Sodium nitrite preconditioning protects against ethanol-induced damage in human hepatoma SMMC-7721 cells

This study is to investigate the cytoprotective role of NaNO₂ preconditioning against ethanol   induced damage in human hepatoma SMMC-7721 cells.  The cells were preconditioned with NaNO₂ (0.25 mmol·L⁻¹) for 24 hours or 4 weeks, and then exposed to ethanol (200 mmol·L⁻¹) for additional 12 h and untreated cells served as control.  Both temporal and chronic NaNO₂ preconditioning could prevent ethanol elicited   cytotoxicity as evidenced by thiazolyl blue (MTT).  NaNO₂ preconditioning also could inhibit ethanol-induced apoptosis, which was confirmed by FITC-Annexin V/PI flow cytometer and Hoechst 33258 and PI staining.  Further, simultaneous NaNO₂ preconditioning treatment along with ethanol showed protection against ethanol mediated cellular damage as indicated by significantly decreased levels of malondialdehyde (MDA) and elevated activities of superoxide dismutase (SOD) and catalase (CAT).  Western blotting analysis revealed that in   ethanol treated cells preconditioned with NaNO₂, the HIF-1α and Bcl-2 increased obviously, while the expression of pro-apoptotic proteins, including Bax, Caspase-9, Caspase-3 decreased.  The results showed that low doses of NaNO₂ preconditioning resistant to ethanol-induced human hepatoma SMMC-7721 cells apoptosis, which mechanism may be related to increased expression of HIF-1α in the cells.