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中华细胞与干细胞杂志(电子版) ›› 2018, Vol. 08 ›› Issue (04) : 218 -223. doi: 10.3877/cma.j.issn.2095-1221.2018.04.005

所属专题： 文献；

论著

硫化氢通过调控SIRT1抑制阿霉素诱导的H9c2细胞损伤
哈斯高娃1,†(), 曹中朝1, 刘东华1   
  1. 1. 010059　内蒙古医科大学附属医院老年病科
  • 收稿日期:2018-03-12 出版日期:2018-08-01
  • 通信作者: 哈斯高娃

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

Hasigaowa1,†(), Zhongchao Cao1, Donghua Liu1   

  1. 1. Department of Geriatrics, Affiliated Hospital of Inner Mongolia Medical University, Hohhot, Inner Mongolia, 010059, China
  • Received:2018-03-12 Published:2018-08-01
  • Corresponding author: Hasigaowa
  • About author:
    Corresponding author:Hasigaowa, Email:
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哈斯高娃, 曹中朝, 刘东华. 硫化氢通过调控SIRT1抑制阿霉素诱导的H9c2细胞损伤[J]. 中华细胞与干细胞杂志(电子版), 2018, 08(04): 218-223.

Hasigaowa, Zhongchao Cao, Donghua Liu. Hydrogen sulfide inhibits doxorubicin-induced cell injury of H9c2 cells by regulating SIRT1 expression[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2018, 08(04): 218-223.

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?<?0.001）。SIRT1抑制剂Sirtinol预处理能明显逆转H2S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。

关键词: 硫化氢, 阿霉素, SIRT1, H9c2细胞
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
表1 H2S对DOX诱导的H9c2细胞活力和氧化应激的影响（ ± s）
分组 样本量 细胞相对活力OD值（﹪） ROS相对荧光强度（﹪） MDA（μmol/g） SOD（U/mg）
Control组 3 100.00±0.00 100.00±0.00 34.18±1.56 53.69±1.44
A组 3 54.58±1.58a 174.90±12.65a 72.65±2.66a 31.80±2.05a
B组 3 85.05±4.31b 126.08±6.25b 44.59±1.92b 48.06±1.56b
C组 3 100.22±4.46 91.86±1.66 32.93±1.56 55.93±1.58
F值 ? 134.9 83.26 271.4 127.0
P值 ? 0.000 0.000 0.000 0.000
图1 H2S和DOX对H9c2细胞SIRT1蛋白表达的影响
表2 Sirtinol减弱H2S抑制DOX导致的H9c2细胞活力降低和氧化应激（ ± s）
分组 样本量 细胞相对活力OD值（﹪） ROS相对荧光强度（﹪） MDA（μmol/g） SOD（U/mg）
Control组 3 100±0.00 100.00±0.00 35.84±2.22 53.03±3.16
F组 3 54.41±1.87a 184.60±11.33a 74.78±5.30a 29.26±0.85a
G组 3 85.37±3.62b 126.50± 7.57b 41.95±3.43b 52.61±2.26b
H组 3 71.11±2.11c 174.70± 5.50c 67.69±1.52c 35.33±1.95c
I组 3 97.53±1.45 98.03± 2.86 37.66±2.49 51.14±1.55
F值 ? 238.20 112.00 93.73 84.92
P值 ? 0.00 0.00 0.00 0.00
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