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目的探讨热休克蛋白90(heat shock protein 90,HSP90)在对抗氯化钴(cobalt chloride,CoCl2)诱导H9C2心肌细胞损伤中的作用。方法应用不同浓度的CoCl2处理H9C2心肌细胞,建立化学性缺氧诱导心肌细胞损伤的实验模型。应用CCK-8比色法检测细胞存活率;Western-blot法检测HSP90的表达;双氯荧光素染色荧光显微镜照相检测细胞内活性氧(reactive oxygen species,ROS)水平;超氧化物歧化酶(superoxidedismutase,SOD)检测试剂盒分析SOD活性抑制率。结果在400～1000μmol/L浓度范围内,CoCl2处理H9C2心肌细胞36 h,呈剂量依赖性地抑制细胞存活率。在0.5～36 h时间范围内,600μmol/L CoCl2呈时间依赖性地促进H9C2心肌细胞HSP90的表达。2～16μmol/L HSP90抑制剂17-丙烯胺基-17-去甲氧基格尔德霉素(17-allylamino-17demethoxygeldanamycin,17AAG)呈剂量依赖性地加重600μmol/L CoCl2对H9C2细胞存活率的抑制。2μmol/L 17AAG本身不损伤H9C2心肌细胞,但明显地加强CoCl2增加H9C2心肌细胞内ROS生成的作用,并增加CoCl2对SOD活性的抑制。结论HSP90表达上调可能是H9C2心肌细胞对抗化学性缺氧的内在防御机制之一。
Objective To investigate the effect of heat shock protein 90 (HSP90) on the injury of H9C2 cardiomyocytes induced by cobalt chloride (CoCl2). Methods Co-treatment of H9C2 cardiomyocytes with different concentrations of CoCl2 established an experimental model of chemical hypoxia-induced cardiomyocyte injury. The cell viability was detected by CCK-8 colorimetric assay, the expression of HSP90 was detected by Western-blot assay, the level of reactive oxygen species (ROS) was measured by fluorescence microscopy with dicrofluorescein staining, the superoxide dismutase , SOD) detection kit analysis of SOD activity inhibition rate. Results H9C2 cardiomyocytes were treated with CoCl2 for 36 h in a concentration range of 400 ~ 1000 μmol / L, and the cell survival rate was inhibited in a dose - dependent manner. In the time range of 0.5 ~ 36 h, 600 μmol / L CoCl 2 promoted the expression of HSP90 in H9C2 cardiomyocytes in a time-dependent manner. 2 ~ 16μmol / L HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) in a dose-dependent manner increased the survival rate of H9C2 cells with 600μmol / L CoCl2 inhibition. 2μmol / L 17AAG itself did not damage H9C2 cardiomyocytes, but significantly enhanced the role of CoCl2 in increasing ROS production in H9C2 cardiomyocytes and increased the inhibition of SOD activity by CoCl2. Conclusion The up-regulation of HSP90 may be one of the intrinsic defense mechanisms of H9C2 cardiomyocytes against chemical hypoxia.