硫化氢通过调控SIRT1抑制阿霉素诱导的H9c2细胞损伤

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

目的探讨硫化氢(H_2S)对阿霉素(DOX)诱导的H9c2细胞损伤的影响及其作用机制。方法 H_2S对DOX心肌毒性保护作用的实验分组为:对照组(Control组),5μmol/L DOX处理组(A组),5μmol/L DOX和400μmol/L NaHS共同处理组(B组),400μmol/L NaHS单独处理组(C组),5μmol/L DOX、400μmol/L NaHS和15μmol/L Sirtinol共同处理组(D组),15μmol/L Sirtinol单独处理组(E组)。SIRT1是否参与H_2S抗DOX心肌毒性作用机制的实验分组为:对照组(Control组),5μmol/L DOX处理组(F组),5μmol/L DOX和400μmol/L NaHS共同处理组(G组),5μmol/L DOX、400μmol/L NaHS和15μmol/L Sirtinol共同处理组(H组),15μmol/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 <0.001)。SIRT1抑制剂Sirtinol预处理能明显逆转H_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H_2S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 H_2S通过调控SIRT1抑制DOX诱导的H9c2细胞损伤。

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 400μmol/L NaHS co-treated group B,400μmol/L NaHS treated group C,5 mol/L DOX,400μmol/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 400μmol/L NaHS co-treated group G,400μmol/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.