Hydrogen sulfide inhibits doxorubicin-induced cell injury of H9c2 cells by regulating SIRT1 expression

Objective To investigate the effect of hydrogen sulfide(H2S) on doxorubicin(DOX)-induced cell injury of H9c2 cells and its possible mechanism. Methods The experimental groups are as follows: Control group, 5 mol/L DOX treated group A, 5 mol/L DOX and 400 μmol/L NaHS co-treated group B, 400 μmol/L NaHS treated group C, 5 mol/L DOX, 400 μmol/ L NaHS and 15 mol/L Sirtinol co-treated group D, 15 mol/ L Sirtinol treated group E. Another expression level of SIRT1 groups are as follows: Control group, 5 mol/L DOX treated group F, 5 mol/L DOX and 400 μmol/L NaHS co-treated group G, 400 μmol/L NaHS and 15 mol/ L Sirtinol co-treated group H, 15 mol/ L Sirtinol treated group I. The viability of H9c2 cells was measured by MTT assay. The levels of MDA and SOD were detected by Elisa. ROS level were measured using DCFH-DA fluorescent probe. The expression of SIRT1 protein were detected by Western Blot. Results NaHS pretreatment significantly inhibited DOX-induced cell death: the viability in Control group, A group, B group, C group was 100﹪, (54.58±1.58)﹪, (85.05±4.31)﹪ and (100.22±4.46)﹪ respectively (F = 134.9, P < 0.001). NaHS pretreatment significantly prevented DOX-induced ROS and MDA levels and increased SOD levels in H9c2 cells: the levels of ROS, MDA and SOD in Control group were 100﹪, (34.18±1.56) μmol/g, (53.69±1.44) U/mg respectively; the levels of ROS, MDA and SOD in A group are (174.90±12.65)﹪, (72.65±2.66) μmol/g, and (31.80±2.05) U/ mg respectively; the levels of ROS, MDA and SOD in B group were (126.08±6.25)﹪, (44.59±1.92) μmol/ g, (48.06±1.56) U/mg respectively; the levels of ROS, MDA and SOD in C group were (91.86±1.66)﹪, (32.93±1.56) μmol/ g, (55.93±1.58) U/mg respectively (F = 83.26, P < 0.001; F = 271.40, P < 0.001; F = 127.00, P < 0.001). The expression level of SIRT1 protein was markedly decreased after treatment with DOX for 6 h, 12 h or 24 h (F = 611.10, P < 0.001), which were prevented by NaHS pretreatment: the expression level of SIRT1 in Control group, F group, G group, and H group were 1.00±0.00, 0.31±0.03, 0.60±0.04, and 1.09±0.09 respectively (F = 123.40, P < 0.001). Sirtinol, the inhibitor of SIRT1, reversed the inhibitory effect of H2S on DOX-induced cell death: cell viability in Control group, F group, G group, H group, and I group were 100﹪, (54.58±1.58)﹪, (85.37±3.62)﹪, (71.11±2.11)﹪, and (97.53±1.45)﹪ respectively (F = 238.20, P < 0.001). Sirtinol pretreatment markedly reversed the inhibitory effect of H2S on DOX-induced increases in ROS as well as MDA levels and decrease in SOD levels: the levels of ROS, MDA and SOD in Control group were 100﹪, (35.84±2.22) μmol/ g, (53.03±3.16) U/mg respectively; the levels of ROS, MDA and SOD in F group were (184.6±11.33)﹪, (74.78±5.30) μmol/g, and (29.26±0.85) U/ mg respectively; the levels of ROS, MDA and SOD in G group were (126.5±7.57)﹪, (41.95±3.43) μmol/g, (52.61±2.26) U/mg respectively; the levels of ROS, MDA and SOD in H group were (174.7±5.50)﹪, (67.69±1.52) μmol/g, (35.33±1.95) U/mg respectively, the levels of ROS, MDA and SOD in I group were (98.03±2.86)﹪, (37.66±2.49) μmol/ g, (51.14 U/mg) respectively (F = 112.00, P < 0.001; F = 93.73, P < 0.001; F = 84.92, P < 0.001). Conclusion H2S inhibits DOX-induced cell injury of H9c2 cells by modulation of SIRT1 expression. Key words: Hydrogen sulfide; Doxorubicin; SIRT1; H9c2 cells