The Role Of Bcl-2 In HSP70-Mediated Protection Against Oxidative Stress-Induce Apoptosis

Studies in recent years have shown that apoptosis is an important mechanism of cardiac myocytes injury,and myocardial apoptosis has been demonstrated to be closely related to the occurrence and development of many cardiovascular disease,such as myocardial ischemia/reperfusion injury,myocardial infarction,cardial remodeling and heart failure.Heat Shock Protein(HSP) is a necessary protein in physiological condition and was up-regulated in stress(particular in heat stress).It was found in our previous study that heat shock pretreatment and HSP70 overexpression could inhibit the release of Smac.However, the molecular mechanism by which HSP70 interferes with Smac release and apoptosis induced by oxidative-stress is not well understood.Bcl-2 is one of the first identified anti-apoptotic protein,which has the function of keeping mitochondria membrane stability.Some studies have shown that Bcl-2 could reduce the release of Smac from mitochndria.However,it is still not clear whether the function that HSP70 inhibited Smac release and apoptosis is related to Bcl-2.In order to detect these questions,firstly,we use HSP70 gene transfection and antisense oligonucleotide technology to explore the effect of HSP70 on Bcl-2 expression.Secondary,with the C2C12 cells HSP70 overexpressing,we build a model of apoptosis by dealing with H₂O₂(0.5mmol/L).Under the condition of up-regulating and inhibiting of Bcl-2 expression,by detecting the release of Smac from mitochondria and by testing caspase-9,3 activation,we explore the role of Bcl-2 during HSP70 against apoptosis and try to reveal the anti-apoptosis molecular mechanism of HSP70 furtherly.The main results are as follows:â‘ Western blot analyses demonstrated higher expression of HSP70 in C2C12 cells transfected with HSP70 gene was associated with higher Bcl-2 expression compared to that in cells which had less HSP70 exprssion.Transfection of As-HSP70 was able to selectively inhibit HSP70 expression and lower expression of Bcl-2 in C2C12 cells that transfected with As-HSP70 compared to that in cells which transfected with Scr-HSP70.â‘¡The C2C12 cells overexpressing HSP70 was transfected with As-Bcl2 or Scr-Bcl2,then exposed to H₂O₂ for 2 hour, mitochondria and cytosolic fractions was analysed by western blot,and shown HSP70 overexpression could inhibit the release of Smac from mitochondria in the cells which transfected with Scr-Bcl2,but the overexpressing HSP70 was no longer able to inhibit oxidative stress-induced Smac release in the cells transfected with As-Bcl2.â‘¢The C2C12 cells overexpressing HSP70 was transfected with As-Bcl2 or Scr-Bcl2,then exposed to H₂O₂(0.5mmol/L)for 8 hour,the activation of caspase-9,3 were analyzed by an substrate-cleaving reaction in vitro,the HSP70 mediated protection against oxidative stress-induced activation of caspase-9,3 was inhibited significantly in the cells that transfected with As-Bcl2 compared to those transfected with Scr-Bcl2.â‘£As-Bcl2 or the control Scr-Bcl2 was introduced into C2C12 cells overexpressing HSP70, the cells were then exposed H₂O₂(0.5mmol/L) for 24 hour,the percentage of apoptosis was higher in the cells that transfected with As-Bcl2 compared to that which transfected with Scr-Bcl2.It was concluded that:â‘ HSP70 contributed to the up-regulation of Bcl-2 expression.â‘¡Bcl-2 played an important role in HSP70-mediated protection against oxidative-stress-induced release of Smac and apoptosis.