Ozone Induces Tolerance Against Cardiomyocytes Oxygen-glucose Deprivation/Reperfusion Through Inhibition Of Autophagy Pathway

Objective: Previous studies have shown that excess activation of autophagy in cardiomyocytes is associated with the increase of myocardial oxygen-glucose deprivation/reperfusion(OGD/R)injury.Ozone therapy affords significant cardioprotection against myocardial OGD/R injury.The present study was designed to determine whether ozone-induced tolerance to myocardial OGD/R injury was mediated by inhibiting autophagy.Methods: The rat cardio myoblast cell line H9C2 was used in this study.The model of H9C2 cells under OGD/R was established.The cells were incubated with different concentrations of ozone(10-60 μg/ml)during reperfusion.Furthermore,to investigate the role of autophagy in OGD/R-induced injury,the autophagy inducer rapamycin and inhibitor 3-MA were applied.Cell viability was detected by CCK-8 assay.Cell apoptosis was evaluated by flow cytometry.Oxidative stress was examined by Superoxide dismutase(SOD),Lactate dehydrogenase(LDH)and Malondialdehyde(MDA)levels.The expressions of apoptosis regulator B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(BAX),cleaved caspase-3,markers of autophagy microtubleassociated protein 1 light chain 3(LC3),Autophagy-related protein 5(Atg5)and Beclin-1 were measured by Western blotting assays.Results: As a result,OGD/R obviously decreased cell viability and induced apoptosis in H9C2 cells,whereas ozone(10-40 μg/ml)can reverse the noxious effects of OGD/R on H9C2 cells,and 20μg/ml ozone was the most effective.It can inhibited the decrease in the ratio of Bcl-2/BAX and the expression of cleaved caspase-3,and inhibited the increase in the ratio of LC3-II/LC3-I and the expression of Atg5 and Beclin-1 elicited by OGD/R,and it dose-dependently prevented OGD/R-induced oxidative stress.Along with this,rapamycin markedly reversed the effects of ozone(20μg/ml)on OGD/Rinduced expressions of autophagy marker proteins and 3-Methyladenine(3-MA)further improve the effect of ozone.Conclusion: Taken together,the data presented here provides a credible mechanism by which ozone treatment at low concentration could protect myocardium from OGD/R-induced injury by inhibiting autophagy.