The Role Of Autophagy Mediated By PINK1/Parkin Pathway On Cardiomyocytes Subjected To Anoxia/Reoxygenation Injury

Objective:To explore the role of autophagy mediated by PINK1/Parkin pathway in H9c2 cells exposed to anoxia/reoxygenation(A/R)injury.Methods:In this study,H9c2 cells were used to establish A/R model,which simulated myocardial ischemia-reperfusion(I/R)injury.The H9c2 cells were divided into five groups:Control group,A/R group,PLKO + A/R group,PLKO-Parkin+A/R group,3-MA+A/R group.Each group of data repeated three times.The activity of lactate dehydrogenase(LDH)and the opening degree of mitochondrial permeability transition pore(MPTP)were measured by spectrophotometry.Western blotting analyzed the protein expression of the Beclin1,LC3II/I,Parkin,PINK1,VDAC1 and other protein levels.Flow cytometry determined the reactive oxygen species(ROS)generation,mitochondrial membrane potential(ΔΨm)and cell apoptosis.MDC staining detected the autophagy vacuoles formation,and the immunofluorescence observed the Parkin localization in the H9c2 cells.Results:1.After A/R treatment,ROS burst,mitochondrial membrane potential collapsed,LDH activity and cell apoptosis increased.Western blot results showed the ratio of LC3II/I,Beclin1 expression significantly up-regulated.Compared to A/R group,the autophagy inhibitor(3-MA)could significantly improved the damage when H9c2 cells were subjected to A/R injury.2.After A/R treatment,compared with the control group,the Parkin and PINK1 protein expression increased.Laser confocal immunofluorescence method detected that the mitochondrial recruitment of Parkin were obviously added.However,when downregulated the Parkin expression via RNAi plasmid(p LKO-Parkin),it showed the PINK1 protein levels declined.In addition,the LC3II/I ratio and the Beclin1 expression decreased,and the MDC staining showed the autophagy vacuoles generated number also reduced significantly.3.The results showed that to downregulated the Parkin protein expression could improve the MPTP opening and MMP,reduce ROS production,and descend the LDH activity and the apoptosis level,indicating the Parkin protein expression downregulated could improve A/R injury.Conclusion:The A/R treatment could activate the Parkin/PINK1 pathway and enhance autophagy.Down-regulation of the Parkin expression via RNAi could inhibit the autophagy level and improve A/R injury.