Research On Resveratrol's Anti-hypoxia/reoxygenation Damage Of Cardiomyocytes Based On Autophagy Flow Regulation

ObjectiveMyocardial ischemia reperfusion injury(MIRI)includes a series of m olecular mechanism,like calcium overload,inflammatory response,mitocho ndrial damage,apoptosis and autophagy,among which autophagy is a hot research topic recently.Autophagy is a dynamic process,which is called a utophagic flux.Recently,it is founded that resveratrol,a polyphenolic compound can alleviate MIRI.Our previous study have proved that resveratrol can inhibit the leakage of lactate dehydrogenase in hypoxia myocardial ce Ils,increase the expression of antioxidase GSH-Px,and alleviate hypoxia i njury.Further studies showed that the protection of resveratrol may attribu te to the regulation of autophagy.Based on these findings,this study is ai med to explore the efficacy of resveratrol and investigate the connection b etween efficacy and autophagy through detecting autophagic flux in myoca rdial cells subjected to hypoxia-reoxygenation(H/R)injury,and finally to provide pharmacological evidences for resveratrol against MIRI.Methods and ResultsFirstly,the neonatal rat myocardial cells were separated and cultured.After the cell density reach 80%,myocardial cells will undergo 12 h hypo xiare and 12 h oxygenation treatment to build the H/R injury model.1.The autophagic flux in myocardial cells after hypoxia-reoxygenationi njury in myocardial cellsAfter the cell density reach 80%,myocardial cells were divided into 4 groups:control group,control + chloroquine group(10 ?mol/L chloroqui ne,Control + CQ group),hypoxia/reoxygenation group(H/R group),and h ypoxia/reoxygenation + chloroquine group(10 ?mol/L chloroquine,H/R +CQ group).Chloroquine was exited in the whole H/R phase for 24h.The protein expression level of Beclin 1,LC3 and p62 was measured by We stern Blot,and the location and distribution of LC3 was detected by imm unofluorescence technique.These methods can evaluate the state of autoph agic flux in myocardial cells.The results showed that:Compared with the control group,the expres sion of Beclin 1 was increased in the H/R group,indicating that autophag y may be activated after undergoing H/R injury,but the expression level of LC3-?(LC3-? represent LC3)and p62 are decreased,suggesting that a utophagy was inhibited after H/R injury,or that,autophagic flux was acce lerated.Compared with the Control + CQ group,the expression level of LC3-II and p62 rise up in H/R + CQ group,and the LC3 immunofluores cence expression in myocardial cells significantly enhanced,indicating that autophagy was induced in H/R injured myocardial cells.So the low expr ession level of LC3 and p62 in H/R group is due to the over-activated au tophagic flux.In summary,these results show that H/R injury can activate autophagy and lead to an over-activated autophagic flux.1.The pharmacodynamic study of Resveratrol against hypoxia-reoxygen ation injury of myocardial cellsMyocardial cells were divided into 10 groups:Control group,hypoxia/reoxygenation group(H/R group),and 1,2.5,5,10,20,40,80 and 160?moI/L resveratrol treated group(1,2.5,5,10,20,40,60 and 180 ?mol/L resveratrol,Res-1 group,Res-2.5 group,Res-5 group,Res-10 group,Res-20 group,Res-40 group,Res-80 group and Res-160 group).After the pret reatment of resveratrol,the H/R injury model was built.The cell viability and release rate of lactate dehydrogenase were detected by CCK-8 and L DH assay to determine the effective concentration of resveratrol.Then cho ose appropriate concentration of resveratrol to further conduct the pharmac odynamic research,adding morphological observation by light microscopeand apoptosis assay by flow cytometry.The myocardial cells were divided into 5 groups:Control group,hypoxia-reoxygenation group(H/R group),low concentration of resveratrol treat group(5 ?mol/L,Res-5 group),hig h concentration of resveratrol treat group(20 ?mol/L,Res-20 group)and r esveratrol + chloroquine treat group(20 ?mol/L resveratrol,10 ?mol/L chl oroquine,Res-20 + CQ group).The results showed that:Compared with the control group,the myoca rdial cell viability and the myocardial cells viability decreased obviously,a nd the LDH activity increased in H/R group(P<0.005,P<0.01 vs.Control group),indicating the treatment of hypoxia-reoxygenation cause significant damage to myocardial cells.5?40 ?mol/L resveratrol can increase myocar dial cell viability,especially in Res-20 group,the myocardial cell viability increase to 25%(P<0.05 vs.H/R group).20 pmol/L and 40 ?mol/L resv eratrol can decrease LDH activity(P<0.05 vs.H/R group).However,when the drug concentration increaseto 80 ?mol/L,resveratrol aggravated H/R i njury contrarily.We choose 5 ?mol/L and 20 ?mol/L as the final drug co ncentration.Compared with H/R group,Res-5 and Res-20 group can allevi ate hypoxia/reoxygenation injury,improve myocardial cell morphology and inhibit apoptosis.In Res-20 + CQ group,compared with Res-20 group,t he myocardial cell viability decrease,LDH activity and the rate of apoptos is rise.In summary,chloroquine can reverse the protective effects of resve ratrol,suggesting that mechanism of resveratrol may attribute to autophagy flux.2.The regulation of Resveratrol on autophagic flux by hypoxia-reoxyge nation injury of myocardial cellsFirstly,we explore the pretreatment of resveratrol in normal myocardi alcells,and find that resveratrol can activate autophagy before H/R injury.Based on the conclusion,the myocardial cells were divided into 5 groups:Control group,H/R group,Res-5 group,Res-20 group and Res-20 + C Q group.The protein expression level of Beclin 1,LC3 and p62 was m easured by Western Blot,and the location of LC3 was detected by immu nofluorescence technique.These methods can evaluate the state of autoph agic flux in myocardial cells.The result showed that:In resveratrol treat group,compared with H/R group,the expression level of Beclin 1 and the LC3 fluoresence signal were enhanced,which indicates that resveratrol may activate autophagy.Si milarly,the expression of LC3-?and p62 increased in resveratrol treat gr oup,there are two cause for the result.First,resveratrol promote autophag y.Second,resveratrol only suppress the degradation of autolysosome,but not activate autophagy.After incubated with chloroquine,the.expression le vel of LC3-? and p62 become higher in resveratrol treat group,indicating that resveratrol can activate autophagy.In summary,resveratrol can impro ve autophagic flux and alleviate the overactive autophagy in H/R injury m yocardial cells.Conclusions1.The autophagic flux is over-actived in H/R injury myocardial cells,whi ch aggravates the damage.2.Low dose of resveratrol protects myocardial cells from H/R injury,but resveratrol makes poisonous in haigh concentration,and the effect of resve ratrol is related to regulating autophagy flux.3.Resveratrol can improve autophagic flux and alleviate the overactived au tophagy in H/R injury myocaedial cells.