drugs for the treatmentof diseases Rhododendron simsii Planch flower a Chineseherbal medicine has been used for treating patients withbronchitis in China for thousands year Total flavones fromRhododendron simsii Planch flower (TFR) an effective partextracted from Rhododendron simsii Planch flower is com-prised of flavones such as rutin hyperin quercetin and otherflavonoids [13 14] Our previous studies have shown thatTFR has significant protective effects against myocardial orcerebral ischemic injuries in rabbit and rat [10ndash12] Howeverits mechanism of cardioprotection remains poorly under-stoodTherefore in the present study the mechanism of TFRon myocardial IR and AR injury was investigated in ratmodel of IR-induced myocardial infarction and cultured ratcardiomyocytes with AR injury By using the examination ofROCK protein expression whole-cell patch clamp recordingand other approaches the main focus in this study is on theroles of ROCK and K+ channels in the cardioprotection ofTFR
2 Material and Methods
21 Drugs and Reagents TFR (content of flavones greaterthan 85) was provided by Hefei Heyuan Medicine Tech-nology Co Ltd (Hefei China) nifedipine and BaCl
2were
purchased from Sigma Co USA Y-27632 was purchasedfrom Santa Cruz USA malondialdehyde (MDA) and lac-tate dehydrogenase (LDH) assay kits were purchased fromNanjing Jiancheng Biological Co Nanjing China cardiac
troponin T (cTnT) ELISA kits were provided by ShanghaiYuanye Biological Co Shanghai China ROCK
1and ROCK
2
were provided by Nanjing Enogene Biological Co NanjingChina
22 Animals Neonatal Sprague-Dawley rats (1sim2 d old halfmale and half female) and adult male Sprague-Dawley rats(weighing 300 to 350 g) were purchased from the Experimen-tal Animal Center of Anhui Medical University Rats werehoused at 22 plusmn 2∘C and relative humidity of 40 plusmn 5 undera 12-hour lightdark cycle This investigation conforms to theregulations stipulated by Anhui Medical University animalcare committee which follows the protocol outlined in theGuide for the Care and Use of Laboratory Animals publishedby the US National Institutes of Health (NIH Publication no85-23 revised 1996)
23 Rat Myocardial IR Injury Model Rat myocardial IRinjury model was carried out according to previous method[10] with some modifications Adult male Sprague-Dawleyrat was anesthetized with 10 chloral hydrate (3mLkg)by peritoneal injection and placed in a supine positionThoracotomy was performed by removing the left third ribto expose the heart a 5-0 suture silk was placed around theleft anterior descending coronary artery (LAD) which was1-2mm under the boundary of pulmonary conus and leftauricle and the ends of this ligature were passed through asmall plastic tube to form a snare The rat underwent 30minof ischemia and then was released allowing reperfusion for aperiod of 90min At the end of reperfusion LADwas ligatedand 025 Evans blue was administrated through femoralveinThen the hearts were harvested and frozen at minus20∘C in afreezer The heart was sectioned into five 2sim3mm transverseslices and incubated in 1 TTC in phosphate buffer (pH 7437∘C) for 15min Infarct size (IS) area at risk (AAR) and leftventricle size of each slice were analyzed by ImageJ version16 (National Institutes of Health Bethesda Md USA)
The rats were randomly divided into the following 7groups sham group IR group 16mgkg verapamil group30mgkg Y27632 group 20mgkg TFR group 40mgkg TFRgroup and 80mgkg TFR group Y27632 and verapamil wereadministrated by intravenous injection once a day for 5 daysand TFR was treated by gavage once a day for 5 days
24 Primary Culture of Neonatal Rat Cardiomyocytes Pri-mary culture of cardiomyocytes was prepared from neonatalSprague-Dawley rat Briefly rat heart was harvested andplaced in ice-cold Ca2+- and bicarbonate-free Hanksrsquo bufferThe ventricle was excised and minced Minced ventriculartissues were dissociated by treatment with 009 trypsin 5times at 37∘C for 10min The supernatants of the first diges-tion were discarded but other four supernatants were savedin Dulbeccorsquos modified Eaglersquos medium (DMEM) containing10 fetal bovine serum (FBS Hyclone Corp South America)and then centrifuged for 5min at 900timesg Resuspended cellswere then placed in a culture bottle at 37∘C in a 5 CO
2ndash95
O2incubator for 15 h to adhere fibroblastThen nonadherent
cells were counted with a hemocytometer and the finalmyocyte cultures with over 90 cardiomyocytes were used
Evidence-Based Complementary and Alternative Medicine 3
for further experiments One part of cardiomyocytes werecultured in 35 mm culture dishes at 37∘C in a 5 CO
2ndash
95 O2incubator and the 3ndash5 d cultured cells were used for
patch clamp recording And other cardiomyocytes culturedin gelatin-coated plates incubated for 3ndash5 days were used forthe experiment of AR injury
25 AR Injury Model The prepared cardiomyocytes wererandomly divided into 9 groups Sham anoxia group ARgroup 1mM nifedipine group 1 120583MY-27632 group and TFR37 111 333 100 and 300mgL groups Except that shamanoxia group was incubated under normoxic conditionsother groupswere subjected to anoxia followed by reoxygena-tion Anoxia was induced by changing the air content with95 N
2and 5 CO
2gas mixture in a metabolic chamber
for 3 h Then the cultures were reoxygenated for 12 h byincubating the cells in 95 O
2and 5 CO
2 Nifedipine Y-
27632 and TFR were respectively added into the DMEM at24 h before anoxia
Another set of experiment was performed for Westernblot assay under the same conditions and grouping
26 Measurement of MTT LDH MDA and cTnT LevelsAfter AR model 20120583L MTT solution (5mgmL) was addedinto each well and incubated for an additional 4 h Then100 120583L DMSO was added into cardiomyocyte cultures todissolve the formazan particulates Finally absorbance at490 nmwasmeasured using amicroplate reader for theMTTassay
Supernatants of cardiomyocyte cultures were collectedMDA content and LDH activity were respectively detectedat 532 nm and 450 nmby spectrophotometry according to theprocedures provided by the assay kits
The level of cTnT in supernatants was measured by themethod of immunoassay [15]
27 Western Blot Analysis Cardiomyocytes were lysed bythe solution containing 04mmol Lminus1 phenylmethanesulfonylfluoride (pH 74) separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis on 10 polyacrylamide-Trisgels (Beyotime China) and then transferred to a polyvinyli-dene difluoride membrane Membranes were blocked bybuffer (5 skim milk and 005 Tween 20 in Tris-bufferedsaline) at room temperature for 2 h and then incubated(4∘C overnight) with the same buffer containing rabbitpolyclonal antibodies against ROCK1 or ROCK2 (EnogeneChina) or monoclonal antibody against 120573-actin (Bioworld)After incubation with anti-rabbit second antibody (ZSGBBio company China 1 10000 dilution in 5 skim milk) for1 h at room temperature the bands were visualized usingan enhanced chemiluminescence kit (Thermo USA) Theintensity of immunoreactive bands was quantified with theuse of an imaging densitometer
28 Whole-Cell Patch Clamp Recording As previously de-scribed [16] K+ currents in single rat cardiomyocyte wererecorded with an EPC 10 patch clamp amplifier (HEKA Elek-tronik LambrechtPfalz Germany) with Pulse and Pulse-fit software 500 120583L of rat cardiomyocytes suspension was
placed in a perfusion chamber on a microscope stage andsuperfused with Tyrodersquos solution (mmolsdotLminus1 143 NaCl 54KCl 033 NaH
2PO4 18 CaCl
2 05 MgCl
2 5 HEPES and 11
glucose adjusted pH to 74 with NaOH) Micropipette patchpipette (resistance 25sim3MΩ) was filled with intercellularfluid (mmol Lminus1 135 KCl 5 NaCl 10 HEPES 5 EGTA10 120583M 441015840-diisothiocyano-221015840-stilbenedisulfonic acid and5 Mg-ATP adjusted pH to 72 with KOH) K+ current wasnormalized through dividing current by cell capacitance toget current density (pApF) Rat cardiomyocyte was held atminus80mV and voltage steps ranging from minus130 to + 70mVwereapplied for 500ms in 20mV increments After rupture of themembrane cardiomyocyte capacitance was estimated by acomposite of the capacitance current and compensation ofcell capacitor and pipette series resistanceThemeasurementscan be made only if seals are in the gigaohm range BaCl
2or
TFR was dissolved in the extracellular solution and directlygiven to cardiomyocyte using injection port setting Changesin K+ currents were observed in the same cell before and afteradministration of BaCl
2or TFR
29 Statistical Analysis Data are presented as means plusmn SDStatistical analyses were performed with one-way ANOVAfollowed by the Duncan test to determine the differencesbetween groups A value of 119875 lt 005 was regarded as statis-tically significant
3 Result
31 TFR Inhibits IR-Induced Myocardial Infarction Asshown in Figure 1 occlusion of LAD followed by reperfusioninduced obvious myocardial injury as indicated by measure-ment of the ISARR ratio The ISAAR ratio was 463 plusmn62 in IR group (lowastlowast119875 lt 001 versus sham group) 20 40and 80mgkg TFR significantly decreased the ISARR ratiorespectively (119875 lt 001 versus sham group) Similarly vera-pamil 16mgkg and Y27632 30mgkg also reduced the ISARR ratio (119875 lt 001 versus sham group)
32 Effect of TFR onViability of Rat Cardiomyocytes Viabilityof rat cardiomyocytes was validated by MTT assay Figure 2showed that AR induced a significant reduction of viabilityof neonatal rat cardiomyocytes compared to that in shamanoxia group (lowastlowast119875 lt 001 versus sham anoxia group) Inthe range of 37sim300mgL TFRmarkedly and concentration-dependently increased the viability of neonatal rat cardiomy-ocytes with an EC
50of 159mgL Similar to TFR 1mmolL
nifedipine also increased the viability of rat cardiomyocytes(119875 lt 001 versus AR group)
33 Effect of TFR on LDH Activity and cTnT Level Leakageof LDH or cTnT from cell into the culture medium is a majorindicator of myocardial AR injury Significant increasesof LDH activity and cTnT level in the culture medium ofneonatal rat cardiomyocytes were detected in AR group(lowastlowast119875 lt 001 versus sham anoxia group) Treatment withTFR (37 111 333 100 and 300mgL) or nifedipine 1mmolLmarkedly inhibited AR-induced increases of LDH activity
4 Evidence-Based Complementary and Alternative Medicine
60
40
20
0
Sham IR
Vera
pam
il
ISA
AR
()
Y27632
TFR20
mg
kg
TFR40
mg
kg
TFR80
mg
kg
lowastlowast
Figure 1 Effects of total flavones of Rhododendron simsii Planchflower (TFR)verapamilandY27632onischemiareperfusion-(IR-)induced myocardial infarction in rat Infarct size was expressed as apercentage of the area at risk (ISAAR) Results are means plusmn SD of6 experiments lowastlowast119875 lt 001 compared to sham 119875 lt 001 comparedto IR
and cTnT level in culture medium (119875 lt 005 or 119875 lt 001
versus AR group) (Figures 3(a) and 3(b))
34 Effect of TFR onProduction ofMDA There is a significantincrease of MDA content in AR group (lowastlowast119875 lt 001 versussham anoxia group) 37 111 333 100 and 300mgL TFRobviously reduced the MDA content compared with ARgroup Treatment of nifedipine 1mmolL had a similar effectin reducing MDA production (119875 lt 005 versus AR group)(Figure 4)
35 Effect of Y27632 on AR-Induced Injury of Rat Cardiomy-ocyte As shown in Figures 3(a) and 4 treatment with Y276321 120583molL dramatically inhibited AR-induced increases ofLDH activity in culture medium (119875 lt 001 versus ARgroup) And Y27632 also obviously decreased the MDAcontent compared with AR group (119875 lt 001 versus ARgroup)
36 Effect of TFR on ROCKs Protein Expression The expres-sions of both ROCK
1and ROCK
2proteins were found in
each group (Figure 5(a)) and levels of ROCK1and ROCK
2
proteins were quantified by using the densitometry (Figures5(b) and 5(c)) Exposure to AR significantly increased bothROCK
1and ROCK
2protein levels (lowastlowast119875 lt 001 versus sham
anoxia group) The increases of ROCK1and ROCK
2were
markedly inhibited by ROCK inhibitor Y27632 1120583molL orTFR 333 100 and 300mgL (119875 lt 005 or
119875 lt 001 versusAR group) 1mmolL nifedipine had a similar effect on theexpressions of ROCK
1and ROCK
2
lowastlowast
Sham AR
10
08
06
04
02
00
MTT
(OD
A490)
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Figure 2 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on the viability of rat car-diomyocytes subjected to anoxiareoxygenation (AR) (MTT assay)Results are means plusmn SD of 5 experiments lowast119875 lt 001 compared tosham anoxia 119875 lt 001 compared to AR
37 K+ Currents in Neonatal Rat Ventricular CardiomyocytesTo explore protectivemechanisms of TFR onmyocardial ARinjury the effect of TFR on K+ channel in rat cardiomyocyteswas investigated using the patch clamp method Undercurrent clamp mode outward K+ current was evoked in ratcardiomyocytes from a holding of 10 or 30mV to 70mV andinward K+ current was elicited from a holding of minus30 orminus50mV to minus130mV (Figure 6(a)) Both outward and inwardK+ currents were voltage-dependent Exposure of cardiomy-ocytes to BaCl
2(100 120583M) a relatively selective inward rectifier
K+ (Kir) channel inhibitor obviously suppressed the inwardK+ current (lowast119875 lt 005 versus control group) without effecton outward K+ current (Figures 6(a) and 6(b)) The resultsindicate inward K+ current evoked in rat cardiomyocytes wascarried by Kir channel
Figure 7 displays that 300mgL TFR markedly aug-mented the inward current and outward K+ currents (lowast119875 lt005 versus control group) suggesting that TFR could activateKir channel and other types of K+ channels to cause K+currents in rat cardiomyocytes
4 Discussion
In the present studywe have found that (1) TFRhas protectiveeffects against myocardial IR and AR injury in rat (2)ROCKs mediate protective effect of TFR on rat cardiomy-ocytes AR injury (3) TFR could promote opening of Kirchannel and other types of K+ channels and increases K+currents in rat cardiomyocytes
The increase in infarct size is documented to be a reliableindex of myocardial IR injury In this study 30min ofischemia followed by 90min of reperfusion was noted toinducemyocardial injury as assessed in terms of the increasedISARR ratio in rat heart Like calcium channel blocker
Evidence-Based Complementary and Alternative Medicine 5
LDH
(UL
)1500
1000
500
0
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowastlowast
(a)
cTnT
(ng
mL)
1000
200
600
800
400
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
lowastlowast
(b)
Figure 3 Effects of total flavones of Rhododendron simsii Planch flower (TFR) nifedipine (Nif) and Y27632 on lactate dehydrogenase (LDH)activity and cardiac troponin T (cTnT) level in the culture medium of neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR)(a) LDH activity (b) cTnT level Results are means plusmn SD of 5 experiments lowastlowast119875 lt 001 compared to sham anoxia 119875 lt 005 119875 lt 001compared to AR
MD
A (n
mol
L)
5
1
0
3
4
2
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowast
Figure 4 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on lactate dehydrogenase mal-ondialdehyde (MDA) in the culture medium of neonatal rat car-diomyocytes subjected to anoxiareoxygenation (AR) Results aremeans plusmn SD of 5 experiments lowast119875 lt 005 compared to sham anoxia119875 lt 005 compared to AR
verapamil TFR markedly reduced the ISARR ratio theresult indicates that TFR has a protective effect against myo-cardial IR injury in rat
LDH serves as an important metabolic enzyme in car-diomyocytes and could be leaked from injured cardiomy-ocytes Hence LDH level in the culture medium is a primary
index to evaluate cell damage [17] Troponin I is an inhibitorysubunit of troponin that binds to actin in thin myofilamentsto hold the actin-tropomyosin complex in place There arethree isoforms of troponin I cardiac troponin I (cTnI) fasttwitch skeletal muscle troponin I (fTnI) and slow twitchskeletal muscle troponin I (sTnI) cTnI is not present inserum from healthy people but it can be detected in serumfrom patients with acute myocardium damage Thus cTnI inthe culture medium is a sensitive and specific biochemicalmarker for detecting cardiomyocytes injury [9 18ndash20] In thepresent study AR-induced rat cardiomyocytes injury wasdetected as indicated by the decrease of cell viability andthe increases of LDH and cTnT in culture medium whiletreatment of TFR in the range of 37 to 300mgL significantlyimproved the aforementioned indexes including the increaseof cell viability and reductions of LDH and cTnT in culturemedium Calcium antagonist nifedipine had comparableeffects These results indicate that TFR has a significantprotective effect on AR-injured rat cardiomyocytes
AR injury can produce large amounts of oxygen-freeradicals in cardiomyocytes and subsequently causes lipid per-oxidation and leads to cell damage Thus lipid peroxidationis one of mechanisms of cellular damage MDA a productof lipid peroxidation has been applied to assess oxygen-free radicals-mediated myocardial IR injury [21] Our studyrevealed that 100 and 300mgL TFR significantly decreasedthe MDA level in culture medium the result not only furtherindicated that TFR has protection on myocardial AR injurybut also proposed that inhibition of lipid peroxidation maybe at least partially involved in cardioprotective mechanismof TFR against myocardial AR injury
6 Evidence-Based Complementary and Alternative Medicine
ROCK1
120573-actin
ROCK2
(a)
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
10
08
06
04
02
00
lowastlowast
ROCK
1120573
-act
in (
)
(b)
10
08
06
04
02
00
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
lowastlowast
ROCK
2120573
-act
in (
)
(c)
Figure 5 Effects of total flavones of Rhododendron simsii Planch flower (TFR) and Y23672 on expressions of ROCK1and ROCK
2proteins in
neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR) (a) Representative western blot analysis of expressions of ROCK1and
ROCK2 (b) Quantification of ROCK
1expression (c) Quantification of ROCK
2expression ROCK
1 ROCK
2 and 120573-actin were analyzed and
quantified by densitometric analysis Data are means plusmn SD of ROCK1120573-actin or ROCK
2120573-actin from 3 experiments lowastlowast119875 lt 001 compared
to sham anoxia 119875 lt 005 119875 lt 001 compared to AR
RhoA is one of the effecters of the small GTP-bindingprotein Rho ROCK is ubiquitously expressed it is the best-known downstream effector of RhoA Both ROCK
1and
ROCK2are expressed in vascular smooth muscle and in
myocardium [22ndash24] but their differential effects are still notwell characterized Increasing evidences have demonstratedthat the RhoA-ROCK pathway plays a pivotal role in car-diovascular pathogenesis such as IR injury vascular smoothmuscle cell (VSMC) proliferation cardiac hypertrophy heartfailure and ventricular remodeling [25 26] ROCK plays animportant role in myocardial AR damage and inhibitionof the RhoA-ROCK pathway has beneficial effects on bothheart and vasculature functions Treatment with the ROCKinhibitor Y-27632 or fasudil protected the heart against IRinjury and enhanced postischemia cardiac function [4 24]Our data indicate that ROCK inhibitor Y27632 markedlyinhibited AR injury-induced leakage of LDH and MDAproduction demonstrating that Y27632 had protective effectson myocardial AR injury In vivo rat model of myocardialIR Y27632 also significantly reducedmyocardial infarction
Oxidative species activate the ROCK pathway [27ndash29]RhoA expression is upregulated in ischemicmyocardium andlater activation of ROCKs occurs during reperfusion [4 30]
Our results show that exposure to AR injury significantlyincreased both ROCK
1and ROCK
2protein levels in rat
cardiomyocytes and the increases were markedly attenuatedby treatment of 333 100 and 300mgL TFR Together withthe facts that ROCK inhibitor Y27632 had similar suppressiononAR-induced increases of ROCK
1andROCK
2expressions
and protective effect on myocardial AR injury our resultssuggest that activation of ROCK during rat myocardialAR injury and inhibition of ROCK were involved in thecardioprotection of TFR against myocardial AR injury
Accumulating studies indicate ROCK is amajor regulatorof the contractile proteins including myosin light chainphosphatase (MLCP) myosin phosphatase target subunitCPI-17 and myosin light chain (MLC) ROCK is responsiblefor VSMCs contraction in various vascular beds [23] andsimilarmechanismwasmade for cardiac contractility ROCKinhibitor Y-27632 significantly suppressed the contractionin rabbit ventricular myocardium [31] This suggests thatthe ROCK activation may at least partly contribute tocardiac contractility and ROCK inhibition could decreasecardiac contractility It is a known fact that the decreaseof cardiac contractility could result in decreased cardiacwork and reduction in myocardial oxygen demand Thus
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
2 Evidence-Based Complementary and Alternative Medicine
inflammatory responses [5]These studies suggest that ROCKinhibitionmay be a novel therapeutic target for the treatmentof ischemic cardiovascular diseases
Multiple types of K+ channels are present in the cardio-vascular system and play crucial roles It is a known factthat K+ channel blocker can block the infarct-limiting effectsof ischemic preconditioning and K+ channel openers mimicthe protective effect [6] The opening of ATP-sensitive K+channel (KATP channel) could protect cardiac myocytesagainst ischemic injuries The cardiac KATP channel consistsof two distinct proteins an inwardly rectifying potassiumchannel pore subunit (Kir62) and the sulfonylurea receptor(SUR2A) 51015840-AMP-activated protein kinase (AMPK) is anenergy sensor protein kinase that takes part in regulatingcellular energy it interacted with Kir62 subsequently trig-gers and promotesKir62KATP channel opening and inducescardioprotection However Kir62 knockout could eliminatethe cardioprotective effect of AMPK [4 7] Adenosine-mediated protection against IR injury was found to beabolished by KATP channel inhibition in humanmyocardium[8] Inward rectifierK+ channel is distinct fromKATP channelwhich plays an important role in the repolarization of cardiacmyocytes Inhibition of inward rectifier K+ channels (Kir21or Kir22) abolished protection of ischemic preconditioningin rabbit cardiomyocytes [9]
Flavonoid compounds are widely distributed in manyChinese herbs and natural plants and have various bio-logical activities and pharmacological functions includingvasorelaxing as well as cardioprotection against myocardialIR injury [10ndash12] Therefore Chinese herbs are importantresources to develop valid and safe drugs for the treatmentof diseases Rhododendron simsii Planch flower a Chineseherbal medicine has been used for treating patients withbronchitis in China for thousands year Total flavones fromRhododendron simsii Planch flower (TFR) an effective partextracted from Rhododendron simsii Planch flower is com-prised of flavones such as rutin hyperin quercetin and otherflavonoids [13 14] Our previous studies have shown thatTFR has significant protective effects against myocardial orcerebral ischemic injuries in rabbit and rat [10ndash12] Howeverits mechanism of cardioprotection remains poorly under-stoodTherefore in the present study the mechanism of TFRon myocardial IR and AR injury was investigated in ratmodel of IR-induced myocardial infarction and cultured ratcardiomyocytes with AR injury By using the examination ofROCK protein expression whole-cell patch clamp recordingand other approaches the main focus in this study is on theroles of ROCK and K+ channels in the cardioprotection ofTFR
2 Material and Methods
21 Drugs and Reagents TFR (content of flavones greaterthan 85) was provided by Hefei Heyuan Medicine Tech-nology Co Ltd (Hefei China) nifedipine and BaCl
2were
purchased from Sigma Co USA Y-27632 was purchasedfrom Santa Cruz USA malondialdehyde (MDA) and lac-tate dehydrogenase (LDH) assay kits were purchased fromNanjing Jiancheng Biological Co Nanjing China cardiac
troponin T (cTnT) ELISA kits were provided by ShanghaiYuanye Biological Co Shanghai China ROCK
1and ROCK
2
were provided by Nanjing Enogene Biological Co NanjingChina
22 Animals Neonatal Sprague-Dawley rats (1sim2 d old halfmale and half female) and adult male Sprague-Dawley rats(weighing 300 to 350 g) were purchased from the Experimen-tal Animal Center of Anhui Medical University Rats werehoused at 22 plusmn 2∘C and relative humidity of 40 plusmn 5 undera 12-hour lightdark cycle This investigation conforms to theregulations stipulated by Anhui Medical University animalcare committee which follows the protocol outlined in theGuide for the Care and Use of Laboratory Animals publishedby the US National Institutes of Health (NIH Publication no85-23 revised 1996)
23 Rat Myocardial IR Injury Model Rat myocardial IRinjury model was carried out according to previous method[10] with some modifications Adult male Sprague-Dawleyrat was anesthetized with 10 chloral hydrate (3mLkg)by peritoneal injection and placed in a supine positionThoracotomy was performed by removing the left third ribto expose the heart a 5-0 suture silk was placed around theleft anterior descending coronary artery (LAD) which was1-2mm under the boundary of pulmonary conus and leftauricle and the ends of this ligature were passed through asmall plastic tube to form a snare The rat underwent 30minof ischemia and then was released allowing reperfusion for aperiod of 90min At the end of reperfusion LADwas ligatedand 025 Evans blue was administrated through femoralveinThen the hearts were harvested and frozen at minus20∘C in afreezer The heart was sectioned into five 2sim3mm transverseslices and incubated in 1 TTC in phosphate buffer (pH 7437∘C) for 15min Infarct size (IS) area at risk (AAR) and leftventricle size of each slice were analyzed by ImageJ version16 (National Institutes of Health Bethesda Md USA)
The rats were randomly divided into the following 7groups sham group IR group 16mgkg verapamil group30mgkg Y27632 group 20mgkg TFR group 40mgkg TFRgroup and 80mgkg TFR group Y27632 and verapamil wereadministrated by intravenous injection once a day for 5 daysand TFR was treated by gavage once a day for 5 days
24 Primary Culture of Neonatal Rat Cardiomyocytes Pri-mary culture of cardiomyocytes was prepared from neonatalSprague-Dawley rat Briefly rat heart was harvested andplaced in ice-cold Ca2+- and bicarbonate-free Hanksrsquo bufferThe ventricle was excised and minced Minced ventriculartissues were dissociated by treatment with 009 trypsin 5times at 37∘C for 10min The supernatants of the first diges-tion were discarded but other four supernatants were savedin Dulbeccorsquos modified Eaglersquos medium (DMEM) containing10 fetal bovine serum (FBS Hyclone Corp South America)and then centrifuged for 5min at 900timesg Resuspended cellswere then placed in a culture bottle at 37∘C in a 5 CO
2ndash95
O2incubator for 15 h to adhere fibroblastThen nonadherent
cells were counted with a hemocytometer and the finalmyocyte cultures with over 90 cardiomyocytes were used
Evidence-Based Complementary and Alternative Medicine 3
for further experiments One part of cardiomyocytes werecultured in 35 mm culture dishes at 37∘C in a 5 CO
2ndash
95 O2incubator and the 3ndash5 d cultured cells were used for
patch clamp recording And other cardiomyocytes culturedin gelatin-coated plates incubated for 3ndash5 days were used forthe experiment of AR injury
25 AR Injury Model The prepared cardiomyocytes wererandomly divided into 9 groups Sham anoxia group ARgroup 1mM nifedipine group 1 120583MY-27632 group and TFR37 111 333 100 and 300mgL groups Except that shamanoxia group was incubated under normoxic conditionsother groupswere subjected to anoxia followed by reoxygena-tion Anoxia was induced by changing the air content with95 N
2and 5 CO
2gas mixture in a metabolic chamber
for 3 h Then the cultures were reoxygenated for 12 h byincubating the cells in 95 O
2and 5 CO
2 Nifedipine Y-
27632 and TFR were respectively added into the DMEM at24 h before anoxia
Another set of experiment was performed for Westernblot assay under the same conditions and grouping
26 Measurement of MTT LDH MDA and cTnT LevelsAfter AR model 20120583L MTT solution (5mgmL) was addedinto each well and incubated for an additional 4 h Then100 120583L DMSO was added into cardiomyocyte cultures todissolve the formazan particulates Finally absorbance at490 nmwasmeasured using amicroplate reader for theMTTassay
Supernatants of cardiomyocyte cultures were collectedMDA content and LDH activity were respectively detectedat 532 nm and 450 nmby spectrophotometry according to theprocedures provided by the assay kits
The level of cTnT in supernatants was measured by themethod of immunoassay [15]
27 Western Blot Analysis Cardiomyocytes were lysed bythe solution containing 04mmol Lminus1 phenylmethanesulfonylfluoride (pH 74) separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis on 10 polyacrylamide-Trisgels (Beyotime China) and then transferred to a polyvinyli-dene difluoride membrane Membranes were blocked bybuffer (5 skim milk and 005 Tween 20 in Tris-bufferedsaline) at room temperature for 2 h and then incubated(4∘C overnight) with the same buffer containing rabbitpolyclonal antibodies against ROCK1 or ROCK2 (EnogeneChina) or monoclonal antibody against 120573-actin (Bioworld)After incubation with anti-rabbit second antibody (ZSGBBio company China 1 10000 dilution in 5 skim milk) for1 h at room temperature the bands were visualized usingan enhanced chemiluminescence kit (Thermo USA) Theintensity of immunoreactive bands was quantified with theuse of an imaging densitometer
28 Whole-Cell Patch Clamp Recording As previously de-scribed [16] K+ currents in single rat cardiomyocyte wererecorded with an EPC 10 patch clamp amplifier (HEKA Elek-tronik LambrechtPfalz Germany) with Pulse and Pulse-fit software 500 120583L of rat cardiomyocytes suspension was
placed in a perfusion chamber on a microscope stage andsuperfused with Tyrodersquos solution (mmolsdotLminus1 143 NaCl 54KCl 033 NaH
2PO4 18 CaCl
2 05 MgCl
2 5 HEPES and 11
glucose adjusted pH to 74 with NaOH) Micropipette patchpipette (resistance 25sim3MΩ) was filled with intercellularfluid (mmol Lminus1 135 KCl 5 NaCl 10 HEPES 5 EGTA10 120583M 441015840-diisothiocyano-221015840-stilbenedisulfonic acid and5 Mg-ATP adjusted pH to 72 with KOH) K+ current wasnormalized through dividing current by cell capacitance toget current density (pApF) Rat cardiomyocyte was held atminus80mV and voltage steps ranging from minus130 to + 70mVwereapplied for 500ms in 20mV increments After rupture of themembrane cardiomyocyte capacitance was estimated by acomposite of the capacitance current and compensation ofcell capacitor and pipette series resistanceThemeasurementscan be made only if seals are in the gigaohm range BaCl
2or
TFR was dissolved in the extracellular solution and directlygiven to cardiomyocyte using injection port setting Changesin K+ currents were observed in the same cell before and afteradministration of BaCl
2or TFR
29 Statistical Analysis Data are presented as means plusmn SDStatistical analyses were performed with one-way ANOVAfollowed by the Duncan test to determine the differencesbetween groups A value of 119875 lt 005 was regarded as statis-tically significant
3 Result
31 TFR Inhibits IR-Induced Myocardial Infarction Asshown in Figure 1 occlusion of LAD followed by reperfusioninduced obvious myocardial injury as indicated by measure-ment of the ISARR ratio The ISAAR ratio was 463 plusmn62 in IR group (lowastlowast119875 lt 001 versus sham group) 20 40and 80mgkg TFR significantly decreased the ISARR ratiorespectively (119875 lt 001 versus sham group) Similarly vera-pamil 16mgkg and Y27632 30mgkg also reduced the ISARR ratio (119875 lt 001 versus sham group)
32 Effect of TFR onViability of Rat Cardiomyocytes Viabilityof rat cardiomyocytes was validated by MTT assay Figure 2showed that AR induced a significant reduction of viabilityof neonatal rat cardiomyocytes compared to that in shamanoxia group (lowastlowast119875 lt 001 versus sham anoxia group) Inthe range of 37sim300mgL TFRmarkedly and concentration-dependently increased the viability of neonatal rat cardiomy-ocytes with an EC
50of 159mgL Similar to TFR 1mmolL
nifedipine also increased the viability of rat cardiomyocytes(119875 lt 001 versus AR group)
33 Effect of TFR on LDH Activity and cTnT Level Leakageof LDH or cTnT from cell into the culture medium is a majorindicator of myocardial AR injury Significant increasesof LDH activity and cTnT level in the culture medium ofneonatal rat cardiomyocytes were detected in AR group(lowastlowast119875 lt 001 versus sham anoxia group) Treatment withTFR (37 111 333 100 and 300mgL) or nifedipine 1mmolLmarkedly inhibited AR-induced increases of LDH activity
4 Evidence-Based Complementary and Alternative Medicine
60
40
20
0
Sham IR
Vera
pam
il
ISA
AR
()
Y27632
TFR20
mg
kg
TFR40
mg
kg
TFR80
mg
kg
lowastlowast
Figure 1 Effects of total flavones of Rhododendron simsii Planchflower (TFR)verapamilandY27632onischemiareperfusion-(IR-)induced myocardial infarction in rat Infarct size was expressed as apercentage of the area at risk (ISAAR) Results are means plusmn SD of6 experiments lowastlowast119875 lt 001 compared to sham 119875 lt 001 comparedto IR
and cTnT level in culture medium (119875 lt 005 or 119875 lt 001
versus AR group) (Figures 3(a) and 3(b))
34 Effect of TFR onProduction ofMDA There is a significantincrease of MDA content in AR group (lowastlowast119875 lt 001 versussham anoxia group) 37 111 333 100 and 300mgL TFRobviously reduced the MDA content compared with ARgroup Treatment of nifedipine 1mmolL had a similar effectin reducing MDA production (119875 lt 005 versus AR group)(Figure 4)
35 Effect of Y27632 on AR-Induced Injury of Rat Cardiomy-ocyte As shown in Figures 3(a) and 4 treatment with Y276321 120583molL dramatically inhibited AR-induced increases ofLDH activity in culture medium (119875 lt 001 versus ARgroup) And Y27632 also obviously decreased the MDAcontent compared with AR group (119875 lt 001 versus ARgroup)
36 Effect of TFR on ROCKs Protein Expression The expres-sions of both ROCK
1and ROCK
2proteins were found in
each group (Figure 5(a)) and levels of ROCK1and ROCK
2
proteins were quantified by using the densitometry (Figures5(b) and 5(c)) Exposure to AR significantly increased bothROCK
1and ROCK
2protein levels (lowastlowast119875 lt 001 versus sham
anoxia group) The increases of ROCK1and ROCK
2were
markedly inhibited by ROCK inhibitor Y27632 1120583molL orTFR 333 100 and 300mgL (119875 lt 005 or
119875 lt 001 versusAR group) 1mmolL nifedipine had a similar effect on theexpressions of ROCK
1and ROCK
2
lowastlowast
Sham AR
10
08
06
04
02
00
MTT
(OD
A490)
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Figure 2 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on the viability of rat car-diomyocytes subjected to anoxiareoxygenation (AR) (MTT assay)Results are means plusmn SD of 5 experiments lowast119875 lt 001 compared tosham anoxia 119875 lt 001 compared to AR
37 K+ Currents in Neonatal Rat Ventricular CardiomyocytesTo explore protectivemechanisms of TFR onmyocardial ARinjury the effect of TFR on K+ channel in rat cardiomyocyteswas investigated using the patch clamp method Undercurrent clamp mode outward K+ current was evoked in ratcardiomyocytes from a holding of 10 or 30mV to 70mV andinward K+ current was elicited from a holding of minus30 orminus50mV to minus130mV (Figure 6(a)) Both outward and inwardK+ currents were voltage-dependent Exposure of cardiomy-ocytes to BaCl
2(100 120583M) a relatively selective inward rectifier
K+ (Kir) channel inhibitor obviously suppressed the inwardK+ current (lowast119875 lt 005 versus control group) without effecton outward K+ current (Figures 6(a) and 6(b)) The resultsindicate inward K+ current evoked in rat cardiomyocytes wascarried by Kir channel
Figure 7 displays that 300mgL TFR markedly aug-mented the inward current and outward K+ currents (lowast119875 lt005 versus control group) suggesting that TFR could activateKir channel and other types of K+ channels to cause K+currents in rat cardiomyocytes
4 Discussion
In the present studywe have found that (1) TFRhas protectiveeffects against myocardial IR and AR injury in rat (2)ROCKs mediate protective effect of TFR on rat cardiomy-ocytes AR injury (3) TFR could promote opening of Kirchannel and other types of K+ channels and increases K+currents in rat cardiomyocytes
The increase in infarct size is documented to be a reliableindex of myocardial IR injury In this study 30min ofischemia followed by 90min of reperfusion was noted toinducemyocardial injury as assessed in terms of the increasedISARR ratio in rat heart Like calcium channel blocker
Evidence-Based Complementary and Alternative Medicine 5
LDH
(UL
)1500
1000
500
0
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowastlowast
(a)
cTnT
(ng
mL)
1000
200
600
800
400
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
lowastlowast
(b)
Figure 3 Effects of total flavones of Rhododendron simsii Planch flower (TFR) nifedipine (Nif) and Y27632 on lactate dehydrogenase (LDH)activity and cardiac troponin T (cTnT) level in the culture medium of neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR)(a) LDH activity (b) cTnT level Results are means plusmn SD of 5 experiments lowastlowast119875 lt 001 compared to sham anoxia 119875 lt 005 119875 lt 001compared to AR
MD
A (n
mol
L)
5
1
0
3
4
2
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowast
Figure 4 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on lactate dehydrogenase mal-ondialdehyde (MDA) in the culture medium of neonatal rat car-diomyocytes subjected to anoxiareoxygenation (AR) Results aremeans plusmn SD of 5 experiments lowast119875 lt 005 compared to sham anoxia119875 lt 005 compared to AR
verapamil TFR markedly reduced the ISARR ratio theresult indicates that TFR has a protective effect against myo-cardial IR injury in rat
LDH serves as an important metabolic enzyme in car-diomyocytes and could be leaked from injured cardiomy-ocytes Hence LDH level in the culture medium is a primary
index to evaluate cell damage [17] Troponin I is an inhibitorysubunit of troponin that binds to actin in thin myofilamentsto hold the actin-tropomyosin complex in place There arethree isoforms of troponin I cardiac troponin I (cTnI) fasttwitch skeletal muscle troponin I (fTnI) and slow twitchskeletal muscle troponin I (sTnI) cTnI is not present inserum from healthy people but it can be detected in serumfrom patients with acute myocardium damage Thus cTnI inthe culture medium is a sensitive and specific biochemicalmarker for detecting cardiomyocytes injury [9 18ndash20] In thepresent study AR-induced rat cardiomyocytes injury wasdetected as indicated by the decrease of cell viability andthe increases of LDH and cTnT in culture medium whiletreatment of TFR in the range of 37 to 300mgL significantlyimproved the aforementioned indexes including the increaseof cell viability and reductions of LDH and cTnT in culturemedium Calcium antagonist nifedipine had comparableeffects These results indicate that TFR has a significantprotective effect on AR-injured rat cardiomyocytes
AR injury can produce large amounts of oxygen-freeradicals in cardiomyocytes and subsequently causes lipid per-oxidation and leads to cell damage Thus lipid peroxidationis one of mechanisms of cellular damage MDA a productof lipid peroxidation has been applied to assess oxygen-free radicals-mediated myocardial IR injury [21] Our studyrevealed that 100 and 300mgL TFR significantly decreasedthe MDA level in culture medium the result not only furtherindicated that TFR has protection on myocardial AR injurybut also proposed that inhibition of lipid peroxidation maybe at least partially involved in cardioprotective mechanismof TFR against myocardial AR injury
6 Evidence-Based Complementary and Alternative Medicine
ROCK1
120573-actin
ROCK2
(a)
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
10
08
06
04
02
00
lowastlowast
ROCK
1120573
-act
in (
)
(b)
10
08
06
04
02
00
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
lowastlowast
ROCK
2120573
-act
in (
)
(c)
Figure 5 Effects of total flavones of Rhododendron simsii Planch flower (TFR) and Y23672 on expressions of ROCK1and ROCK
2proteins in
neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR) (a) Representative western blot analysis of expressions of ROCK1and
ROCK2 (b) Quantification of ROCK
1expression (c) Quantification of ROCK
2expression ROCK
1 ROCK
2 and 120573-actin were analyzed and
quantified by densitometric analysis Data are means plusmn SD of ROCK1120573-actin or ROCK
2120573-actin from 3 experiments lowastlowast119875 lt 001 compared
to sham anoxia 119875 lt 005 119875 lt 001 compared to AR
RhoA is one of the effecters of the small GTP-bindingprotein Rho ROCK is ubiquitously expressed it is the best-known downstream effector of RhoA Both ROCK
1and
ROCK2are expressed in vascular smooth muscle and in
myocardium [22ndash24] but their differential effects are still notwell characterized Increasing evidences have demonstratedthat the RhoA-ROCK pathway plays a pivotal role in car-diovascular pathogenesis such as IR injury vascular smoothmuscle cell (VSMC) proliferation cardiac hypertrophy heartfailure and ventricular remodeling [25 26] ROCK plays animportant role in myocardial AR damage and inhibitionof the RhoA-ROCK pathway has beneficial effects on bothheart and vasculature functions Treatment with the ROCKinhibitor Y-27632 or fasudil protected the heart against IRinjury and enhanced postischemia cardiac function [4 24]Our data indicate that ROCK inhibitor Y27632 markedlyinhibited AR injury-induced leakage of LDH and MDAproduction demonstrating that Y27632 had protective effectson myocardial AR injury In vivo rat model of myocardialIR Y27632 also significantly reducedmyocardial infarction
Oxidative species activate the ROCK pathway [27ndash29]RhoA expression is upregulated in ischemicmyocardium andlater activation of ROCKs occurs during reperfusion [4 30]
Our results show that exposure to AR injury significantlyincreased both ROCK
1and ROCK
2protein levels in rat
cardiomyocytes and the increases were markedly attenuatedby treatment of 333 100 and 300mgL TFR Together withthe facts that ROCK inhibitor Y27632 had similar suppressiononAR-induced increases of ROCK
1andROCK
2expressions
and protective effect on myocardial AR injury our resultssuggest that activation of ROCK during rat myocardialAR injury and inhibition of ROCK were involved in thecardioprotection of TFR against myocardial AR injury
Accumulating studies indicate ROCK is amajor regulatorof the contractile proteins including myosin light chainphosphatase (MLCP) myosin phosphatase target subunitCPI-17 and myosin light chain (MLC) ROCK is responsiblefor VSMCs contraction in various vascular beds [23] andsimilarmechanismwasmade for cardiac contractility ROCKinhibitor Y-27632 significantly suppressed the contractionin rabbit ventricular myocardium [31] This suggests thatthe ROCK activation may at least partly contribute tocardiac contractility and ROCK inhibition could decreasecardiac contractility It is a known fact that the decreaseof cardiac contractility could result in decreased cardiacwork and reduction in myocardial oxygen demand Thus
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
Evidence-Based Complementary and Alternative Medicine 3
for further experiments One part of cardiomyocytes werecultured in 35 mm culture dishes at 37∘C in a 5 CO
2ndash
95 O2incubator and the 3ndash5 d cultured cells were used for
patch clamp recording And other cardiomyocytes culturedin gelatin-coated plates incubated for 3ndash5 days were used forthe experiment of AR injury
25 AR Injury Model The prepared cardiomyocytes wererandomly divided into 9 groups Sham anoxia group ARgroup 1mM nifedipine group 1 120583MY-27632 group and TFR37 111 333 100 and 300mgL groups Except that shamanoxia group was incubated under normoxic conditionsother groupswere subjected to anoxia followed by reoxygena-tion Anoxia was induced by changing the air content with95 N
2and 5 CO
2gas mixture in a metabolic chamber
for 3 h Then the cultures were reoxygenated for 12 h byincubating the cells in 95 O
2and 5 CO
2 Nifedipine Y-
27632 and TFR were respectively added into the DMEM at24 h before anoxia
Another set of experiment was performed for Westernblot assay under the same conditions and grouping
26 Measurement of MTT LDH MDA and cTnT LevelsAfter AR model 20120583L MTT solution (5mgmL) was addedinto each well and incubated for an additional 4 h Then100 120583L DMSO was added into cardiomyocyte cultures todissolve the formazan particulates Finally absorbance at490 nmwasmeasured using amicroplate reader for theMTTassay
Supernatants of cardiomyocyte cultures were collectedMDA content and LDH activity were respectively detectedat 532 nm and 450 nmby spectrophotometry according to theprocedures provided by the assay kits
The level of cTnT in supernatants was measured by themethod of immunoassay [15]
27 Western Blot Analysis Cardiomyocytes were lysed bythe solution containing 04mmol Lminus1 phenylmethanesulfonylfluoride (pH 74) separated by sodium dodecyl sulfate poly-acrylamide gel electrophoresis on 10 polyacrylamide-Trisgels (Beyotime China) and then transferred to a polyvinyli-dene difluoride membrane Membranes were blocked bybuffer (5 skim milk and 005 Tween 20 in Tris-bufferedsaline) at room temperature for 2 h and then incubated(4∘C overnight) with the same buffer containing rabbitpolyclonal antibodies against ROCK1 or ROCK2 (EnogeneChina) or monoclonal antibody against 120573-actin (Bioworld)After incubation with anti-rabbit second antibody (ZSGBBio company China 1 10000 dilution in 5 skim milk) for1 h at room temperature the bands were visualized usingan enhanced chemiluminescence kit (Thermo USA) Theintensity of immunoreactive bands was quantified with theuse of an imaging densitometer
28 Whole-Cell Patch Clamp Recording As previously de-scribed [16] K+ currents in single rat cardiomyocyte wererecorded with an EPC 10 patch clamp amplifier (HEKA Elek-tronik LambrechtPfalz Germany) with Pulse and Pulse-fit software 500 120583L of rat cardiomyocytes suspension was
placed in a perfusion chamber on a microscope stage andsuperfused with Tyrodersquos solution (mmolsdotLminus1 143 NaCl 54KCl 033 NaH
2PO4 18 CaCl
2 05 MgCl
2 5 HEPES and 11
glucose adjusted pH to 74 with NaOH) Micropipette patchpipette (resistance 25sim3MΩ) was filled with intercellularfluid (mmol Lminus1 135 KCl 5 NaCl 10 HEPES 5 EGTA10 120583M 441015840-diisothiocyano-221015840-stilbenedisulfonic acid and5 Mg-ATP adjusted pH to 72 with KOH) K+ current wasnormalized through dividing current by cell capacitance toget current density (pApF) Rat cardiomyocyte was held atminus80mV and voltage steps ranging from minus130 to + 70mVwereapplied for 500ms in 20mV increments After rupture of themembrane cardiomyocyte capacitance was estimated by acomposite of the capacitance current and compensation ofcell capacitor and pipette series resistanceThemeasurementscan be made only if seals are in the gigaohm range BaCl
2or
TFR was dissolved in the extracellular solution and directlygiven to cardiomyocyte using injection port setting Changesin K+ currents were observed in the same cell before and afteradministration of BaCl
2or TFR
29 Statistical Analysis Data are presented as means plusmn SDStatistical analyses were performed with one-way ANOVAfollowed by the Duncan test to determine the differencesbetween groups A value of 119875 lt 005 was regarded as statis-tically significant
3 Result
31 TFR Inhibits IR-Induced Myocardial Infarction Asshown in Figure 1 occlusion of LAD followed by reperfusioninduced obvious myocardial injury as indicated by measure-ment of the ISARR ratio The ISAAR ratio was 463 plusmn62 in IR group (lowastlowast119875 lt 001 versus sham group) 20 40and 80mgkg TFR significantly decreased the ISARR ratiorespectively (119875 lt 001 versus sham group) Similarly vera-pamil 16mgkg and Y27632 30mgkg also reduced the ISARR ratio (119875 lt 001 versus sham group)
32 Effect of TFR onViability of Rat Cardiomyocytes Viabilityof rat cardiomyocytes was validated by MTT assay Figure 2showed that AR induced a significant reduction of viabilityof neonatal rat cardiomyocytes compared to that in shamanoxia group (lowastlowast119875 lt 001 versus sham anoxia group) Inthe range of 37sim300mgL TFRmarkedly and concentration-dependently increased the viability of neonatal rat cardiomy-ocytes with an EC
50of 159mgL Similar to TFR 1mmolL
nifedipine also increased the viability of rat cardiomyocytes(119875 lt 001 versus AR group)
33 Effect of TFR on LDH Activity and cTnT Level Leakageof LDH or cTnT from cell into the culture medium is a majorindicator of myocardial AR injury Significant increasesof LDH activity and cTnT level in the culture medium ofneonatal rat cardiomyocytes were detected in AR group(lowastlowast119875 lt 001 versus sham anoxia group) Treatment withTFR (37 111 333 100 and 300mgL) or nifedipine 1mmolLmarkedly inhibited AR-induced increases of LDH activity
4 Evidence-Based Complementary and Alternative Medicine
60
40
20
0
Sham IR
Vera
pam
il
ISA
AR
()
Y27632
TFR20
mg
kg
TFR40
mg
kg
TFR80
mg
kg
lowastlowast
Figure 1 Effects of total flavones of Rhododendron simsii Planchflower (TFR)verapamilandY27632onischemiareperfusion-(IR-)induced myocardial infarction in rat Infarct size was expressed as apercentage of the area at risk (ISAAR) Results are means plusmn SD of6 experiments lowastlowast119875 lt 001 compared to sham 119875 lt 001 comparedto IR
and cTnT level in culture medium (119875 lt 005 or 119875 lt 001
versus AR group) (Figures 3(a) and 3(b))
34 Effect of TFR onProduction ofMDA There is a significantincrease of MDA content in AR group (lowastlowast119875 lt 001 versussham anoxia group) 37 111 333 100 and 300mgL TFRobviously reduced the MDA content compared with ARgroup Treatment of nifedipine 1mmolL had a similar effectin reducing MDA production (119875 lt 005 versus AR group)(Figure 4)
35 Effect of Y27632 on AR-Induced Injury of Rat Cardiomy-ocyte As shown in Figures 3(a) and 4 treatment with Y276321 120583molL dramatically inhibited AR-induced increases ofLDH activity in culture medium (119875 lt 001 versus ARgroup) And Y27632 also obviously decreased the MDAcontent compared with AR group (119875 lt 001 versus ARgroup)
36 Effect of TFR on ROCKs Protein Expression The expres-sions of both ROCK
1and ROCK
2proteins were found in
each group (Figure 5(a)) and levels of ROCK1and ROCK
2
proteins were quantified by using the densitometry (Figures5(b) and 5(c)) Exposure to AR significantly increased bothROCK
1and ROCK
2protein levels (lowastlowast119875 lt 001 versus sham
anoxia group) The increases of ROCK1and ROCK
2were
markedly inhibited by ROCK inhibitor Y27632 1120583molL orTFR 333 100 and 300mgL (119875 lt 005 or
119875 lt 001 versusAR group) 1mmolL nifedipine had a similar effect on theexpressions of ROCK
1and ROCK
2
lowastlowast
Sham AR
10
08
06
04
02
00
MTT
(OD
A490)
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Figure 2 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on the viability of rat car-diomyocytes subjected to anoxiareoxygenation (AR) (MTT assay)Results are means plusmn SD of 5 experiments lowast119875 lt 001 compared tosham anoxia 119875 lt 001 compared to AR
37 K+ Currents in Neonatal Rat Ventricular CardiomyocytesTo explore protectivemechanisms of TFR onmyocardial ARinjury the effect of TFR on K+ channel in rat cardiomyocyteswas investigated using the patch clamp method Undercurrent clamp mode outward K+ current was evoked in ratcardiomyocytes from a holding of 10 or 30mV to 70mV andinward K+ current was elicited from a holding of minus30 orminus50mV to minus130mV (Figure 6(a)) Both outward and inwardK+ currents were voltage-dependent Exposure of cardiomy-ocytes to BaCl
2(100 120583M) a relatively selective inward rectifier
K+ (Kir) channel inhibitor obviously suppressed the inwardK+ current (lowast119875 lt 005 versus control group) without effecton outward K+ current (Figures 6(a) and 6(b)) The resultsindicate inward K+ current evoked in rat cardiomyocytes wascarried by Kir channel
Figure 7 displays that 300mgL TFR markedly aug-mented the inward current and outward K+ currents (lowast119875 lt005 versus control group) suggesting that TFR could activateKir channel and other types of K+ channels to cause K+currents in rat cardiomyocytes
4 Discussion
In the present studywe have found that (1) TFRhas protectiveeffects against myocardial IR and AR injury in rat (2)ROCKs mediate protective effect of TFR on rat cardiomy-ocytes AR injury (3) TFR could promote opening of Kirchannel and other types of K+ channels and increases K+currents in rat cardiomyocytes
The increase in infarct size is documented to be a reliableindex of myocardial IR injury In this study 30min ofischemia followed by 90min of reperfusion was noted toinducemyocardial injury as assessed in terms of the increasedISARR ratio in rat heart Like calcium channel blocker
Evidence-Based Complementary and Alternative Medicine 5
LDH
(UL
)1500
1000
500
0
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowastlowast
(a)
cTnT
(ng
mL)
1000
200
600
800
400
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
lowastlowast
(b)
Figure 3 Effects of total flavones of Rhododendron simsii Planch flower (TFR) nifedipine (Nif) and Y27632 on lactate dehydrogenase (LDH)activity and cardiac troponin T (cTnT) level in the culture medium of neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR)(a) LDH activity (b) cTnT level Results are means plusmn SD of 5 experiments lowastlowast119875 lt 001 compared to sham anoxia 119875 lt 005 119875 lt 001compared to AR
MD
A (n
mol
L)
5
1
0
3
4
2
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowast
Figure 4 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on lactate dehydrogenase mal-ondialdehyde (MDA) in the culture medium of neonatal rat car-diomyocytes subjected to anoxiareoxygenation (AR) Results aremeans plusmn SD of 5 experiments lowast119875 lt 005 compared to sham anoxia119875 lt 005 compared to AR
verapamil TFR markedly reduced the ISARR ratio theresult indicates that TFR has a protective effect against myo-cardial IR injury in rat
LDH serves as an important metabolic enzyme in car-diomyocytes and could be leaked from injured cardiomy-ocytes Hence LDH level in the culture medium is a primary
index to evaluate cell damage [17] Troponin I is an inhibitorysubunit of troponin that binds to actin in thin myofilamentsto hold the actin-tropomyosin complex in place There arethree isoforms of troponin I cardiac troponin I (cTnI) fasttwitch skeletal muscle troponin I (fTnI) and slow twitchskeletal muscle troponin I (sTnI) cTnI is not present inserum from healthy people but it can be detected in serumfrom patients with acute myocardium damage Thus cTnI inthe culture medium is a sensitive and specific biochemicalmarker for detecting cardiomyocytes injury [9 18ndash20] In thepresent study AR-induced rat cardiomyocytes injury wasdetected as indicated by the decrease of cell viability andthe increases of LDH and cTnT in culture medium whiletreatment of TFR in the range of 37 to 300mgL significantlyimproved the aforementioned indexes including the increaseof cell viability and reductions of LDH and cTnT in culturemedium Calcium antagonist nifedipine had comparableeffects These results indicate that TFR has a significantprotective effect on AR-injured rat cardiomyocytes
AR injury can produce large amounts of oxygen-freeradicals in cardiomyocytes and subsequently causes lipid per-oxidation and leads to cell damage Thus lipid peroxidationis one of mechanisms of cellular damage MDA a productof lipid peroxidation has been applied to assess oxygen-free radicals-mediated myocardial IR injury [21] Our studyrevealed that 100 and 300mgL TFR significantly decreasedthe MDA level in culture medium the result not only furtherindicated that TFR has protection on myocardial AR injurybut also proposed that inhibition of lipid peroxidation maybe at least partially involved in cardioprotective mechanismof TFR against myocardial AR injury
6 Evidence-Based Complementary and Alternative Medicine
ROCK1
120573-actin
ROCK2
(a)
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
10
08
06
04
02
00
lowastlowast
ROCK
1120573
-act
in (
)
(b)
10
08
06
04
02
00
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
lowastlowast
ROCK
2120573
-act
in (
)
(c)
Figure 5 Effects of total flavones of Rhododendron simsii Planch flower (TFR) and Y23672 on expressions of ROCK1and ROCK
2proteins in
neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR) (a) Representative western blot analysis of expressions of ROCK1and
ROCK2 (b) Quantification of ROCK
1expression (c) Quantification of ROCK
2expression ROCK
1 ROCK
2 and 120573-actin were analyzed and
quantified by densitometric analysis Data are means plusmn SD of ROCK1120573-actin or ROCK
2120573-actin from 3 experiments lowastlowast119875 lt 001 compared
to sham anoxia 119875 lt 005 119875 lt 001 compared to AR
RhoA is one of the effecters of the small GTP-bindingprotein Rho ROCK is ubiquitously expressed it is the best-known downstream effector of RhoA Both ROCK
1and
ROCK2are expressed in vascular smooth muscle and in
myocardium [22ndash24] but their differential effects are still notwell characterized Increasing evidences have demonstratedthat the RhoA-ROCK pathway plays a pivotal role in car-diovascular pathogenesis such as IR injury vascular smoothmuscle cell (VSMC) proliferation cardiac hypertrophy heartfailure and ventricular remodeling [25 26] ROCK plays animportant role in myocardial AR damage and inhibitionof the RhoA-ROCK pathway has beneficial effects on bothheart and vasculature functions Treatment with the ROCKinhibitor Y-27632 or fasudil protected the heart against IRinjury and enhanced postischemia cardiac function [4 24]Our data indicate that ROCK inhibitor Y27632 markedlyinhibited AR injury-induced leakage of LDH and MDAproduction demonstrating that Y27632 had protective effectson myocardial AR injury In vivo rat model of myocardialIR Y27632 also significantly reducedmyocardial infarction
Oxidative species activate the ROCK pathway [27ndash29]RhoA expression is upregulated in ischemicmyocardium andlater activation of ROCKs occurs during reperfusion [4 30]
Our results show that exposure to AR injury significantlyincreased both ROCK
1and ROCK
2protein levels in rat
cardiomyocytes and the increases were markedly attenuatedby treatment of 333 100 and 300mgL TFR Together withthe facts that ROCK inhibitor Y27632 had similar suppressiononAR-induced increases of ROCK
1andROCK
2expressions
and protective effect on myocardial AR injury our resultssuggest that activation of ROCK during rat myocardialAR injury and inhibition of ROCK were involved in thecardioprotection of TFR against myocardial AR injury
Accumulating studies indicate ROCK is amajor regulatorof the contractile proteins including myosin light chainphosphatase (MLCP) myosin phosphatase target subunitCPI-17 and myosin light chain (MLC) ROCK is responsiblefor VSMCs contraction in various vascular beds [23] andsimilarmechanismwasmade for cardiac contractility ROCKinhibitor Y-27632 significantly suppressed the contractionin rabbit ventricular myocardium [31] This suggests thatthe ROCK activation may at least partly contribute tocardiac contractility and ROCK inhibition could decreasecardiac contractility It is a known fact that the decreaseof cardiac contractility could result in decreased cardiacwork and reduction in myocardial oxygen demand Thus
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
4 Evidence-Based Complementary and Alternative Medicine
60
40
20
0
Sham IR
Vera
pam
il
ISA
AR
()
Y27632
TFR20
mg
kg
TFR40
mg
kg
TFR80
mg
kg
lowastlowast
Figure 1 Effects of total flavones of Rhododendron simsii Planchflower (TFR)verapamilandY27632onischemiareperfusion-(IR-)induced myocardial infarction in rat Infarct size was expressed as apercentage of the area at risk (ISAAR) Results are means plusmn SD of6 experiments lowastlowast119875 lt 001 compared to sham 119875 lt 001 comparedto IR
and cTnT level in culture medium (119875 lt 005 or 119875 lt 001
versus AR group) (Figures 3(a) and 3(b))
34 Effect of TFR onProduction ofMDA There is a significantincrease of MDA content in AR group (lowastlowast119875 lt 001 versussham anoxia group) 37 111 333 100 and 300mgL TFRobviously reduced the MDA content compared with ARgroup Treatment of nifedipine 1mmolL had a similar effectin reducing MDA production (119875 lt 005 versus AR group)(Figure 4)
35 Effect of Y27632 on AR-Induced Injury of Rat Cardiomy-ocyte As shown in Figures 3(a) and 4 treatment with Y276321 120583molL dramatically inhibited AR-induced increases ofLDH activity in culture medium (119875 lt 001 versus ARgroup) And Y27632 also obviously decreased the MDAcontent compared with AR group (119875 lt 001 versus ARgroup)
36 Effect of TFR on ROCKs Protein Expression The expres-sions of both ROCK
1and ROCK
2proteins were found in
each group (Figure 5(a)) and levels of ROCK1and ROCK
2
proteins were quantified by using the densitometry (Figures5(b) and 5(c)) Exposure to AR significantly increased bothROCK
1and ROCK
2protein levels (lowastlowast119875 lt 001 versus sham
anoxia group) The increases of ROCK1and ROCK
2were
markedly inhibited by ROCK inhibitor Y27632 1120583molL orTFR 333 100 and 300mgL (119875 lt 005 or
119875 lt 001 versusAR group) 1mmolL nifedipine had a similar effect on theexpressions of ROCK
1and ROCK
2
lowastlowast
Sham AR
10
08
06
04
02
00
MTT
(OD
A490)
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Figure 2 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on the viability of rat car-diomyocytes subjected to anoxiareoxygenation (AR) (MTT assay)Results are means plusmn SD of 5 experiments lowast119875 lt 001 compared tosham anoxia 119875 lt 001 compared to AR
37 K+ Currents in Neonatal Rat Ventricular CardiomyocytesTo explore protectivemechanisms of TFR onmyocardial ARinjury the effect of TFR on K+ channel in rat cardiomyocyteswas investigated using the patch clamp method Undercurrent clamp mode outward K+ current was evoked in ratcardiomyocytes from a holding of 10 or 30mV to 70mV andinward K+ current was elicited from a holding of minus30 orminus50mV to minus130mV (Figure 6(a)) Both outward and inwardK+ currents were voltage-dependent Exposure of cardiomy-ocytes to BaCl
2(100 120583M) a relatively selective inward rectifier
K+ (Kir) channel inhibitor obviously suppressed the inwardK+ current (lowast119875 lt 005 versus control group) without effecton outward K+ current (Figures 6(a) and 6(b)) The resultsindicate inward K+ current evoked in rat cardiomyocytes wascarried by Kir channel
Figure 7 displays that 300mgL TFR markedly aug-mented the inward current and outward K+ currents (lowast119875 lt005 versus control group) suggesting that TFR could activateKir channel and other types of K+ channels to cause K+currents in rat cardiomyocytes
4 Discussion
In the present studywe have found that (1) TFRhas protectiveeffects against myocardial IR and AR injury in rat (2)ROCKs mediate protective effect of TFR on rat cardiomy-ocytes AR injury (3) TFR could promote opening of Kirchannel and other types of K+ channels and increases K+currents in rat cardiomyocytes
The increase in infarct size is documented to be a reliableindex of myocardial IR injury In this study 30min ofischemia followed by 90min of reperfusion was noted toinducemyocardial injury as assessed in terms of the increasedISARR ratio in rat heart Like calcium channel blocker
Evidence-Based Complementary and Alternative Medicine 5
LDH
(UL
)1500
1000
500
0
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowastlowast
(a)
cTnT
(ng
mL)
1000
200
600
800
400
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
lowastlowast
(b)
Figure 3 Effects of total flavones of Rhododendron simsii Planch flower (TFR) nifedipine (Nif) and Y27632 on lactate dehydrogenase (LDH)activity and cardiac troponin T (cTnT) level in the culture medium of neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR)(a) LDH activity (b) cTnT level Results are means plusmn SD of 5 experiments lowastlowast119875 lt 001 compared to sham anoxia 119875 lt 005 119875 lt 001compared to AR
MD
A (n
mol
L)
5
1
0
3
4
2
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowast
Figure 4 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on lactate dehydrogenase mal-ondialdehyde (MDA) in the culture medium of neonatal rat car-diomyocytes subjected to anoxiareoxygenation (AR) Results aremeans plusmn SD of 5 experiments lowast119875 lt 005 compared to sham anoxia119875 lt 005 compared to AR
verapamil TFR markedly reduced the ISARR ratio theresult indicates that TFR has a protective effect against myo-cardial IR injury in rat
LDH serves as an important metabolic enzyme in car-diomyocytes and could be leaked from injured cardiomy-ocytes Hence LDH level in the culture medium is a primary
index to evaluate cell damage [17] Troponin I is an inhibitorysubunit of troponin that binds to actin in thin myofilamentsto hold the actin-tropomyosin complex in place There arethree isoforms of troponin I cardiac troponin I (cTnI) fasttwitch skeletal muscle troponin I (fTnI) and slow twitchskeletal muscle troponin I (sTnI) cTnI is not present inserum from healthy people but it can be detected in serumfrom patients with acute myocardium damage Thus cTnI inthe culture medium is a sensitive and specific biochemicalmarker for detecting cardiomyocytes injury [9 18ndash20] In thepresent study AR-induced rat cardiomyocytes injury wasdetected as indicated by the decrease of cell viability andthe increases of LDH and cTnT in culture medium whiletreatment of TFR in the range of 37 to 300mgL significantlyimproved the aforementioned indexes including the increaseof cell viability and reductions of LDH and cTnT in culturemedium Calcium antagonist nifedipine had comparableeffects These results indicate that TFR has a significantprotective effect on AR-injured rat cardiomyocytes
AR injury can produce large amounts of oxygen-freeradicals in cardiomyocytes and subsequently causes lipid per-oxidation and leads to cell damage Thus lipid peroxidationis one of mechanisms of cellular damage MDA a productof lipid peroxidation has been applied to assess oxygen-free radicals-mediated myocardial IR injury [21] Our studyrevealed that 100 and 300mgL TFR significantly decreasedthe MDA level in culture medium the result not only furtherindicated that TFR has protection on myocardial AR injurybut also proposed that inhibition of lipid peroxidation maybe at least partially involved in cardioprotective mechanismof TFR against myocardial AR injury
6 Evidence-Based Complementary and Alternative Medicine
ROCK1
120573-actin
ROCK2
(a)
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
10
08
06
04
02
00
lowastlowast
ROCK
1120573
-act
in (
)
(b)
10
08
06
04
02
00
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
lowastlowast
ROCK
2120573
-act
in (
)
(c)
Figure 5 Effects of total flavones of Rhododendron simsii Planch flower (TFR) and Y23672 on expressions of ROCK1and ROCK
2proteins in
neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR) (a) Representative western blot analysis of expressions of ROCK1and
ROCK2 (b) Quantification of ROCK
1expression (c) Quantification of ROCK
2expression ROCK
1 ROCK
2 and 120573-actin were analyzed and
quantified by densitometric analysis Data are means plusmn SD of ROCK1120573-actin or ROCK
2120573-actin from 3 experiments lowastlowast119875 lt 001 compared
to sham anoxia 119875 lt 005 119875 lt 001 compared to AR
RhoA is one of the effecters of the small GTP-bindingprotein Rho ROCK is ubiquitously expressed it is the best-known downstream effector of RhoA Both ROCK
1and
ROCK2are expressed in vascular smooth muscle and in
myocardium [22ndash24] but their differential effects are still notwell characterized Increasing evidences have demonstratedthat the RhoA-ROCK pathway plays a pivotal role in car-diovascular pathogenesis such as IR injury vascular smoothmuscle cell (VSMC) proliferation cardiac hypertrophy heartfailure and ventricular remodeling [25 26] ROCK plays animportant role in myocardial AR damage and inhibitionof the RhoA-ROCK pathway has beneficial effects on bothheart and vasculature functions Treatment with the ROCKinhibitor Y-27632 or fasudil protected the heart against IRinjury and enhanced postischemia cardiac function [4 24]Our data indicate that ROCK inhibitor Y27632 markedlyinhibited AR injury-induced leakage of LDH and MDAproduction demonstrating that Y27632 had protective effectson myocardial AR injury In vivo rat model of myocardialIR Y27632 also significantly reducedmyocardial infarction
Oxidative species activate the ROCK pathway [27ndash29]RhoA expression is upregulated in ischemicmyocardium andlater activation of ROCKs occurs during reperfusion [4 30]
Our results show that exposure to AR injury significantlyincreased both ROCK
1and ROCK
2protein levels in rat
cardiomyocytes and the increases were markedly attenuatedby treatment of 333 100 and 300mgL TFR Together withthe facts that ROCK inhibitor Y27632 had similar suppressiononAR-induced increases of ROCK
1andROCK
2expressions
and protective effect on myocardial AR injury our resultssuggest that activation of ROCK during rat myocardialAR injury and inhibition of ROCK were involved in thecardioprotection of TFR against myocardial AR injury
Accumulating studies indicate ROCK is amajor regulatorof the contractile proteins including myosin light chainphosphatase (MLCP) myosin phosphatase target subunitCPI-17 and myosin light chain (MLC) ROCK is responsiblefor VSMCs contraction in various vascular beds [23] andsimilarmechanismwasmade for cardiac contractility ROCKinhibitor Y-27632 significantly suppressed the contractionin rabbit ventricular myocardium [31] This suggests thatthe ROCK activation may at least partly contribute tocardiac contractility and ROCK inhibition could decreasecardiac contractility It is a known fact that the decreaseof cardiac contractility could result in decreased cardiacwork and reduction in myocardial oxygen demand Thus
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
Evidence-Based Complementary and Alternative Medicine 5
LDH
(UL
)1500
1000
500
0
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowastlowast
(a)
cTnT
(ng
mL)
1000
200
600
800
400
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
lowastlowast
(b)
Figure 3 Effects of total flavones of Rhododendron simsii Planch flower (TFR) nifedipine (Nif) and Y27632 on lactate dehydrogenase (LDH)activity and cardiac troponin T (cTnT) level in the culture medium of neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR)(a) LDH activity (b) cTnT level Results are means plusmn SD of 5 experiments lowastlowast119875 lt 001 compared to sham anoxia 119875 lt 005 119875 lt 001compared to AR
MD
A (n
mol
L)
5
1
0
3
4
2
Sham AR
Nif1
mM
TFR37
mg
L
TFR111
mg
L
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y276321120583
M
lowast
Figure 4 Effects of total flavones of Rhododendron simsii Planchflower (TFR) and nifedipine (Nif) on lactate dehydrogenase mal-ondialdehyde (MDA) in the culture medium of neonatal rat car-diomyocytes subjected to anoxiareoxygenation (AR) Results aremeans plusmn SD of 5 experiments lowast119875 lt 005 compared to sham anoxia119875 lt 005 compared to AR
verapamil TFR markedly reduced the ISARR ratio theresult indicates that TFR has a protective effect against myo-cardial IR injury in rat
LDH serves as an important metabolic enzyme in car-diomyocytes and could be leaked from injured cardiomy-ocytes Hence LDH level in the culture medium is a primary
index to evaluate cell damage [17] Troponin I is an inhibitorysubunit of troponin that binds to actin in thin myofilamentsto hold the actin-tropomyosin complex in place There arethree isoforms of troponin I cardiac troponin I (cTnI) fasttwitch skeletal muscle troponin I (fTnI) and slow twitchskeletal muscle troponin I (sTnI) cTnI is not present inserum from healthy people but it can be detected in serumfrom patients with acute myocardium damage Thus cTnI inthe culture medium is a sensitive and specific biochemicalmarker for detecting cardiomyocytes injury [9 18ndash20] In thepresent study AR-induced rat cardiomyocytes injury wasdetected as indicated by the decrease of cell viability andthe increases of LDH and cTnT in culture medium whiletreatment of TFR in the range of 37 to 300mgL significantlyimproved the aforementioned indexes including the increaseof cell viability and reductions of LDH and cTnT in culturemedium Calcium antagonist nifedipine had comparableeffects These results indicate that TFR has a significantprotective effect on AR-injured rat cardiomyocytes
AR injury can produce large amounts of oxygen-freeradicals in cardiomyocytes and subsequently causes lipid per-oxidation and leads to cell damage Thus lipid peroxidationis one of mechanisms of cellular damage MDA a productof lipid peroxidation has been applied to assess oxygen-free radicals-mediated myocardial IR injury [21] Our studyrevealed that 100 and 300mgL TFR significantly decreasedthe MDA level in culture medium the result not only furtherindicated that TFR has protection on myocardial AR injurybut also proposed that inhibition of lipid peroxidation maybe at least partially involved in cardioprotective mechanismof TFR against myocardial AR injury
6 Evidence-Based Complementary and Alternative Medicine
ROCK1
120573-actin
ROCK2
(a)
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
10
08
06
04
02
00
lowastlowast
ROCK
1120573
-act
in (
)
(b)
10
08
06
04
02
00
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
lowastlowast
ROCK
2120573
-act
in (
)
(c)
Figure 5 Effects of total flavones of Rhododendron simsii Planch flower (TFR) and Y23672 on expressions of ROCK1and ROCK
2proteins in
neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR) (a) Representative western blot analysis of expressions of ROCK1and
ROCK2 (b) Quantification of ROCK
1expression (c) Quantification of ROCK
2expression ROCK
1 ROCK
2 and 120573-actin were analyzed and
quantified by densitometric analysis Data are means plusmn SD of ROCK1120573-actin or ROCK
2120573-actin from 3 experiments lowastlowast119875 lt 001 compared
to sham anoxia 119875 lt 005 119875 lt 001 compared to AR
RhoA is one of the effecters of the small GTP-bindingprotein Rho ROCK is ubiquitously expressed it is the best-known downstream effector of RhoA Both ROCK
1and
ROCK2are expressed in vascular smooth muscle and in
myocardium [22ndash24] but their differential effects are still notwell characterized Increasing evidences have demonstratedthat the RhoA-ROCK pathway plays a pivotal role in car-diovascular pathogenesis such as IR injury vascular smoothmuscle cell (VSMC) proliferation cardiac hypertrophy heartfailure and ventricular remodeling [25 26] ROCK plays animportant role in myocardial AR damage and inhibitionof the RhoA-ROCK pathway has beneficial effects on bothheart and vasculature functions Treatment with the ROCKinhibitor Y-27632 or fasudil protected the heart against IRinjury and enhanced postischemia cardiac function [4 24]Our data indicate that ROCK inhibitor Y27632 markedlyinhibited AR injury-induced leakage of LDH and MDAproduction demonstrating that Y27632 had protective effectson myocardial AR injury In vivo rat model of myocardialIR Y27632 also significantly reducedmyocardial infarction
Oxidative species activate the ROCK pathway [27ndash29]RhoA expression is upregulated in ischemicmyocardium andlater activation of ROCKs occurs during reperfusion [4 30]
Our results show that exposure to AR injury significantlyincreased both ROCK
1and ROCK
2protein levels in rat
cardiomyocytes and the increases were markedly attenuatedby treatment of 333 100 and 300mgL TFR Together withthe facts that ROCK inhibitor Y27632 had similar suppressiononAR-induced increases of ROCK
1andROCK
2expressions
and protective effect on myocardial AR injury our resultssuggest that activation of ROCK during rat myocardialAR injury and inhibition of ROCK were involved in thecardioprotection of TFR against myocardial AR injury
Accumulating studies indicate ROCK is amajor regulatorof the contractile proteins including myosin light chainphosphatase (MLCP) myosin phosphatase target subunitCPI-17 and myosin light chain (MLC) ROCK is responsiblefor VSMCs contraction in various vascular beds [23] andsimilarmechanismwasmade for cardiac contractility ROCKinhibitor Y-27632 significantly suppressed the contractionin rabbit ventricular myocardium [31] This suggests thatthe ROCK activation may at least partly contribute tocardiac contractility and ROCK inhibition could decreasecardiac contractility It is a known fact that the decreaseof cardiac contractility could result in decreased cardiacwork and reduction in myocardial oxygen demand Thus
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
6 Evidence-Based Complementary and Alternative Medicine
ROCK1
120573-actin
ROCK2
(a)
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
10
08
06
04
02
00
lowastlowast
ROCK
1120573
-act
in (
)
(b)
10
08
06
04
02
00
Sham AR
Nif1
mM
TFR333
mg
L
TFR100
mg
L
TFR300
mg
L
Y27632
lowastlowast
ROCK
2120573
-act
in (
)
(c)
Figure 5 Effects of total flavones of Rhododendron simsii Planch flower (TFR) and Y23672 on expressions of ROCK1and ROCK
2proteins in
neonatal rat cardiomyocytes subjected to anoxiareoxygenation (AR) (a) Representative western blot analysis of expressions of ROCK1and
ROCK2 (b) Quantification of ROCK
1expression (c) Quantification of ROCK
2expression ROCK
1 ROCK
2 and 120573-actin were analyzed and
quantified by densitometric analysis Data are means plusmn SD of ROCK1120573-actin or ROCK
2120573-actin from 3 experiments lowastlowast119875 lt 001 compared
to sham anoxia 119875 lt 005 119875 lt 001 compared to AR
RhoA is one of the effecters of the small GTP-bindingprotein Rho ROCK is ubiquitously expressed it is the best-known downstream effector of RhoA Both ROCK
1and
ROCK2are expressed in vascular smooth muscle and in
myocardium [22ndash24] but their differential effects are still notwell characterized Increasing evidences have demonstratedthat the RhoA-ROCK pathway plays a pivotal role in car-diovascular pathogenesis such as IR injury vascular smoothmuscle cell (VSMC) proliferation cardiac hypertrophy heartfailure and ventricular remodeling [25 26] ROCK plays animportant role in myocardial AR damage and inhibitionof the RhoA-ROCK pathway has beneficial effects on bothheart and vasculature functions Treatment with the ROCKinhibitor Y-27632 or fasudil protected the heart against IRinjury and enhanced postischemia cardiac function [4 24]Our data indicate that ROCK inhibitor Y27632 markedlyinhibited AR injury-induced leakage of LDH and MDAproduction demonstrating that Y27632 had protective effectson myocardial AR injury In vivo rat model of myocardialIR Y27632 also significantly reducedmyocardial infarction
Oxidative species activate the ROCK pathway [27ndash29]RhoA expression is upregulated in ischemicmyocardium andlater activation of ROCKs occurs during reperfusion [4 30]
Our results show that exposure to AR injury significantlyincreased both ROCK
1and ROCK
2protein levels in rat
cardiomyocytes and the increases were markedly attenuatedby treatment of 333 100 and 300mgL TFR Together withthe facts that ROCK inhibitor Y27632 had similar suppressiononAR-induced increases of ROCK
1andROCK
2expressions
and protective effect on myocardial AR injury our resultssuggest that activation of ROCK during rat myocardialAR injury and inhibition of ROCK were involved in thecardioprotection of TFR against myocardial AR injury
Accumulating studies indicate ROCK is amajor regulatorof the contractile proteins including myosin light chainphosphatase (MLCP) myosin phosphatase target subunitCPI-17 and myosin light chain (MLC) ROCK is responsiblefor VSMCs contraction in various vascular beds [23] andsimilarmechanismwasmade for cardiac contractility ROCKinhibitor Y-27632 significantly suppressed the contractionin rabbit ventricular myocardium [31] This suggests thatthe ROCK activation may at least partly contribute tocardiac contractility and ROCK inhibition could decreasecardiac contractility It is a known fact that the decreaseof cardiac contractility could result in decreased cardiacwork and reduction in myocardial oxygen demand Thus
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
Evidence-Based Complementary and Alternative Medicine 7
Control BaCl2
70mV
minus80mV
minus130mV
100ms100pA
(a)
30
20
10
0
minus10
minus20
minus30
ControlBaCl2
Curr
ent d
ensit
y (p
Ap
F)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
Voltage (mV)lowast
lowast
lowast
(b)
Figure 6 K+ currents in neonatal rat ventricular cardiomyocytes and effect of BaCl2on the currents (a) Traces of outward and inward K+
currents without or with 100 120583molL BaCl2 (b) Curves of current-voltage relationships of outward and inward K+ currents Results are means
plusmn SD of 5 experiments lowast119875 lt 005 compared to control
ROCK inhibitor-induced decrease of cardiac contractilitymay be also associatedwith its cardioprotection on IR injuryHyperin one of the main effective components of TFR wasfound to inhibit contraction of papillary muscles isolatedfrom rabbit heart [32] the study suggests that TFR mightlead to a decrease of cardiac contractility through ROCKinhibition which might aid in its cardioprotection
K+ channel plays an important roles in diverse phys-iological processes Multiple K+ channels such as voltage-dependent K+ (Kv) channel Ca2+-activated K+ (KCa) chan-nel KATP channel and Kir channel have been identified incardiomyocytes RhoA-ROCK pathway also participates inmodulation of K+ channel function Activation of ROCKelicited Kv channel endocytosis and consequently attenuatedKv current in human embryonic kidney 293 cells andthis channel endocytosis was inhibited by ROCK inhibitorY27632 [33] It was noted that selective inhibition of ROCKcould protect KCa channel function in rat cerebral arteries[28] Thus TFR might be at least having an indirect effecton the K+ channel via ROCK inhibition In the presentstudy both inward K+ current and outward K+ currentwere elicited in neonatal rat cardiomyocytes It is well-known that the inward K+ current is mediated by Kir
channel and our study shows that this inward K+ currentwas obviously suppressed by 100 120583molL BaCl
2 a selective
Kir channel inhibitor The result indicates that this inwardK+ current in neonatal rat cardiomyocytes is Kir currentwhich is mediated by Kir channel Kir current plays a keyrole in setting up the resting membrane potentials andthe repolarization in cardiomyocytes [34] Inhibition of Kircould abolish ischemic preconditioning-induced protectionin rabbit ventricular cardiomyocytes [9] Our data revealthat TFR significantly increased inward K+ current in ratcardiomyocytes suggesting that TFR could promote theopening of Kir channel Outward K+ current results in cellmembrane hyperpolarization which could cause the closureof voltage-dependentCa2+ channel and subsequent reductionin Ca2+ influx Our data indicate that TFR also markedlyincreased outward K+ current in rat cardiomyocytes thisimplies that TFR could also open other types of K+ channelssuch as KCa channel or KATP channelTherefore the openingof K+ channels may partly contribute to cardioprotectionof TFR on myocardial AR injury in rat However outwardK+ current is conducted by several types of K+ channelsincluding KCa channel Kv channel KATP channel and otherK+ channels It is necessary in future study to determine
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
8 Evidence-Based Complementary and Alternative Medicine
70mV
minus80mV
minus130mV
100ms
100pAControl
TFR 333mgL
TFR 100mgL
TFR 300mgL
(a)
minus130
minus110
minus90
minus70
minus30
minus50
minus10
10
30
50
70
60
40
20
0
minus20
minus40
minus60
Curr
ent d
ensit
y (p
Ap
F)
ControlTFR 333mgL
TFR 100mgLTFR 300mgL
Voltage (mV)
lowast
lowast
(b)
Figure 7 Effect of total flavones of Rhododendron simsii Planch flower (TFR) on K+ currents in neonatal rat ventricular cardiomyocytes (a)Traces of outward and inward K+ currents without or with 333 100 and 300mgL TFR (b) Curve of current-voltage relationships of outwardand inward K+ currents Results are means plusmn SD of 5 experiments lowast119875 lt 005 compared to control
which types of K+ channels are responsible for TFR-increasedoutward K+ current and whether TFR activates K+ channelsdirectly or indirectly via ROCK inhibition
In summary the present study shows that TFR has asignificant protective effect on rat cardiomyocytes AR injurythrough the improvement of cell viability decreases of LDHand cTnT releases and inhibition of MDA production Ourstudy is the first attempt to investigate the mechanisms ofTFR against myocardial AR injury It was found that ROCKsinhibition and activation of K+ channels might mediate thecardioprotective effect of TFR
Abbreviations
TFR Total flavones from Rhododendron simsiiPlanch flower
AR Anoxia and reoxygenation
IR Ischemia and reperfusionROCK Rho-associated coiled-coil forming protein kinaseKir Inward rectifier potassium channel
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by the National Natural Sci-ence Foundation of China under Grant no 81374002 no30840104 and no 81173596 the Natural Science Foundationof Anhui province under Grant no 11040606M196 andthe Anhui provincial Science Foundation for Young Talents2012QRL267
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
Evidence-Based Complementary and Alternative Medicine 9
References
[1] M Kambe R Bessho M Fujii M Ochi and K ShimizuldquoSivelestat reduces myocardial ischemia and reperfusion injuryin rat hearts even when administered after onset of myocardialischemiardquo Interactive Cardiovascular and Thoracic Surgery vol8 no 6 pp 629ndash634 2009
[2] MDong B P Yan J K Liao Y-Y LamGW K Yip and C-MYu ldquoRho-kinase inhibition a novel therapeutic target for thetreatment of cardiovascular diseasesrdquo Drug Discovery Todayvol 15 no 15-16 pp 622ndash629 2010
[3] T Hattori H Shimokawa M Higashi et al ldquoLong-terminhibition of rho-kinase suppresses left ventricular remodelingafter myocardial infarction inmicerdquoCirculation vol 109 no 18pp 2234ndash2239 2004
[4] S A Hamid H S Bower and G F Baxter ldquoRho kinase acti-vation plays a major role as a mediator of irreversible injury inreperfused myocardiumrdquoTheAmerican Journal of PhysiologymdashHeart and Circulatory Physiology vol 292 no 6 pp H2598ndashH2606 2007
[5] W Bao E Hu L Tao et al ldquoInhibition of Rho-kinase protectsthe heart against ischemiareperfusion injuryrdquo CardiovascularResearch vol 61 no 3 pp 548ndash558 2004
[6] T Sato B OrsquoRourke and E Marban ldquoModulation of mito-chondrial ATP-dependent K+ channels by protein kinase CrdquoCirculation Research vol 83 no 1 pp 110ndash114 1998
[7] R-H Du T Dai W-J Cao M Lu J-H Ding and G HuldquoKir62-containing ATP-sensitive K+ channel is required forcardioprotection of resveratrol in micerdquo Cardiovascular Dia-betology vol 13 no 1 article 35 2014
[8] J C Cleveland Jr D R Meldrum R T Rowland A Banerjeeand A H Harken ldquoAdenosine preconditioning of humanmyocardium is dependent upon the ATP-sensitive K+ channelrdquoJournal of Molecular and Cellular Cardiology vol 29 no 1 pp175ndash182 1997
[9] R J Diaz C Zobel H C Cho et al ldquoSelective inhibition ofinward rectifier k+ channels (kir21 or kir22) abolishes pro-tection by ischemic preconditioning in rabbit ventricular car-diomyocytesrdquo Circulation Research vol 95 no 3 pp 325ndash3322004
[10] J-H Zhang Z-W Chen and Z Wu ldquoLate protective effectof pharmacological preconditioning with total flavones ofrhododendra against myocardial ischemia-reperfusion injuryrdquoCanadian Journal of Physiology and Pharmacology vol 86 no3 pp 131ndash138 2008
[11] L P Yuan Z W Chen F Li L Y Dong and F H ChenldquoProtective effect of total flavones of rhododendra on ischemicmyocardial injury in rabbitsrdquo The American Journal of ChineseMedicine vol 34 no 3 pp 483ndash492 2006
[12] J Han G-W He and Z-W Chen ldquoProtective effect and mech-anism of total flavones from rhododendron simsii planchon endothelium-dependent dilatation and hyperpolarizationin cerebral ischemia-reperfusion and correlation to hydrogensulphide release in ratsrdquo Evidence-Based Complementary andAlternativeMedicine vol 2014 Article ID 904019 11 pages 2014
[13] S-J Dai R-Y Chen and D-Q Yu ldquoStudies on the flavonoidcompounds of Rhododendron anthopogonoidesrdquo China Jour-nal of Chinese Materia Medica vol 29 no 1 pp 44ndash47 2004
[14] Y Huang P Yin D F Jiang et al ldquoQuality standard ofrhododendron flosrdquo World Science and Technology vol 16 no1 pp 151ndash155 2014
[15] E Missov C Calzolari and B Pau ldquoCirculating cardiac tro-ponin I in severe congestive heart failurerdquo Circulation vol 96no 9 pp 2953ndash2958 1997
[16] D-J Sung J-G Kim K J Won et al ldquoBlockade of K+ and Ca2+channels by azole antifungal agents in neonatal rat ventricularmyocytesrdquo Biological and Pharmaceutical Bulletin vol 35 no 9pp 1469ndash1475 2012
[17] Y Yan O Wuliji X Zhao et al ldquoEffect of essential oil ofSyringa pinnatifolia Hemsl var alashanensis on ischemia ofmyocardium hypoxia and platelet aggregationrdquo Journal ofEthnopharmacology vol 131 no 2 pp 248ndash255 2010
[18] T Omland J A De Lemos M S Sabatine et al ldquoA sensitivecardiac troponin T assay in stable coronary artery diseaserdquoTheNewEngland Journal ofMedicine vol 361 no 26 pp 2538ndash25472009
[19] K Thygesen J S Alpert A S Jaffe et al ldquoThird universaldefinition of myocardial infarctionrdquoCirculation vol 126 no 16pp 2020ndash2035 2012
[20] Y-E Zhang J-N Wang J-M Tang et al ldquoIn Vivo proteintransduction delivery of PEP-1-SOD1 fusion protein intomyocardium efficiently protects against ischemic insultrdquoMolecules and Cells vol 27 no 2 pp 159ndash166 2009
[21] M K Ozer H Parlakpinar N Vardi Y Cigremis M Ucar andA Acet ldquoMyocardial ischemiareperfusion-induced oxidativerenal damage in rats protection by caffeic acid phenethyl ester(Cape)rdquo Shock vol 24 no 1 pp 97ndash100 2005
[22] A Wibberley Z Chen E Hu J P Hieble and T D WestfallldquoExpression and functional role of Rho-kinase in rat urinarybladder smooth musclerdquo British Journal of Pharmacology vol138 no 5 pp 757ndash766 2003
[23] G Loirand P Guerin and P Pacaud ldquoRho kinases in cardiovas-cular physiology and pathophysiologyrdquo Circulation Researchvol 98 no 3 pp 322ndash334 2006
[24] S L M Peters and M C Michel ldquoThe RhoARho kinase path-way in the myocardiumrdquo Cardiovascular Research vol 75 no 1pp 3ndash4 2007
[25] V Q Chau F N Salloum N N Hoke A Abbate and R CKukreja ldquoMitigation of the progression of heart failure withsildenafil involves inhibition of RhoARho-kinase pathwayrdquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 300 no 6 pp H2272ndashH2279 2011
[26] A Yatani K Irie T Otani M Abdellatif and L Wei ldquoRhoAGTPase regulates L-type Ca2+ currents in cardiac myocytesrdquoThe American Journal of Physiology mdashHeart and CirculatoryPhysiology vol 288 no 2 pp H650ndashH659 2005
[27] A J McNeish F Jimenez-Altayo G S Cottrell and C JGarland ldquoStatins and selective inhibition of rho kinase protectsmall conductance calcium-activated potassium channel func-tion (k(ca)23) in cerebral arteriesrdquo PLoS ONE vol 7 no 10Article ID e46735 2012
[28] S Chandra M J Romero A Shatanawi A M Alkilany RB Caldwell and R W Caldwell ldquoOxidative species increasearginase activity in endothelial cells through the rhoarhokinase pathwayrdquo British Journal of Pharmacology vol 165 no2 pp 506ndash519 2012
[29] K Noma C Goto K Nishioka et al ldquoRoles of rho-associatedkinase and oxidative stress in the pathogenesis of aortic stiff-nessrdquo Journal of the American College of Cardiology vol 49 no6 pp 698ndash705 2007
[30] J Zhang X-X Li H-J Bian X-B Liu X-P Ji and Y ZhangldquoInhibition of the activity of Rho-kinase reduces cardiomyocyte
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
10 Evidence-Based Complementary and Alternative Medicine
apoptosis in heart ischemiareperfusion via suppressing JNK-mediated AIF translocationrdquo Clinica Chimica Acta vol 401 no1-2 pp 76ndash80 2009
[31] L Chu I Norota andM Endoh ldquoDifferential inhibition by theRho kinase inhibitor Y-27632 of the increases in contractilityand Ca2+ transients induced by endothelin-1 in rabbit ventric-ular myocytesrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacol-ogy vol 371 no 3 pp 185ndash194 2005
[32] ZWChenCGMaM Fang and S Y Xu ldquoTheblocking effectof hyperin on the inward flow of calcium ionrdquo Yao Xue Xue Baovol 29 no 1 pp 15ndash19 1994
[33] L Stirling M R Williams and A D Morielli ldquoDual rolesfor rhoarho-kinase in the regulated trafficking of a voltage-sensitive potassium channelrdquoMolecular Biology of the Cell vol20 no 12 pp 2991ndash3002 2009
[34] Y Liu X-H Xu Z Liu et al ldquoEffects of the natural flavonetrimethylapigenin on cardiac potassium currentsrdquo BiochemicalPharmacology vol 84 no 4 pp 498ndash506 2012
