热休克蛋白90在硫化氢对抗化学性缺氧引起的心肌细胞损伤中的作用

Role of Heat-Shock Protein90 in Hydrogen Sulfide-induced Protection Against Cardiomyocytes Injuries Elicited by Chemical Hypoxia

目的探讨热休克蛋白90在硫化氢保护H9C2心肌细胞对抗氯化钴诱导的损伤中的作用。方法应用不同浓度的氯化钴处理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。硫氢化钠(硫化氢的供体)在氯化钴处理H9C2心肌细胞前30min加入培养基中,作为预处理。应用细胞计数试剂盒8检测细胞存活率;Hoechst33258染色荧光显微镜照相术检测凋亡心肌细胞的形态学改变;免疫印迹法检测热休克蛋白90的表达。结果在600～1000μmol/L浓度范围内,氯化钴处理H9C2心肌细胞24h,呈剂量依赖性地抑制细胞存活率。在应用不同浓度氯化钴处理H9C2心肌细胞前30min,应用400μmol/L硫氢化钠可分别显著地抑制600、800、1000μmol/L氯化钴对心肌细胞的损伤作用,使细胞存活率明显升高。400μmol/L硫氢化钠可促进H9C2心肌细胞的HSP90表达,在硫氢化钠作用6～9h时,HSP90表达最多,作用18h时表达恢复到基础水平。2μmol/L热休克蛋白90抑制剂17-丙烯胺基-17-去甲氧基格尔德霉素自身对H9C2心肌细胞无损伤作用,但是能加重氯化钴对心肌细胞的损伤作用,并能明显地阻断硫化氢抑制氯化钴对心肌细胞的损伤作用,使H9C2心肌细胞存活率降低,凋亡细胞数量增多。结论硫化氢能保护心肌细胞对抗氯化钴诱导的损伤作用,并能上调热休克蛋白90表达。热休克蛋白90介导硫化氢的心肌细胞保护作用。

Aim To explore the role of heat-shock protein 90 in protective effect of hydrogen sulfide against H9C2 cardiac ceils injuries induced by cobalt chloride. Methods H9C2 cells were exposed to cobalt chloride at different doses to set up the chemical hypoxia-induced cardiomyocyte injury model. Sodium hydrosulfide （a hydrogen sulfide donor） was added into cell medium for 30 min before cobalt chloride treatment. Cell viability was tested by using cell counter kit-8. Morphological changes in apoptotic cardiomyocytes were detected by Hoechst 33258 staining and photofluo-rography. The expression of heat-shock protein 90 was evaluated by Western blot. Results H9C2 cell viability was inhibited by cobalt chloride at the concentrations from 600 to 1000μmol/L for 24 h in a dose-dependent manner. Pre-treatment with 400μmol/L sodium hydrosulfide 30 rain before exposure to cobalt chloride significantly blocked the cardio-myocyte cell injuries induced by cobalt chloride at 600,800 and 1000μmol/L respectively, leading to an increase in cell viability. Heat-shock protein 90 expression was upregulated after treatment with 400μmol/L sodium hydrosulfide for 30 min, peaking at 6 h to 9 h, returning to the basal level at 18 h. 17-allylamino-17-demethoxygeldanamyein （2μmol/L）, an inhibitor of beat-shock protein 90, not only enhanced H9C2 cells injury induced by cobalt chloride, but also obviously blocked the inhibition of hydrogen sulfide on cobalt chloride-induced cardiomyoeyte damage, reducing viability of H9C2 cells, increasing number of apoptotic cells, which didn＇ t damage H9C2 cells alone. Conclusion Hydrogen sulfide can protect H9C2 cells against cobalt chloride-induced injury and upregulate expression of heat-shock protein 90. 17-allyl- amino-17-demetbexygeldanamycin not only increases cobalt chloride-induced H9C2 cell injury, but also significantly inhibits the cardioprotection of hydrogen sulfide, suggesting that heat-shock protein 90 may mediate the cardioprotoctive effect afforded by hydrogen sulfide.