Synergistic Effect And Mechanism Of Celastrol Enhance Cardiomyocyte Apoptosis Induced By Palmitic Acid

Objective: Evaluating synergistic effect of celastrol under palmitic acid induced cardiomyocyte apoptosis and exploring the possible mechanism,which will provide theoretical foundation for the clinical application of celastrol in the future.Methods: H9c2 cardiac myoblast cells were cultured in DMEM medium supplemented with 10% FBS.Upon reaching 80% confluence,the cells were incubated with different concentrations of celastrol or different concentrations of celastrol and palmitic acid(0.1mmol/L)combination,subsequently measured H9c2 cells viability with CCK-8 kit,detected the production of intracellular Cleaved-caspase3,p-AMPKα,AMPKα,p-JNK,JNK,and LC3 with Western blotting and ROS with immunofluorescence and microplate reader.Results: H9c2 cells were treated with celastrol at a final concentration of 800nmol/L or less for 12 h,there were not significant change found in H9c2 cells viability,the result was the same as the production of intracellular cleaved caspase 3in H9c2 treated for 24 h,but the significant cytotoxicity were revealed at a final concentration 1600 nmol/L.When H9c2 myocardial cells were treated by the combination of celastrol and palmitic acid,celastrol could decrease H9c2 cells viability at a concentration of 200 nmol/L or more,and increased the accumulation of Cleaved-caspase3 with celastrol dose-dependence more than palmitic acid alone.Each group with p<0.05,the difference was statistically significant.Exploring the behind mechanisms,we found that celastrol combination with palmitic acid increased the production of ROS in H9c2 cells more than palmitic acid alone.Five times higher than celastrol alone,and three times higher than palmitic acid alone,and p<0.05.H9c2 cells were treated with celastrol for 24 hours,there were significant increases of the intracellular activation of caspase3 accumulation and the levels of LC3-II,which were suppressed significantly by autophagy inhibitors CQ and 3-MA,and p<0.05.When the time shorter to 6 hours,celastrol inhibited phosphorylation of AMPKα,JNK,so that,decreased phosphorylation levels of AMPKα,JNK induced by palmitic acid at 6h by celastrol,palmitic acid or celastrol combination with palmitic acid.Conclusion: Low dose of celastrol itself does not induce cardiomyocyte apoptosis.Celastrol possesses synergistic toxicity with palmitic acid,enhances myocardial apoptosis dependent on activation of caspase signaling pathway in H9c2 cells,and the possible mechanism is that celastrol enhanced significant accumulation of ROS and regulated activation of p-AMPKα,p-JNK in the initial stage of palmitic acid-induced apoptosis.On the other hand,increasing of autophagy induced by celastrol in H9c2 cells enhances the activation of caspase-dependent apoptosis signaling to promote H9c2 cells apoptosis.