Roles Of MiR-21 In Machanisms Of Total Flavonids From Carya Cathayensis Leaves Suppressing The Myocardial Ischemia-reperfusion

Objective:Myocardia ischaemia/reperfusion injury remains a significant and unsolved clinical problem,which seriously affects human health.Cell apoptosis play very improtant role in ischemia/reperfusion(I/R)injury.Protective meansures on cell apotosis can avoid from I/R injury,meantime,the expression of miR-21 have changed in I/R injury.Our prevous findings had indicated that the total flavonids(TFs)from Carya cathayensis leaves induced a significant protection against ischaemia injury,TFs can inhibit the expression of Bcl-2 and improve the erpression of miR-21.As a result,We make a hypothesis that TFs supresses the injury of myocardial ischaemia/reperfusion by inhibiting cell apoptosis through the regulation of miR-21.Methods:1、In vivo experiment:All rats were randomly divided into 6 groups,including three drug treatment groups(50mg/kg、100mg/kg、200mg/kg),drug positive group,I/R model group and sham group,10 rats each.Cardiac ischemia reperfusion injury animal model achevied by tying left anterior descending coronary artery for 30min and reperfusion for 120min.Infarct size of the myocardium was measured by Evans Blue dye/TTC staning.The influence of TFs on the expression of Bcl-2、Bax and Caspase-3 protein in myocardia was evaluated by western-blot.miR-21 was detected by RT-PCR.Hemodynamic and biochemical indicators were detected at the same time.2、In vitro experiment:Using the method of chemical to constructe Hypoxia reoxygenation(H/R)model.Experiment were divided into five groups randomly,including three drug treatment groups,H/R model group and control group.Cell viability was measured by the modified MTT method using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay.Hoechst-PI staining and Flow cytometry were used to study the effects of apoptosis by TFs.The influence of TFs on the expression of Bcl-2,Bax and Caspase-3 protein in H9C2 cells were evaluated by western-blot.Bcl-2、Bax and Caspase-3 gene were detected by RT-PCR.Biochemical indicators were detected at the same time.3、H9C2 cell were transfected with miR-21 mimic and inhibitor by Lipofectamine? RNAiMAX Reagent,RT-PCR was used to assess the expression level of miR-21.H9C2 were randomly into control,(H/R),TFs+H/R,mimic+H/R,mimic+TFs+H/R,inhibitor+H/R,inhibitor+TFs+H/R.Apoptotic changes in H9C2 cells were observed by using Hoechst 33324/PI staining.PTEN、p-AKT、Bax、Bcl-2 and Caspase-3 protein were detected by western blot.Result:l、TFs can reduce the Infarct size of the myocardium and have protective effeects on myocardium.High dose group decrease the content of MDA significantly(P<0.01).TFs increase the activity of SOD and GSH-PX,decrease the content of LDH.miR-21 was up-regulated in the ischemic area after I/R,and miR-21 expression in the high dose group was significantly increased compared with the sham group(P<0.01).Compared with model group,the level of Bcl-2/Bax was increased when the rats treated with TFs(P<0.05),and the level of caspase-3 protein was decreased(P<0.05).2、TFs can down-regulate the apoptosis rate when H9C2 cell suffered from H/R,meanwhile enhance the capability of the cell clearing the oxygen free radial.Compared with H/R gounp,the cells tread with TFs can decrease the activity of LDH(P<0.05)and the content of MDA(P<0.05),increase the activity of SOD(P<0.05),moreove,the expression of Bcl-2/Bax was up-regulated and the level of caspase-3 was down-regulated.3、Compared with H/R group,the level of PTEN and Caspase-3 protein decreased when miR-21 over expressed(P<0.01),the level of Bcl-2/Bax and p-Akt protein increased(P<0.05).Using the TFs and miR-21 mimic together will further reduce the expression of PTEN and Caspase-3 protein(P<0.01),further increase the expression of Bcl-2/Bax and p-Akt protein(P<0.01),also down-regulate cell apoptosis.Compared with H/R group,the level of PTEN and Caspase-3 protein increased when inhibit the expression of miR-21(P<0.05),the level of Bcl-2/Bax and p-Akt protein decreased(P<0.01).Conclusion:1.TFs can increase the expression of miR-21 and decrease the Infarct size of the myocardium after rat sufferd from I/R.2、TFs can down-regulate cell apoptosis after H9C2 cells sufferd from H/R,the mechanism may be related to decrease of Bax,Caspase-3 gene expression and increase Bcl-2 gene expression.3、Cell apoptosis can be decreased by miR-21 over-expression,otherwise it can be increased when miR-21was inhibited.The mechanism may be related to regulate cell apoptosis through PTEN/Akt/Bcl/Caspase signal pathway.