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目的:探讨热休克蛋白90(HSP90)在硫化氢(H2S)对抗化学性低氧模拟剂氯化钴(CoCl2)诱导H9c2心肌细胞氧化应激损伤中的作用。方法:应用CoCl2处理H9c2心肌细胞,建立化学性缺氧损伤心肌细胞的实验模型。在CoCl2处理H9c2心肌细胞前30min,把硫氢化钠(NaHS,H2S的供体)加入培养基中,作为预处理。应用高效液相色谱法(HPLC)检测细胞内ATP的含量;罗丹明123(Rh123)染色荧光显微镜照相检测线粒体膜电位(MMP);超氧化物歧化酶(SOD)检测试剂盒检测SOD活性;免疫印迹法(Western blotting)检测血红素氧合酶-1(HO-1)的表达。结果:600μmol/LCoCl2明显地降低H9c2心肌细胞内SOD活性、ATP水平及MMP,并增加HO-1表达。400μmol/LNaHS预处理可显著地抑制CoCl2诱导的细胞毒性及氧化应激反应,使SOD活性、ATP水平及MMP提高,HO-1表达减少。热休克蛋白90抑制剂17-丙烯胺基-17去甲氧基格尔德霉素(17AAG)能明显地阻断H2S对CoCl2诱导的细胞毒性和氧化应激反应的抑制作用,使细胞内ATP水平及MMP降低,HO-1表达增多,但对SOD活性的影响不明显。结论:热休克蛋白90可通过抑制化学性缺氧引起的氧化应激反应来介导H2S的心肌保护作用。
AIM: To investigate the role of heat shock protein 90 (HSP90) in the oxidative stress-induced injury of H9c2 cardiomyocytes induced by hydrogen sulfide (H2S), a chemical hypoxia mimics coCl2. Methods: H9c2 cardiomyocytes were treated with cobalt chloride (CoCl2) to establish a model of hypoxia-injured myocardial cells. Thirty minutes prior to CoCl2 treatment of H9c2 cardiomyocytes, sodium hydrosulfide (NaHS, a donor of H2S) was added to the culture medium as a pretreatment. The content of intracellular ATP was detected by HPLC. The mitochondrial membrane potential (MMP) was measured by Rh123 staining fluorescence microscope. The SOD activity was detected by superoxide dismutase (SOD) detection kit. Western blotting was used to detect the expression of heme oxygenase-1 (HO-1). Results: 600μmol / L CoCl2 significantly decreased SOD activity, ATP level and MMP in H9c2 cardiomyocytes and increased HO-1 expression. Pretreatment with 400μmol / L NaHS significantly inhibited the cytotoxicity and oxidative stress induced by CoCl2, and increased the activity of SOD, the level of ATP and the level of MMP, but decreased the expression of HO-1. The heat shock protein 90 inhibitor 17-acrylamido-17-demethoxygeldanamycin (17AAG) significantly blocked the inhibitory effect of H2S on CoCl2-induced cytotoxicity and oxidative stress, allowing intracellular ATP Level and MMP decreased, the expression of HO-1 increased, but the effect on SOD activity was not obvious. Conclusion: Hsp90 can mediate the myocardial protective effect of H2S by inhibiting oxidative stress induced by chemical hypoxia.