硫化氢通过调控SIRT1抑制阿霉素诱导的H9c2细胞损伤

目的探讨硫化氢（H₂S）对阿霉素（DOX）诱导的H9c2细胞损伤的影响及其作用机制。 方法H₂S对DOX心肌毒性保护作用的实验分组为：对照组（Control组），5?μmol/?L DOX处理组（A组），5?μmol/L DOX和400?μmol/L NaHS共同处理组（B组），400?μmol/L NaHS单独处理组（C组），5?μmol/L DOX、400?μmol/L NaHS和15?μmol/L Sirtinol共同处理组（D组），15?μmol/L Sirtinol单独处理组（E组）。SIRT1是否参与H₂S抗DOX心肌毒性作用机制的实验分组为：对照组（Control组），5?μmol/L DOX处理组（F组），5?μmol/L DOX和400?μmol/L NaHS共同处理组（G组），5?μmol/L DOX、400?μmol/L NaHS和15?μmol/L Sirtinol共同处理组（H组），15?μmol/L Sirtinol单独处理组（I组）。使用MTT法检测细胞活力；Elisa法检测细胞MDA以及SOD水平；DCFH-?DA荧光探针法检测ROS水平；采用Western Blot法检测SIRT1蛋白表达。使用单因素方差分析法进行统计学分析。 结果NaHS预处理可抑制DOX导致的H9c2细胞活力下降：Control组，A组、B组、C组细胞活力分别为100﹪、（54.58±1.58）﹪、（85.05±4.31）﹪、（100.22±4.46）﹪ （F = 134.9，P < 0.001）。NaHS预处理可减弱DOX引起的H9c2细胞ROS、MDA水平的增加以及SOD水平的降低：Control组的ROS、MDA和SOD水平分别是100﹪、（34.18±1.56） μmol/g、（53.69±1.44） U/?mg；A组的ROS、MDA和SOD水平分别是（174.90±12.65）﹪、（72.65±2.66） μmol/g、（31.80±2.05） U/?mg；B组的ROS、MDA和SOD水平分别是（126.08±6.25）﹪、（44.59±1.92） μmol/g、（48.06±1.56） U/mg；C组的ROS、MDA和SOD水平分别是（91.86±1.66）﹪、（32.93±1.56）?μmol/?g、（55.93±1.58）?U/?mg （F?= 83.26，P < 0.001；F = 271.4，P < 0.001；F = 127.0，P < 0.001）。F组（6、12、24?h）H9c2细胞SIRT1蛋白表达水平分别是（0.45±0.03）、（0.27±0.02）、（0.25±0.03），较Control组（1.00±0.00）降低（F = 611.1，P < 0.001）。本研究还发现，NaHS预处理H9c2细胞能阻止DOX引起的SIRT1蛋白表达下调：Control组、F组、G组、H组的SIRT1蛋白表达水平分别是（1.00±0.00）、（0.31±0.03）、（0.60±0.04）、（1.09±0.09）（F = 123.4，P?2S对DOX诱导的H9c2细胞活力降低的抑制作用：Control组，F组、G组、H组、I组细胞活力分别为100﹪、（54.58±1.58）﹪、（85.37±3.62）﹪、（71.11±2.11）﹪、（97.53±1.45）﹪ （F = 238.2，P < 0.001）。Sirtinol预处理可明显逆转H₂S对DOX导致的H9c2细胞ROS和MDA含量增加及SOD水平降低的抑制作用：Control组的ROS、MDA和SOD水平分别是100﹪、（35.84±2.22）μmol/?g、（53.03±3.16） U/mg；F组的ROS、MDA和SOD水平分别是（184.6±11.33）﹪、（74.78±5.30）μmol/g、（29.26±0.85）U/mg；G组的ROS、MDA和SOD水平分别是（126.5±7.57）﹪、（41.95±3.43）μmol/g、（52.61±2.26）U/mg；H组的ROS、MDA和SOD水平分别是（174.7±5.50）﹪、（67.69±1.52） μmol/g、（35.33±1.95） U/mg，I组的ROS、MDA和SOD水平分别是（98.03±2.86）﹪、（37.66±2.49）μmol/g、51.14 U/mg（F = 112.0，P < 0.001；F = 93.73，P < 0.001；F = 84.92，P < 0.001）。 结论H₂S通过调控SIRT1抑制DOX诱导的H9c2细胞损伤。

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

ObjectiveTo investigate the effect of hydrogen sulfide(H₂S) on doxorubicin(DOX)-induced cell injury of H9c2 cells and its possible mechanism. MethodsThe 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. ResultsNaHS 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). ConclusionH₂S inhibits DOX-induced cell injury of H9c2 cells by modulation of SIRT1 expression.