Ozone induces tolerance against cardiomyocytes oxygen-glucose deprivation/reperfusion through inhibition of autophagy pathway.

Abstract

Previous studies have reported that excess activation of autophagy in cardiomyocytes is associated with an increase in myocardial oxygen-glucose deprivation/reperfusion (OGD/R) injury. Ozone therapy affords significant cardio-protection 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. Subsequently, the rat cardio myoblast H9C2 cell line was used in the present study. A 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 and inhibitor were applied. Cell viability was detected by Cell Counting kit-8 assay. Cell apoptosis was evaluated by flow cytometry. Oxidative stress was examined by superoxide dismutase, lactate dehydrogenase and malondialdehyde levels. The expressions of apoptosis regulator B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (BAX), cleaved caspase-3, markers of autophagy microtuble-associated protein 1 light chain 3 (LC3), autophagy-related protein 5 (Atg5) and Beclin-1 were measured by western blot analysis. As a result, OGD/R notably decreased cell viability and induced apoptosis in H9C2 cells, while ozone (10-40 µg/ml) reversed the noxious effects of OGD/R on H9C2 cells, and 20 µg/ml ozone was the most effective. Ozone 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, as well as dose-dependently preventing OGD/R-induced oxidative stress. Furthermore, rapamycin markedly reversed the effects of ozone (20 µg/ml) on OGD/R-induced expression of autophagy marker proteins and 3-methyladenine further improved the effect of ozone. Taken together, the results of the present study provided a credible mechanism by which ozone treatment at low concentrations could protect the myocardium from OGD/R-induced injury by inhibiting autophagy.