2,3,5,4'-Tetrahydroxystilbene-2-O-?-D-Glucoside Protects Against Myocardial Ischemia/Reperfusion Injury And Its Underlying Mechanisms

Background:Cardiovascular diseases are the leading contributor to mortality rates worldwide.Myocardial ischemia is one of the main consequences of cardiovascular diseases.For lacking of effective therapies,MI/R injury remains a major medical problem today.Timely reperfusion is essential for recovery of the ischemia myocardium,however,reperfusion itself results in major cardiac damage.The underlying mechanism regulating myocardial injury induced by MI/R remains to be fully elucidated.2,3,5,4?-Tetrahydroxystilbene-2-O-?-D-Glucoside(TSG,molecular formula: C20H22O9,molecular weight: 406),a water-soluble active component extracted from Polygonum multiflorum Thunb,which exhibits a wide spectrum of biological functions,such as anti-inflammatory,anti-oxidant,anti-aging,lipid lowering,neuroprotection,and cardioprotection.Recently,TSG has attracted extensive attention as a therapeutic agent against a number of heart diseases,including atherosclerosis,myocardial fibrosis,cardiac hypertrophy and vascular senescence.Importantly,it has been proven that many organs suffering from ischemic injuries can be ameliorated by TSG.For the cardioprotective properties of TSG,it has elicited much interest as a potential agent to attenuate MI/R injury.Nevertheless,the specific mechanism of TSG's cardioprotection remains unknown.The Notch signaling pathway regulates multiple cellular processes,including cell fate determination,differentiation,development,proliferation,apoptosis and regeneration.In mammals,4 Notch receptors(Notch1-4)and five Notch ligands(Delta-like1/3/4,and Jagged1/2)have been identified.The activation of Notch receptor occurs via binding to Notch ligands.The target genes of Notch signaling include basic helix-loop-helix transcription factors Hairy and enhancer of split 1(Hes1)and Hairy-related transcription(HRT)factor family members,which are found in both developing and adult hearts.In the heart,Notch signaling not only regulates embryonic cardiac development and differentiation,but also stimulates proliferation of immature cardiomyocytes and promotes quiescent cardiomyocytes to reenter the cell cycle.All of these effects promote regeneration and repair of the injured myocardium.Expressions of Notch1,NICD and the downstream target protein Hes1,which decline in cardiomyocytes during cardiac postnatal development,are activated and restored in the adult injured myocardium.ER is a type of organelle in the cells of eukaryotic organisms whose membranes are continuous with the outer membrane of the nuclear envelope.ER participates in the biosynthesis and folding of proteins.Plenty of physiological and pathological stimulations,such as ischemia,hypoxia,and poisons,could induce the overexpression of ER molecular chaperones and an unfolded protein response(UPR)to increase normal protein folding and decrease abnormal protein folding through a process referred to as ER-associated degradation(ERAD).UPR signaling acts to promote survival and adaptation of ER stress.However,when activation of the UPR is excessive or prolonged,the final cellular outcome is pathologic apoptotic cell death.MI/R injury is known to result in the accumulation of unfolded proteins in the ER,which causes a severe UPR.Persistent UPR eventually leads to cellular apoptosis.Therefore,reducing excessive UPR,also referred to as ER stress,is of great importance in ameliorating MI/R injury.Whether TSG protects cardiomyocytes against MI/R-induced ER stress and cell apoptosis is not yet known.Objective:This study was designed to investigate whether TSG could protect against MI/R injury through modulating ER stress level and determine the role of Notch1/Hes1 signaling in TSG's protective effect against MI/R injury.Methods:(1)Myocardial ischemia/reperfusion injury: C57Bl/6 mice were anesthetized by 1% pentobarbital sodium.Myocardial ischemia operation was carried out by exteriorizing the heart through a left thoracic incision,placing a 6–0 silk suture around the left anterior descending coronary artery and tying a slipknot.After 30 min of ischemia,the slipknot was released and the myocardium was reperfused for 4 h(for analysis of protein expression),6 h(for quantification of myocardial apoptosis),and 72 h(for cardiac function determination).Mice of sham group underwent the same operation procedures except that the suture passed under the left coronary artery was left untied.(2)Echocardiography measurement: After the myocardial ischemia/reperfusion injury,two-dimensional and M-mode echocardiographic measurements were carried out with a VEVO 770 high-resolution in vivo imaging system.Left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were calculated with the software.(3)Serum CK and LDH assay: Blood samples were collected after 6 h of reperfusion.Serum CK and LDH levels were measured strictly according to the manufacturer's instruction.(4)Determination of myocardial apoptosis:Myocardial apoptosis was analyzed by TUNEL assay using an in situ cell death detection kit.The index of apoptosis was expressed by number of apoptotic myocytes / the total number of myocytes counted ×100%.(5)Cell viability assay: After H9c2 cardiomyoblasts were seeded in 96-well culture plates and received different treatments,10 ?L CCK-8 solution was added to each well at a 1/10 dilution.After 2 h of incubation,the absorbance was measured using a microtiter plate reader at a wavelength of 450 nm.The cell viability was calculated by dividing the optical density of samples with the optical density of control group.In addition,the cell morphology was observed under inverted/phase contrast microscopy,and images were obtained.(6)Simulated ischemia/reperfusion treatment: The SIR treatment was performed using physiological concentrations of potassium,hydrogen,and lactate.The H9c2 cardiomyoblasts were exposed to an ischemic buffer containing(in mmol/L)137 NaCl,12 KCl,0.49 MgCl2,0.9 CaCl2,and 4 HEPES.This buffer was also supplemented with(in mmol/L)10 deoxyglucose,0.75 sodium dithionate,and 20 lactate.The buffer pH was 6.5,and the cells were incubated for 50 min in a humidified cell culture incubator(21% O2,5% CO2,37°C).Reperfusion was initiated by returning the cells to normal culture medium for 4 h in a humidified cell culture incubator(21% O2,5% CO2,37°C).(7)Determination of cellular apoptosis: After H9c2 cardiomyoblasts were fixed in paraformaldehyde(4%)for 24 h,the cellular apoptosis was also analyzed by performing a TUNEL assay using the in situ cell death detection kit according to the manufacturer's instructions.The apoptotic index was expressed as the number of positively stained apoptotic cells / the total number of cells counted × 100%.(8)Western blot analysis: Myocardial tissue and cultured cells were harvested and lysed after the indicated treatments.The protein samples was separated by SDS-PAGE and then transferred to a poly vinylidene difluoride membrane.The membrane was blocked with 5% free-fat milk,then incubated with primary antibodies and corresponding secondary antibodies.The positive protein bands were developed using a chemiluminescent system,and the bands were scanned and quantified by densitometric analysis.(9)Statistical analysis: Data are expressed as mean ± SEM.Differences were compared by ANOVA followed by Bonferroni correction for post hoc t test,where appropriate.P< 0.05 was considered to be statistically significant.All of the statistical tests were performed with the GraphPad Prism software version 5.0(GraphPad Software).Result:(1)In vivo study,TSG significantly improved cardiac function through increasing left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)in MI/R injury mice.TSG could protect against MI/R injury by reducing myocardial apoptosis,inhibiting apoptosis pathway marker protein,downregulating creatine kinase and lactate dehydrogenase levels.In this process,TSG activated the Notch1/Hes1 signaling pathway,inhibited PERK-eIF2?-ATF4-CHOP-mediated endoplasmic reticulum stress signaling pathway,and thus reducing MI/R injury in mice.(2)In vitro study,TSG treatment attenuated H9c2 cardiomyoblasts apoptosis by decreasing apoptotic index.Western blotting analysis indicated that TSG treatment activated Notch1/Hes1 signaling and reduced PERK-e IF2?-ATF4-CHOP-mediated endoplasmic reticulum stress.Further investigation showed that DAPT,a Notch1/Hes1 signaling inhibitor,could significantly attenuated TSG's protective effect against SIR injury.These date also indicated that Notch1/Hes1 signaling activation by TSG reduced the ER stress level.However,blocking this pathway by DAPT attenuated this effect.Although further study is needed to investigate the detailed mechanisms,we did demonstrate that TSG reduced the myocardial ER stress by activating Notch1/Hes1 signaling.Conclusion:(1)In this study,we utilized in vivo and in vitro models to investigate the protective effect of TSG against MI/R-induced cardiac damage.We found that TSG treatment conferred cardioprotective effects,as evidenced by improved post-MI/R cardiac functional recovery,attenuated ER stress and reduced myocardial apoptosis.(2)Importantly,Notch1/Hes1 signaling was proven to play a key role in this process.TSG could activated Notch1/Hes1 signaling and reduced PERK-e IF2?-ATF4-CHOP-mediated endoplasmic reticulum stress.This study could help to further understand the pharmacology of TSG and highlighted a novel strategy against ischemic heart disease.