Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes

Tert-butylhydroquinone (tBHQ), an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2), has been demonstrated to attenuate oxidative stress-induced injury and the apoptosis of human neural stem cells and other cell types. However, whether tBHQ is able to exert a protective effect against oxidative stress and the apoptosis of cardiomyocytes has not yet been determined. Thus, the objective of the present study was to determine whether tBHQ protects H9c2 cardiomyocytes against ethanol-induced apoptosis. For this purpose, four sets of experiments were performed under standard culture conditions as follows: i) untreated control cells; ii) cell treatment with 200 mM ethanol; iii) cell treatment with 5 µM tBHQ; and iv) cell pre-treatment with 5 µM tBHQ for 24 h, followed by medium change and co-culture with 200 mM ethanol containing 5 µM tBHQ for a further 24 h. The viability of the cardiomyocytes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of intracellular reactive oxygen species (ROS) and apoptosis were assessed by flow cytometry. Protein expression was measured by western blot analysis, and Nrf2 nuclear localization was observed by immunofluorescence. Exposure to ethanol led to a decrease in the protein expression of Nrf2 and its downstream antioxidant enzymes, accompanied by an increase in ROS generation and in the apoptosis of H9c2 cells. Pre-treatment with tBHQ significantly prevented the H9c2 cells from undergoing ethanol-induced apoptosis. tBHQ also increased the expression of B-cell lymphoma-2 (Bcl-2), whereas Bcl-2-associated X protein (Bax) expression was decreased. tBHQ promoted Nrf2 nuclear localization and increased the expression of Nrf2, superoxide dismutase (SOD), catalase (CAT) and heme oxygenase-1 (HO-1), and simultaneously inhibited the ethanol-induced overproduction of intracellular ROS. Therefore, tBHQ confers protection against the ethanol-induced apoptosis of and activates the Nrf2 antioxidant pathway in H9c2 cardiomyocytes.