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目的 :探讨热休克预处理 (heatshockpretreatment,HS)对过氧化氢 (hydrogenperoxide ,H2 O2 )所致心肌细胞凋亡及促凋亡分子线粒体释放的第二种Caspase激活物 (thesecondmitochondria derivedactivatorofcaspases ,Smac)从线粒体释放的影响。方法 :①采用H2 O2 (0 .5mmol/L)导致心肌细胞凋亡 ;②采用热休克预处理 (4 2℃ ,1h ,恢复 12h)诱导热休克蛋白表达 ;③采用Hoechst荧光染色 ,观察细胞凋亡发生并计算凋亡核百分率 ;④采用cas pase活性定量检测及Western blotting观察caspase 3,9的活化 ;采用免疫荧光及Western blotting检测细胞成分分离后Smac从线粒体的释放。结果 :①H2 O2 处理 2h ,Smac从心肌细胞线粒体释放入胞浆 ;处理 4hcaspase 3,9活性升高 ,12h达高峰 ;处理 2 4h心肌细胞明显出现凋亡 ,凋亡核百分率明显升高 ;②热休克预处理可诱导心肌细胞中HSP90 ,HSP70及αB 晶状体蛋白的表达增高 ;抑制Smac释放、caspase 3,9的活化及细胞凋亡的发生。结论 :线粒体信号通路在H2 O2 所致的心肌细胞凋亡中发挥重要作用 ;热休克预处理通过抑制上述通路而减轻H2 O2 所致的细胞凋亡 ,其机制与其诱导热休克蛋白表达、阻断Smac从线粒体向胞浆释放、抑制caspase 3,9的活化有关。
OBJECTIVE: To investigate the effect of heat shock pretreatment (HS) on the apoptosis of cardiomyocytes induced by hydrogen peroxide (H2 O2) and the expression of thesecondmitochondria derived activator of caspases (Smac) from the mitochondria The impact of release. Methods ①H2 O2 (0.5 mmol / L) induced apoptosis of cardiomyocytes; ② Heat shock preconditioning (4 2 ℃, 1 h, recovery 12h) induced heat shock protein expression; ③ Hoechst staining was used to observe the changes of cell apoptosis The percentage of apoptotic nuclei was calculated and the percentage of apoptotic nuclei was calculated.4. The caspase 3 and 9 were detected by quantitative detection of caspase activity and Western blotting. The release of Smac from mitochondria was detected by immunofluorescence and Western blotting. Results: ①H2 O2 treatment, Smac released into the cytoplasm from the mitochondria of cardiomyocytes, the activity of 4 h caspase 3 and 9 increased and reached the peak at 12 h. The apoptotic cells were significantly increased after 24 h treatment, and the percentage of apoptotic nuclei increased significantly. Shock preconditioning induced the expression of HSP90, HSP70 and αB crystallin in cardiomyocytes, and inhibited the release of Smac, the activation of caspase 3 and 9 and the occurrence of apoptosis. Conclusion: Mitochondrial signal transduction pathway plays an important role in the apoptosis of cardiomyocytes induced by H2O2. Heat shock pretreatment attenuates the H2O2-induced apoptosis by inhibiting these pathways, and its mechanism is related to the induction of heat shock protein expression and blockade Smac release from the mitochondria to the cytoplasm, inhibiting the activation of caspase 3,9.