Hydrogen Sulfied Inhibits Homocysteine-induced Cell Senescence In HT22 Cells By Up-regulating SIRT1

Objective: To investigate whether H2 S inhibits homocysteine-induced cell senescence in HT22 cells and whether SIRT1 mediates this inhibitory role of H2 S.Method: The senescence of HT22 cellular was tested by the beta galactosidase staining(Senescence Associated aging acidic-beta-Galactosidase SA-β-Gal), the expression of detected cell senescence-associated proteins P16INK4 a and P21 CIPL determined by western blotting, and cell growth curve determined by using trypan blue staining. SIRT1 protein levels were assessed by western blotting. Cell activity was tested by CCK8 assay. Apoptosis was assessed by flow cytometry after Annexin V/PI staining and observed by fluorescence microscopy after Hoechst 33258 staining. Mitochondrial membrane potential was evaluated by fluorescence microscopy after Rh123 staining.Result: 1. Hcy(2.5, 5, 10 mmol/L, for 48 h) dose-dependently increased the proportion of SA-β-Gal positive cells, the expressions of senescence related protein P16INK4 a and P21 CIPL protein, and the declining tendency of cell growth curve in HT22 cells, indicating that Hcy induces the senescence of HT22 cells. 2. Na HS(the donor of H2 S, 100、200、400 μmol/L) dose-dependently inhibited the increases in the proportion of SA-β-Gal positive cells, the expressions of senescence related protein P16INK4 a and P21 CIPL protein, and the declining tendency of cell growth curve in HT22 cells induced by treatment of 5 mmol/L of Hcy for 48 h, indicating that H2 S prevents Hcy-triggered senescence of HT22 cells. 3. The expression of SIRT1 in HT22 cells was dose-dependently reduced by treatment of Hcy(2.5, 5, and 10 mmol/L) for 48 h, suggesting that Hcy suppress the expression of Sirt1 in HT22 cells. 4. Treatment with Na HS(100, 200 or 400 μmol/L, for 48 h) not only dose-dependently increased the levels of SIRT1 expression in HT22 cells but also reversed the decrease in SIRT1 expression induced by exposed to 5 mmol/L of Hcy for 48 h, indicating that H2 S up-regulates the expression of SIRT1 in HT22 cells. Sirtinol(a inhibitor of SIRT1, 15μmol/L) blocked the inhibitory actions of Na HS(400 μmol/L) in Hcy(5 mmol/L, for 48 h)-induced increases in the proportion of SA-β-Gal positive cells, the expressions of senescence related key protein P16INK4 a and P21 CIPL protein, and the declining tendency of cell growth curve in HT22 cells, indicating that SIRT1 mediates the inhibitory role of H2 S in Hcy-induced senescence of HT22 cells.Conclusion: H2 S inhibits Hcy-induced senescence of HT22 cells by up-regulating the expression of SIRT1.