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Protective Effect of Tempol Against Hypoxia-Induced Oxidative Stress and Apoptosis in H9c2 Cells

Linlin Jing, Qian Li, Lei He, Wei Sun, Zhengping Jia, Huiping Ma

Department of Pharmacy, Lanzhou General Hospital, Lanzhou, Gansu, China (mainland)

Med Sci Monit Basic Res 2017; 23:159-165

DOI: 10.12659/MSMBR.903764

Available online: 2017-04-21

Published: 2017-04-21


BACKGROUND: Hypoxia-induced oxidant stress and cardiomyocyte apoptosis are considered essential processes in the progression of heart failure. Tempol is a nitroxide compound that scavenges many reactive oxygen species (ROS) and has antioxidant and cardioprotective effects. This study aimed to investigate the protective effect of Tempol against hypoxia-induced oxidative stress and apoptosis in the H9c2 rat cardiomyoblast cell line, in addition to related mechanisms.
MATERIAL AND METHODS: H9c2 cells were pre-treated with Tempol, followed by hypoxia (37°C, 5% CO2, and 95% N2) for 24 h. Cell viability was detected using MTT assay. ROS level was evaluated using DCFH-DA. Lactate dehydrogenase (LDH), creatinine kinase (CK), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were evaluated using the relevant kits. Cell apoptosis was determined by Annexin V/7-AAD double labelling. The expression of apoptosis-related molecules was assessed with RT-PCR analysis and Western blotting.
RESULTS: Tempol protected H9c2 cells against hypoxia-induced injury, with characteristics of increased the cell viability and reduced LDH and CK release. Tempol also reduced oxidant stress by inhibiting ROS generation and lipid peroxidation, as well as enhancing antioxidant enzyme activity. Moreover, Tempol pretreatment upregulated the expression of Bcl-2 and downregulated the expression of Bax and caspase-3, thereby reducing hypoxia-induced apoptosis in H9c2 cells.
CONCLUSIONS: These results indicate that Tempol reduces the hypoxia-induced oxidant stress and apoptosis in H9c2 cells by scavenging free radicals and modulating the expression of apoptosis-related proteins.

Keywords: Cell Hypoxia, Free Radical Scavengers, Oxidative Stress