Protective Effects And Mechanisms Of Ginsenoside Rb2 And Ginsenoside Rg2 On Myocardial Injury Based On SIRT1 Signaling Pathway

The aim of this study was to explore the effects of ginsenoside Rb2 and ginsenoside Rg2 against H2O2-induced stress in H9c2 cells in vitro and on animal model of MI/R injury in vivo and its mechanism.Protective Effects and Mechanisms of Ginsenoside Rb2 on Myocardial Injury Based on SIRT1 Signaling Pathway1.Protective effects of ginsenoside Rb2 against H2O2-induced injury and apoptosis in H9c2 cellsMethods:H9c2 cells were pretreatment with different concentrations of ginsenoside Rb2(1,3 or 10 ?g/mL)for 4 h followed by treatment of 150?M H2O2 for 6 h.Cell viability was measured by MTT assay;The morphological changes of H9c2 cells were detected by fluorescence microscope;The activity of LDH in medium,the activity of SOD and GSH-PX,the content of MDA in H9c2 cells were determined by spectrophotometry;ROS generation in H9c2 cells was measured by flow cytometry;Occurrence of apoptosis was detected by DAPI and Annexin V/PI double staining;Procaspase-3,Procaspase-9,Bcl-2,Bax and SIRT1 protein expression were determined by Western Blot analysis.Results:Pretreatment with ginsenoside Rb2(1,3,10 ?g/mL)for 4 h increased cell viability compared with the model group(P<0.01).H2O2 treatment induced distinctive morphological changes,such as cell shrinkage,irregular shape and a wider intercellular gap.However,the proportion of abnormal cells in 1,3,10 ?g/mL of ginsenoside Rb2-pretreated groups decreased significantly.Ginsenoside Rb2 pretreatment significantly reduced the levels of LDH activity in the culture medium,decreased MDA content and increased SOD and GSH-PX activities compared with the model group(P<0.05 or P<0.01).ROS generation was significantly reduced by ginsenoside Rb2 pretreatment in a dose-dependent manner compared with the model group(P<0.01).The results indicated that ginsenoside Rb2 can extenuate oxidative injury and increase the antioxidant capacity of H9c2 cells.In addition,pretreatment with ginsenoside Rb2 decreased apoptotic nuclei induced by H2O2.These data proposed that ginsenoside Rb2 has a protective role in H2O2-induced apoptotic cell death of H9c2 cells.Furthermore,we also detected the apoptotic rate by Annexin V/PI staining.Pretreatment with ginsenoside Rb2 significantly reduced the percentage of Annexin V/PI positive cells compared with the model group(P<0.01).Ginsenoside Rb2 pretreatment restored protein levels of Procaspase-3,Procaspase-9 and Bcl-2 reduced expression of Bax in H2O2-subjected H9c2 cells.Western Blot demonstrated that,with increasing concentrations of ginsenoside Rb2,protein expression of SIRT1 was increased in a dose-dependent manner compared with the model group(P<0.01).2.Protective effects of ginsenoside Rb2 on myocardial ischemia reperfusion injury in ratsMethods:Wistar rats were randomly assigned to experimental groups:Sham group,MI/R group,Ginsenoside Rb2(10,20 mg/kg)groups and Diltiazem group.Myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 6-0 silk suture and making a slipknot around the left anterior descending coronary artery.After 30 min of ischemia,the slipknot was released and the myocardium was reperfused for 6 h and 72 h.The cardiac function was measured by color Doppler ultrasound;The myocardial infarct size,myocardial histopathology and ultrastructure changes were observed;The activities of LDH,CK-MB and AST in serum,the activities of CAT,SOD and GSH-PX and the content of MDA in myocardial tissues were measured;IL-1?,IL-6 and TNF-? levels were detected by QPCR;The apoptosis rate of myocardial tissue was detected by TUNEL staining;SIRT1 expression was detected by immunohistochemical assay;Expression of Bcl-2,Bax,Caspase family protein,expression of SIRTl/p53 signaling pathway and PI3K/Akt signaling pathway relative protein were determined by Western blot analysis.Results:Pretreatment with ginsenoside Rb2 significantly ameliorated myocardial dysfunction and remodeling,which was verified by increased LVEF,LUFS and decreased LVIDd,LUIDs compared with model group(P<0.05,or P<0.01).Pretreatment with ginsenoside Rb2(10 and 20 mg/kg)resulted in dose-dependent reduction in the infarct size.Pretreatment with ginsenoside Rb2(10 and 20 mg/kg)inhibited the increase of CK-MB,AST and LDH compared with MI/R group(P<0.05 or P<0.01).In addition,it could decrease the level of MDA,but increased the activities of SOD,CAT and GSH-PX in tissues.QPCR detection demonstrated that,with increasing concentrations of ginsenoside Rb2 gene expression of IL-1?,IL-6 and TNF-a was decreased significantly.Myocardial histopathology and ultra-structural observation results shown that ginsenoside Rb2 could improve myocardial pathological changes.Pretreatment with ginsenoside Rb2(10 and 20 mg/kg)significantly decreased apoptotic cells compared with MI/R group(P<0.01).Western Blot demonstrated that pretreatment with ginsenoside Rb2 decreased the expression of Bax and increased the expression of procaspase-3,procaspase-9 and Bcl-2 compared with the MI/R group.These results indicated that pretreatment with ginsenoside Rb2 may activate SIRT1 expression,thus decreasing Ac-p53 expression and inhibit the apoptotic signaling pathway.In addition,pretreatment with ginsenoside Rb2 increased the expression of p-PI3K,p-Akt and p-eNOS compared with the MI/R group.These results indicated that pretreatment with ginsenoside Rb2 may activate PI3K/Akt/eNOS signaling pathway.3.Protective effects and mechanisms of ginsenoside Rb2 on myocardial ischemia reperfusion injury based on SIRT1 signaling pathwayMethods:Wistar rats were randomly assigned to experimental groups:MI/R group,MI/R+Rb2 group,MI/R+Rb2+EX527(a selective SIRT1 inhibitor)group and MI/R+EX527 group.After 30 min of ischemia,the slipknot was released and the myocardium was reperfused for 6 h and 72 h.The cardiac function was measured by color Doppler ultrasound;The myocardial infarct size,myocardial histopathology and ultrastructure changes were observed;The activities of LDH,CK-MB and AST in serum,the activities of CAT,SOD and GSH-PX and the content of MDA in myocardial tissues were measured;IL-1?,IL-6 and TNF-? levels in myocardial tissues were detected by QPCR;The apoptosis rate of myocardial tissue was detected by TUNEL staining;SIRT1 expression was detected by immunohistochemical assay;Expression of Bcl-2,Bax,Caspase family protein,expression of SIRT1/p53 signaling pathway and PI3K/Akt signaling pathway relative protein were determined by Western Blot analysis.Results:Pretreatment with ginsenoside Rb2 significantly ameliorated myocardial dysfunction and remodeling,reduced the infarct size,decreased the activity of myocardial enzymes and improved myocardial pathological changes.However,EX527 treatment abolished the protective effect of ginsenoside Rb2.Previously we have demonstrated that pretreatment with ginsenoside Rb2 reduced oxidative stress in MI/R hearts.We further studied the effect of EX527 on ginsenoside Rb2 treatment.EX527 treatment also abolished anti-oxidative stress effect of ginsenoside Rb2.Pretreatment with ginsenoside Rb2 markedly inhibited the increase in the mRNA levels of pro-inflammatory cytokines.However,EX527 treatment increased the mRNA levels of IL-1?,IL-6 and TNF-?,which was reduced by ginsenoside Rb2 treatment compared with MI/R+Rb2 group(P<0.05 or P<0.01).Western Blot demonstrated that EX527 treatment could increase Ac-p53 expression by decreasing SIRT1 expression.Consistent with the previous observations,we found that EX527 treatment enhanced apoptotic signaling by decreasing procaspase-3,procaspase-9 and Bcl-2 expressions while increasing Bax expression compared with MI/R+Rb2 group.These results suggest that ginsenoside Rb2 reduces apoptosis at least in part,by upregulating SIRT1 expression.In addition,pretreatment with ginsenoside Rb2 increased the expression of p-PI3K,p-Akt,and p-eNOS.However,EX527 treatment inhibited the effect of ginsenoside Rb2.Protective Effects and Mechanisms of Ginsenoside Rg2 on Myocardial Injury Based on SIRT1 Signaling Pathway1.Protective effects of ginsenoside Rg2 against H2O2-induced injury and apoptosis in H9c2 cellsMethods:H9c2 cells were pretreatment with different concentrations of ginsenoside Rg2(1,3 or 10 ?g/mL)for 4 h followed by treatment of 150?M H2O2 for 6 h.Cell viability was measured by MTT assay;The morphological changes of H9c2 cells were detected by fluorescence microscope;The activity of LDH in medium,the activity of SOD and GSH-PX,the content of MDA in H9c2 cells were determined by spectrophotometry;ROS generation in H9c2 cells was measured by flow cytometry;Occurrence of apoptosis was detected by DAPI and Annexin V/PI double staining;Procaspase-3,Procaspase-9,Bcl-2,Bax and SIRT1 protein expression were determined by Western Blot analysis.Results:Pretreatment with ginsenoside Rg2(1,3,10 ?g/mL)for 4 h increased cell viability compared with the model group(P<0.01).H2O2 treatment induced distinctive morphological changes,such as cell shrinkage,irregular shape and a wider intercellular gap.However,the proportion of abnormal cells in 1,3,10 ?g/mL of ginsenoside Rg2-pretreated groups decreased significantly.Ginsenoside Rg2 pretreatment significantly reduced the levels of LDH activity in the culture medium,decreased MDA content and increased SOD and GSH-PX activities compared with the model group(P<0.05 or P<0.01).ROS generation was significantly reduced by ginsenoside Rg2 pretreatment in a dose-dependent manner compared with the model group(P<0.01).The results indicated that ginsenoside Rg2 can extenuate oxidative injury and increase the antioxidant capacity of H9c2 cells.In addition,pretreatment with ginsenoside Rg2 decreased apoptotic nuclei induced by H2O2.These data proposed that ginsenoside Rg2 has a protective role in H2O2-induced apoptotic cell death of H9c2 cells.Furthermore,we also detected the apoptotic rate by Annexin V/PI staining.Pretreatment with ginsenoside Rg2 treatment significantly reduced the percentage of Annexin V/PI positive cells compared with the model group(P<0.01).Ginsenoside Rg2 pretreatment restored protein levels of Procaspase-3,Procaspase-9 and Bcl-2 reduced expression of Bax in H2O2-subjected H9c2 cells.Western Blot demonstrated that,with increasing concentrations of ginsenoside Rg2,protein expression of SIRT1 was increased in a dose-dependent manner compared with the model group(P<0.01).2.Protective effects of ginsenoside Rg2 on myocardial ischemia reperfusion injury in ratsMethods:.Wistar rats were randomly assigned to experimental groups:Sham group,MI/R group,Ginsenoside Rg2(10,20 mg/kg)groups and Diltiazem group.After 30 min of ischemia,the slipknot was released,and the myocardium was reperfused for 6 h and 72 h.The cardiac function was measured by color Doppler ultrasound;The myocardial infarct size,myocardial histopathology and ultrastructure changes were observed;The activities of LDH,CK-MB and AST in serum,the activities of CAT,SOD and GSH-PX and the content of MDA in myocardial tissues were measured;IL-1?,IL-6 and TNF-a levels in myocardial tissues were detected by QPCR;The apoptosis rate of myocardial tissue was detected by TUNEL staining;SIRT1 expression was detected by immunohistochemical assay;Expression of Bcl-2,Bax,Caspase family protein,expression of SIRT1/p53 signaling pathway and PI3K/Akt signaling pathway relative protein were determined by Western Blot analysis.Results:Pretreatment with ginsenoside Rg2 significantly ameliorated myocardial dysfunction and remodeling,which was verified by increased LVEF,LVFS and decreased LVIDd,LVIDs compared with model group(P<0.05 or P<0.01).Pretreatment with ginsenoside Rg2(10 and 20 mg/kg)resulted in dose-dependent reduction in the infarct size.Pretreatment with ginsenoside Rg2(10 and 20 mg/kg)inhibited the increase of CK-MB,AST and LDH compared with MI/R group(P<0.05 or P<0.01).In addition,it could decrease the level of MDA,but increased the activities of SOD,.CAT and GSH-PX in tissues.QPCR detection demonstrated that,pretreatment with ginsenoside Rg2 gene expression of IL-1?,IL-6 and TNF-a was decreased significantly.Myocardial histopathology and ultra-structural observation results shown that ginsenoside Rg2 could improve myocardial pathological changes.Pretreatment with ginsenoside Rg2(10 and 20 mg/kg)significantly decreased apoptotic cells compared with MI/R group(P<0.01).Western Blot analysis demonstrated that pretreatment with ginsenoside Rg2 decreased the expression of Bax and increased the expression of procaspase-3,procaspase-9 and Bcl-2 compared with MI/R group.These results indicated that pretreatment with ginsenoside Rg2 may activate SIRT1 expression,thus decreasing Ac-p53 expression,and inhibit the apoptotic signaling pathway.In addition,pretreatment with ginsenoside Rg2 increased the expression of p-PI3K,p-Akt and p-eNOS compared with MI/R group.These results indicated that pretreatment with ginsenoside Rg2 may activate PI3K/Akt/eNOS signaling pathway.3.Protective effects and mechanisms of ginsenoside Rg2 on myocardial ischemia reperfusion injury based on SIRT1 signaling pathwayMethods:Wistar rats were randomly assigned to experimental groups:MI/R group,MI/R+Rg2 group,MI/R+Rg2+EX527 group and MI/R+EX527 group.After 30 min of ischemia,the slipknot was released,and the myocardium was reperfused for 6 h and 72 h.The cardiac function was measured by color Doppler ultrasound;The myocardial infarct size,myocardial histopathology and ultrastructure changes were observed;The activities of LDH,CK-MB,AST in serum,the activities of CAT,SOD,GSH-PX and the content of MDA in myocardial tissues were measured;IL-1?,IL-6 and TNF-a levels were detected by QPCR;The apoptosis rate of myocardial tissue was detected by TUNEL staining;SIRT1 expression was detected by immunohistochemical assay;Expression of Bcl-2,Bax,Caspase family protein,expression of SIRT1/p53 signaling pathway and PI3K/Akt signaling pathway relative protein were determined by Western Blot analysis.Results:Pretreatment with ginsenoside Rg2 significantly ameliorated myocardial dysfunction and remodeling,reduced the infarct size,decreased the activity of myocardial enzymes and improved myocardial pathological changes.However,EX527 treatment abolished the protective effect of ginsenoside Rg2.We have demonstrated that pretreatment with ginsenoside Rg2 reduced oxidative stress in MI/R hearts.We further studied the effect of EX527 on ginsenoside Rg2 treatment.EX527 treatment also abolished anti-oxidative stress effect of ginsenoside Rg2.Pretreatment with ginsenoside Rg2 markedly inhibited the increase in the mRNA levels of pro-inflammatory cytokines.However,EX527 treatment increased the mRNA levels of IL-1?,IL-6 and TNF-a,which was reduced by ginsenoside Rg2 treatment compared with MI/R+Rg2 group(P<0.05).Western Blot analysis demonstrated that EX527 treatment could increase Ac-p53 expression by decreasing SIRT1 expression.Consistent with the previous observations,we found that EX527 treatment enhanced apoptotic signaling by decreasing procaspase-3,procaspase-9 and Bcl-2 expressions while increasing Bax expression compared with MI/R+Rg2 group.These results suggest that ginsenoside Rg2 reduces apoptosis at least in part,by up regulating SIRT1 expression.In addition,pretreatment with ginsenoside Rg2 increased the expression of p-PI3K,p-Akt and p-eNOS.However,EX527 treatment inhibited the effect of ginsenoside Rg2.In summary,ginsenoside Rb2 and ginsenoside Rg2 exhibited significant protective effects on H2O2-induced injury in H9c2 cells and myocardial ischemia reperfusion injury in rats.Our results demonstrate that ginsenoside Rb2 and ginsenoside Rg2 attenuated MI/R injury by activating SIRT1,activation of PI3K/Akt signaling pathway.