热休克蛋白90对大鼠缺氧心肌细胞丝氨酸苏氨酸蛋白激酶表达的影响

Influence of heat shock protein 90 on protein serine threonine kinases expression in hypoxic cadiomyocytes

目的探讨内源性热休克蛋白90(HSP 90)在缺氧心肌细胞丝氨酸苏氨酸蛋白激酶(AKT)相关信号通路中的作用。方法建立新生Wistar大鼠心肌细胞缺氧模型,将细胞分为正常组、缺氧组、加入HSP 90特异性阻断剂格尔德霉素后再缺氧组(格尔德霉素+缺氧组)。于缺氧后1、3、6、12、24、48 h用噻唑蓝法检测心肌细胞的活力;缺氧24 h,原位缺口末端标记法检测心肌细胞凋亡指数(AI);缺氧1、3、6、12、24 h,蛋白质印迹法检测大鼠心肌细胞中内源性HSP 90及AKT表达水平。结果(1)缺氧24、48 h,缺氧组、格尔德霉素+缺氧组细胞活力均较正常组明显下降(P<0.05);格尔德霉素+缺氧组细胞活力缺氧12 h即开始明显下降,缺氧48 h时明显低于缺氧组(P<0.05)。(2)缺氧24 h,缺氧组细胞AI为(10.7±1.2)%,明显高于正常组[(1.9±0.3)%,P<0.05];格尔德霉素+缺氧组细胞AI为(26.3±5.3)%,明显高于缺氧组(P<0.01)。(3)缺氧12 h,缺氧组心肌细胞内源性HSP 90及AKT表达水平高于正常组与格尔德霉素+缺氧组;缺氧24 h,缺氧组有所下降,格尔德霉素+缺氧组则下降更明显。结论内源性HSP 90对维持心肌细胞的活力有重要作用,缺氧心肌细胞AKT表达水平可受内源性HSP 90表达水平的影响。

Objective To explore the effect of endogenous heat shock protein 90 （HsP90） on the AKT signaling pathway of hypoxic cardiomyocytes. Methods The hypoxia model of neonatal rat cadiomyocyte was established. The cells were randomly divided into normal control, hypoxia, Geldanamycin （ GA, with hypoxia after Geldanamycin treatment） groups. The myocardial cell activity and the expression of endogenous HSP90 and AKT were determined with MTT and Western blotting, respectively at 1, 3, 6, 12, 24 and 48 post-hypoxia hours （ PHH）. The apoptotic index （AI） of cadiomyocytes were determined with TUNEL method at 24 PHH. Results （ 1 ） At 24 and 48 PHH, the activity of cardiomyocytes in hypoxia group and GA group were evidently lower than that in control group（ P 〈0. 05）. The activity of cardiomyocytesin in GA group began to decrease at 12 PHH, and it was obviously lower than that in hypoxia group at 48 PHH （ P 〈 0. 05）. （2）At 24 PHH,the AI in hypoxia group （10.7 ± 1.2） % was obviously higher than that in normal control group[ （ 1.9 ± 0.3 ） % , P 〈 0.05 ] , while it was obviously lower than that in GA group [ （ 26.3± 5.3） % , P 〈0.01 ]. （3）The expression of endogenous HSP90 and AKT in hypoxia and GA groups were markedly increased compared with that of normal controls at 12 PHH, and it decreased at 24 PHH in hypoxia and GA groups, especially in the latter . Conclusion Endogenous HSP90 plays important roles in maintaining the cardiomyocyte activity, and its level might affect the expression of AKT.