Effects And Mechanisms Of Ginsenoside Rc On Ameliorating Myocardial Ischaemia/reperfusion Injury Based On Silent Information Regulator 1

In this study,we examined the protective effects of ginsenoside Rc in myocardial ischemia/reperfusion(MI/R)injury,along with its underlying mechanisms,using an in vitro H9c2 cardiomyocyte model of oxygen-glucose deprivation/reoxygenation(OGD/R)and an in vivo rat model of MI/R injury.Prior to this,the H9c2cardiomyocytes or rats were exposed to ginsenoside Rc with or without silencing information factor 1(SIRT1)small interfering RNA(si RNA)or the selective SIRT1inhibitor EX527.The cardinal research procedures are as below:1.Protective effects of ginsenoside Rc against OGD/R-induced injury H9c2cardiomyocytesTo observe the protective effects of different concentrations of ginsenoside Rc against OGD/R injury in H9c2 cardiomyocytes.The cells were randomly assigned into five groups:(1)Control;(2)OGD/R;(3)OGD/R+Rc 5?M;(4)OGD/R+Rc 10?M;(5)OGD/R+Rc 20?M.To establish an in vitro OGD/R model,H9c2 cardiomyocytes were pre-treated with ginsenoside Rc for 24 h and then subjected to OGD/R injury or left uninjured.Cell viability,the activity of lactate dehydrogenase(LDH)in medium,apoptotic rate,mitochondrial membrane potential(^??m)were measured.To explore the degree of mitochondrial oxidative damage,superoxide dismutase(SOD)activity and the levels of malondialdehyde(MDA)and hydrogen peroxide(H₂O₂)in the mitochondrial samples of H9c2 cardiomyocytes were determined using assay kits.Additionally,the expression levels of mitochondrial succinate dehydrogenase complex subunit B(SDHB)and cytochrome c oxidase(Cyto-c)were assessed using western blot analysis.In addition,the molecular simulation docking technology was used to simulate the crystal structure of SIRT1 protein and the molecular structure of ginsenoside Rc to evaluate the affinity of ginsenoside Rc to SIRT1 protein.To investigate whether ginsenoside Rc ameliorates OGD/R-induced injury by up-regulating SIRT1,we examined SIRT1 activity,SIRT1 protein expression using western blot analysis and IF staining,and SIRT1 m RNA level using quantitative real-time PCR(QPCR).H9c2 cardiomyocytes were pre-treated with ginsenoside Rc(5,10,or 20?M)for24 h and then subjected to OGD/R injury or left uninjured.Ginsenoside Rc pre-treatment(5,10,or 20?M)effectively enhanced cell viability,decreased LDH activity,reduced the percentage of apoptotic H9c2 cardiomyocytes,and increased^??m,suggesting that ginsenoside Rc protected the cardiomyocytes from OGD/R injury(vs.OGD/R group).H9c2 cardiomyocytes pre-treated with ginsenoside Rc exhibited up-regulated mitochondrial SDHB and Cyto-c,increased mitochondrial SOD activity,and decreased mitochondrial MDA and H₂O₂ levels,suggesting that ginsenoside Rc attenuated mitochondrial oxidative stress injury from OGD/R injury(vs.OGD/R group).Molecular docking simulation results showed that ginsenoside Rc has certain binding ability with SIRT1 protein molecule,and the binding energy is-5.2 kcal/mol.In addition,ginsenoside Rc pre-treatment significantly up-regulated SIRT1 activity,protein expression and m RNA level,demonstrating that ginsenoside Rc ameliorated OGD/R-induced apoptosis and mitochondrial damage by up-regulating SIRT1.2.Effects and mechanisms of ginsenoside Rc on ameliorating OGD/R-induced injury based on SIRT1To investigate the effect of SIRT1 knockdown,H9C2 cardiomyocytes were transfected with non-targeting NC or SIRT1 si RNA using Lipofectamine 3000.The cells were randomly assigned into four groups:(1)NC+OGD/R;(2)NC+Rc+OGD/R;(3)SIRT1 si RNA+Rc+OGD/R;(4)SIRT1 si RNA+OGD/R.Next,the cells were transfected with NC or SIRT1 si RNA and the cells in groups 2 and 3 were pre-treated with ginsenoside Rc(20?M)for 24 h prior to the establishment of the OGD/R model.Cell viability,the activity of LDH in medium,AR and^??m were measured.To explore the degree of mitochondrial oxidative damage,SOD activity and the levels of MDA and H₂O₂ in the mitochondrial samples of H9c2 cardiomyocytes were determined using assay kits.Additionally,the expression levels of mitochondrial SDHB and Cyto-c were assessed using western blot analysis.To investigate whether ginsenoside Rc ameliorates OGD/R-induced injury by up-regulating SIRT1,we examined SIRT1 activity,SIRT1protein expression using western blot analysis and IF staining,and SIRT1 m RNA level using QPCR.The expression of SIRT1/FOXO1 signaling pathway and apoptosis-related protein Bcl-2,Bax,and Cleaved Caspase-3 were determined by western blot analysis.The result showed that SIRT1 si RNA significantly reduced the protein expression and m RNA level of SIRT1(P<0.01,vs.NC),indicating that SIRT1 si RNA transfection was successful.Ginsenoside Rc enhanced H9c2 cardiomyocyte viability,decreased the activity of LDH and apoptotic index after OGD/R injury(vs.NC+OGD/R group).However,SIRT1 si RNA blocked most of the protective effects of ginsenoside Rc(vs.NC+Rc+OGD/R group).Furthermore,ginsenoside Rc pre-treatment increased the fluorescence intensity of Rhodamine 123,increased the activity of mitochondrial SOD,decreased the levels of mitochondrial MDA and H₂O₂,and up-regulated the expression of SDHB and Cyto-c(vs.NC+OGD/R group).These anti-oxidative effects of ginsenoside Rc were partly counteracted by SIRT1 si RNA.The activity,protein expression and m RNA level of SIRT1 were up-regulated and the expression of Ac-FOXO1/FOXO1 was down-regulated in OGD/R-injured H9c2 cardiomyocytes pre-treated with ginsenoside Rc(vs.NC+OGD/R group).These changes were partly abrogated by SIRT1 si RNA silencing(vs.NC+Rc+OGD/R group).Moreover,ginsenoside Rc up-regulated Bcl-2,and down-regulated Bax and Cleaved Caspase-3after OGD/R injury,while SIRT1 si RNA counteracted these effects of ginsenoside Rc.These results,together with the increased cell viability,the decreased LDH activity and apoptotic rate,and the inhibition of m PTP opening,suggest that ginsenoside Rc protects cardiomyocytes from OGD/R injury via SIRT1 activation in vitro.3.Protective effects of ginsenoside Rc on MI/R injury in rats3.1 Evaluation of the effects of ginsenoside Rc on normal ratsRats were randomly assigned to:(1)Control group;(2)Ginsenoside Rc 10mg/kg group;and(3)Ginsenoside Rc 20 mg/kg group.The rats were intragastrically(ig)administered ginsenoside Rc(10 or 20 mg/kg;dissolved in distilled water)or distilled water once daily for 7 days.One hour after administration of ginsenoside Rc or distilled water on day 7,echocardiography was used to monitor the effect of ginsenoside Rc on cardiac function.The myocardial infarct size(MIS),the contents of CK-MB and c Tn I,and the activity of LDH in serum were measured.The expression of SIRT1 was determined by western blot analysis.In comparison to the control group,pre-treatment with ginsenoside Rc had no impact on cardiac function(LVIDd,LVIDs,LVEF,and LVFS),myocardial markers(CK-MB,LDH and c Tn I),or MIS.However,ginsenoside Rc up-regulated SIRT1 in a dose-dependent fashion in normal rats.3.2 Evaluation of the effects of ginsenoside Rc on MI/R-injured ratsRats were randomly assigned to:(1)Sham group;(2)MI/R group;(3)MI/R+Rc10 mg/kg group;and(4)MI/R+Rc 20 mg/kg group.The rats were ig administered ginsenoside Rc(10 or 20 mg/kg)or distilled water once daily for 7 days.One hour after administration of ginsenoside Rc or distilled water on day 7,a surgical operation was performed to establish the MI/R injury model.The model was established by ligating the left anterior descending coronary artery to induce ischemia for 30 min followed by reperfusion for 6 h.The cardiac function was measured by color Doppler ultrasound;The MIS,the contents of CK-MB and c Tn I,and the activity of LDH in serum were measured;Myocardial histopathology and ultrastructure changes were observed;The apoptotic rate of myocardial tissue was detected by TUNEL staining.To explore the degree of mitochondrial oxidative damage,SOD activity,the contents of MDA and H₂O₂,and the levels of E_h in myocardial mitochondria were determined using assay kits.Additionally,the expression levels of mitochondrial SDHB and Cyto-c were assessed using western blot analysis.The expression of SIRT1 was detected by immunohistochemistry(IHC)analysis.The m RNA level of SIRT1 was measured by QPCR.The expression of SIRT1/FOXO1 signaling pathway and apoptosis-related protein Bcl-2,Bax,and Cleaved Caspase-3 were determined by western blot analysis.MI/R-injured rats pre-treated with ginsenoside Rc significantly improved cardiac function,which was verified by decreased LVIDd,LVIDs and increased LVEF,LVFS(vs.MI/R group).Pre-treated with ginsenoside Rc markedly reduced the contents of CK-MB and c Tn I,and the activity of LDH in serum,decreased MIS,and attenuated pathophysiological changes in the cardiac tissue.MI/R injury down-regulated SDHB and Cyto-c,induced ROS generation(H₂O₂)and lipid peroxidation(MDA),inhibited antioxidant enzyme activity(SOD),and decreased E_h(GSH/GSSG)in myocardial mitochondria.Ginsenoside Rc pre-treatment remarkably lessened MI/R injury-induced mitochondrial oxidative stress(vs.MI/R group).Consistent with the SIRT1 IHC staining result,western blot and QPCR analysis showed that SIRT1 was markedly down-regulated and Ac-FOXO1/FOXO1 was considerably up-regulated in the myocardium after MI/R injury(vs.Sham group).Ginsenoside Rc pre-treatment notably up-regulated SIRT1 and down-regulated Ac-FOXO1/FOXO1(vs.MI/R group).Moreover,ginsenoside Rc also significantly decreased the myocardial apoptotic rate,up-regulated Bcl-2 and down-regulated Bax and Cleaved Caspase-3(vs.MI/R group).These results suggest that ginsenoside Rc ameliorates MI/R injury by reducing mitochondrial oxidative stress and apoptosis,at least in part,by activating SIRT1.4.Effects and mechanisms of ginsenoside Rc on ameliorating MI/R injury based on SIRT1Based on the above findings,we hypothesized that the cardioprotective effect of ginsenoside Rc is related to the SIRT1 signaling pathway.Hence,we investigated the interaction between ginsenoside Rc and EX527(a selective small-molecule inhibitor of SIRT1).Rats were randomly assigned to:(1)MI/R group;(2)MI/R+Rc group;(3)MI/R+Rc+EX527(a selective small-molecule inhibitor of SIRT1)group;and(4)MI/R+EX527 group.The rats were ig administered ginsenoside Rc(20 mg/kg)or distilled water once daily for 7 days.EX527(5 mg/kg)was intraperitoneally(ip)administered every other day.One hour after administration of ginsenoside Rc or distilled water on day 7,a surgical operation was performed to establish the MI/R injury model.The cardiac function was measured by color Doppler ultrasound;The MIS,the contents of CK-MB and c Tn I,and the activity of LDH in serum were measured;Myocardial histopathology and ultrastructure changes were observed;The apoptotic rate of myocardial tissue was detected by TUNEL staining.To explore the degree of mitochondrial oxidative damage,SOD activity,the contents of MDA and H₂O₂,and the levels of E_h in myocardial mitochondria were determined using assay kits.Additionally,the expressions of mitochondrial SDHB and Cyto-c were assessed using western blot analysis.The expression of SIRT1 was detected by IHC analysis.The m RNA level of SIRT1 was measured by QPCR.The expression of SIRT1/FOXO1 signaling pathway and apoptosis-related protein Bcl-2,Bax,and Cleaved Caspase-3 were determined by western blot analysis.In comparison to the MI/R+Rc group,EX527 administration significantly reduced cardiac function recovery,demonstrated by increased LVIDd and LVIDs,and decreased LVEF and LVFS.Moreover,while ginsenoside Rc pre-treatment significantly decreased levels of myocardial markers and MIS,attenuated pathophysiological changes of the cardiac tissue(vs.MI/R group),EX527 pre-treatment partly abolished the cardioprotective effects of ginsenoside Rc on these indexes(vs.MI/R+Rc group).Furthermore,although ginsenoside Rc attenuated MI/R injury-induced mitochondrial oxidative stress,the effect was mostly abolished by EX527,which down-regulated SDHB and Cyto-c,increased MDA and H₂O₂ levels,inhibited SOD activity,and decreased E_h(GSH/GSSG)(vs.MI/R+Rc group).Ginsenoside Rc pre-treatment up-regulated SIRT1 and down-regulated Ac-FOXO1 in MI/R-injured rats.The opposite trends were observed with EX527 administration,with down-regulated SIRT1 and up-regulated Ac-FOXO1.Additionally,EX527 abolished the anti-apoptotic effect of ginsenoside Rc.EX527 also increased myocardial apoptotic rate,down-regulated Bcl-2 and up-regulated Bax and Cleaved Caspase-3(vs.MI/R+Rc group).Based on these results,we suggest that ginsenoside Rc ameliorates MI/R injury by inhibiting mitochondrial oxidative stress and apoptosis by up-regulating SIRT1 in vivo.In summary,ginsenoside Rc exhibited significant protective effects on OGD/R-induced injury in H9c2 cardiomyocytes and MI/R injury in rats.Our results demonstrate that ginsenoside Rc ameliorated MI/R injury by reducing mitochondrial oxidative stress and apoptosis,at least in part,by activating SIRT1.