Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy via PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway

OBJECTIVE:To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2(PINK1/PARKIN)signaling pathway.METHODS:3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells;the hypoxia/reoxygenation(H/R)model was established in tri-gas incubator;2’,7’-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species(ROS);the changes of mitochondrial membrane potential was determined by 5,5’,6,6’-Tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine iodide staining;the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits;flow cytometry was used to detect the ratio of apoptotic cells;transmission electron microscope was used to observe the ultrastructure of H9C2 cells;Western blot was used to detect the protein changes of mitochondrial 20 k Da outer membrane protein(TOM20),translocase of inner mitochondrial membrane 23(TIM23),presenilins associated rhomboid-like protein(PARL),PINK1,PARKIN and mitofusin 1(Mfn1),mitofusin 2(Mfn2),phosphotyrosine independent ligand for the Lck SH2 domain of 62 k Da(P62),microtubule-associated protein 1 light chain 3 beta(LC3B);the m RNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction;immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin.RESULTS:Resveratrol could inhibit the proliferation of H9C2 cells in a time-and concentration-dependent manner;however,pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels,alleviate the loss of mitochondrial membrane potential induced by H/R,inhibit H/R-induced apoptosis of H9C2 cells,and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage.Resveratrol could further increase the levels of p62,PINK1,PARKIN protein,the expression of PINK1,PARKIN m RNA and the ratio of LC3BⅡ/LC3BⅠin H/Rinduced H9C2 cells,inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells,and further reduce the expression of TOM20,TIM23,PARL,Mfn1 and Mfn2 protein in H/R-induced H9C2 cells.The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells.CONCLUSIONS:Resveratrol can protect H9C2 cells from H/R injury,which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.