The Protective Effcet And Mechanism Of Geniposide And Genipin On Myocardial Ischemia-reperfusion Injury

PART I THE ROLE AND MECHANISM OF AUTOPHAGY IN THE PROTECTION OF GENIPOSIDE AGAINST MYOCARDIAL ISCHEMIA-REPERFUSION INJURYObjective:To investigate the effect of geniposide(GP)on myocardial ischemia/reperfusion(I/R)injury in vivo and hypoxia/reoxygenation(H/R)in vitro respectively,and its mechanism.Methods:In vivo SD rats were subjected to 2 h reperfusion followed by 30 min ischemia to establish the I/R model.The SD rats were randomly divided into three groups such as Sham group,I/R group,and I/R+GP group.Myocardial infract size in rats after I/R was assessed by TTC staining.Myocardial tissue structure was assessed by HE staining.Echocardiography was used to evaluate cardiac function.Cardiomyocyte apoptosis rate was determined by TUNEL staining.The expression of Beclin1 in myocardium was detected by IHC staining.The formation of autophagosomes was observed by TEM.The expression of Bax,Bcl-2,ATG5,ATG7,Beclin1,LC3-I/II,P62,AKT and p-AKT~ser473er473 proteins were detected by Western Blot.In vitro H9c2 cells were subjected to 4 h reoxygenation followed by 12 h hypoxia to establish the H/R model.H9c2cells were divided into seven groups such as Control group,Control+GP group,Control+Rapamycin(RAPA)group,H/R group,H/R+GP group,H/R+RAPA group,and H/R+GP+RAPA group.Cell viability was detected by CCK-8.Cell apoptosis rate was determined by flow cytometry.Autophagosome formation was observed by TEM.The expression of ATG5,Beclin1,LC3-I/II,P62,Bax,Bcl-2,AKT,p-AKT,mTOR and p-mTOR proteins were assessed by Western Blot.Results:In vivo(1)TTC staining:There was no significant myocardial infarction in sham group.The size of infarction in I/R+GP group was smaller than those of I/R group.(2)HE staining:The I/R group showed tissue disarrangement,cellular swelling and nuclear shrinkage compared with the sham group.The acute tissue injury was attenuated by GP administration in I/R+GP group.(3)Echocardiography:EF and FS values of I/R group were decreased compared with the sham group,while the EF and FS were improved in rats receiving GP pretreatment.The SV and IVSs were declined in I/R group compared with the sham group,while the SV and IVSs were increased by GP pretreatment.(4)TUNEL staining:TUNEL-positive nuclei(brown and yellow)of heart tissue sections in I/R group were higher than those of the sham group.GP pretreatment reduced the percentage of apoptotic cells during I/R.(5)IHC staining:GP pretreatment reduced the level of Beclin1 induced by myocardial I/R injury.(6)TEM:GP pretreatment prevented autophagosome accumulation in I/R myocardium.(7)Western Blot:Bax expression was increased in I/R group as compared to the sham group,and that was decreased in I/R+GP group.Bcl-2 expression and the ratio of Bcl-2/Bax were declined after I/R and restored by GP pretreatment.Compared with I/R group,GP pretreatment inhibited the expression of ATG5,ATG7,Beclin-1 and the ratio of LC3-II/LC3-I,and enhanced expression of p62.GP pretreatment also increased the expression of p-AKT during I/R.In vitro(1)CCK-8:GP pre-incubation enhanced the cell viability of H9c2 cells subjected to H/R.The protective effect of GP was counteracted by RAPA cotreatment during H/R.(2)Flow cytometry:No difference was observed in the rate of apoptosis among Control,Control+GP,and Control+RAPA groups.Pre-incubation with RAPA blocked the inhibitory effect of GP on H/R-induced apoptosis.(3)TEM:H/R led to autophagy accumulation.The number of autophagosomes was decreased in H/R+GP group as compared to H/R group.The effect of GP was inhibited by RAPA pre-incubation during H/R.(4)Western Blot:GP pre-incubation inhibited ATG5,Beclin1,LC3-II expression and p62 degradation during H/R.The effects of GP were blocked by RAPA pre-incubation during H/R.Compared with H/R group,GP pre-incubation inhibited the expression of Bax,and enhanced expression of Bcl-2 and Bcl-2/Bax rate.The effects of GP on apoptosis-related proteins were counteracted by RAPA pre-incubation.GP pre-incubation up-regulated the expression of p-AKT and p-mTOR as compared to the control group during H/R.Compared with H/R+GP group,the expression of p-mTOR in H/R+GP+RAPA group was reduced,but expression of p-AKT was increased.No difference was observed in total AKT and mTOR expression among groups.Conclusions:GP protected against myocardial I/R injury by inhibiting excessive autophagy,which might involve activating AKT/mTOR signaling pathway.PART II EFFECT OF GENIPIN ON CARDIOMYOCYTE APOPTOSIS AND AUTOPHAGY AFTER HYPOXIA REOXYGENATION INJURYObjective: To investigate the effect of genipin(GE)on cardiomyocyte apoptosis and autophagy after hypoxia/reoxygenation(H/R)injury.Methods: The method with hypoxia treatment of H9c2 cells for 12 h and then reoxygenation treatment for 4 h was used in the present study in order to establish H/R model.The H9c2 cells were divided into Control group,GE group,H/R group,and H/R+ GE group.Cell viability was detected by CCK-8.Cell apoptosis was determined by flow cytometry.TEM was used to observe autophagosome formation.The expression of Bax,Bcl-2,P62,Beclin1,LC3-I/II,AKT,p-AKT,m TOR and p-m TOR proteins were assessed by Western Blot.Results:(1)CCK-8: The cell viability of H/R and 1,5,10,20,40,80μM GE groups were obviously decreased as compared to the control group,while the cell viability in 10,20 and 40 μM GE groups were significantly enhanced as compared to the H/R group,and the peak was 20 μM.(2)Flow cytometry: The ratio of apoptosis was reduced by GE pretreatment after H/R injury.(3)TEM: The number of autophagosomes was reduced by GE pretreatment after H/R injury.(4)Western Blot: GE pretreatment decreased the expression of Bax,LC3-II,Beclin1 proteins and increased the expression of Bcl-2,p62,p-AKT and p-m TOR proteins after H/R injury.Conclusions: GE could reduce H/R-induced apoptosis and autophagy,which might involve activating AKT/m TOR signaling pathway.