热休克蛋白70抑制c—Jun氨基末端激酶通路对H2O2诱导心肌细胞凋亡的保护作用

HSP70 inhibiting c-Jun amino-terminus kinase signal transduction and its protective effect on cardiomyocyte apoptosis induced by H2O2

目的 观察热休克蛋白70（HSP70）对H2O2诱导乳鼠心肌细胞凋亡的保护作用及其机制。方法培养大鼠心肌细胞，随机分为5组：对照组、H2O2组、热休克组、c—Jun氨基末端激酶（JNK）抑制剂组、热休克＋JNK抑制剂组。生化法测定各组细胞培养液中乳酸脱氢酶（LDH）、超氧化物歧化酶（SOD）、肌酸激酶（CK）活性和丙二醛（MDA）水平．流式细胞仪分析心肌细胞凋亡，噻唑蓝（MTT）法测定心肌细胞相对活力，Western blot法检测JNK的表达。结果 H2O2组LDH、MDA水平和CK活性明显高于其他组（P〈0．01），而SOD活性则明显低于其他组（P〈0．01）。细胞凋亡率H2O2组明显高于其他各组（P〈0．01）。细胞活力H20：组明显低于对照组（P〈0．01）．而热休克组、JNK抑制剂组、热休克＋JNK抑制剂组的细胞活力明显高于H2O2组（P〈0．01）。JNK只在H2O2组有表达。结论 HSP70对H2O2诱导的心肌细胞凋亡具有保护作用，其机制可能是HSP70大量表达后抑制了JNK信号的转导。

Objective To discuss the relationship between heat shock protein 70（HSP70） and neonatal rat cardiomyocyte apoptosis induced by H2O2. Methods Cardiomyocytes were cultured in vitro and divided into live groups： control group, H2O2 injury group, heat shock group, JNK inhibitor group and heat shock ＋ JNK inhibitor group. Apoptosis of cardiomyocytes was determined by flow cytometry（FCM）. The activities of SOD, LDH, CK and the content of MDA were analyzed in the culture medium of cardiomyocytes. The relative activity of cardiomyocytes was further determined by the way of MTT and the expression of phospho-JNK by Western blot. Results The activities of LDH, MDA and CK in H2O2 group were significantly higher than other groups, JNK inhibitor group and heat shock ＋ JNK inhibitor group（all P 〈 0.01 ）. On the contrary, the activities of SOD in H2O2 injury group was significantly lower than that in other groups（P 〈 0.01 ）. The cell apoptotic rate in H2O2 injury group was significantly higher than other groups （P 〈 0.01 ）. Cell viability in H202 group was significantly lower than other groups（P 〈 0.01 ）. JNK was expressed only in H2O2 injury group. Conclusions Both HSP70 and JNK inhibitor protect cardiomyocytes from apoptosis. The possible mechanisms are as follows： up-regulated HSP70 suppresses JNK signal transduction pathway. Our results can demonstrate the anti-apoptosis function of HSP70.