AMPK信号通路在七氟醚预处理增强大鼠心肌细胞缺氧复氧时抗氧化能力中的作用

摘要

目的 探讨单磷酸腺苷激活蛋白激酶(AMPK)信号通路在七氟醚预处理增强大鼠心肌细胞缺氧复氧时抗氧化能力中的作用.方法 采用随机数字表法,将大鼠胚胎心肌细胞(H9C2细胞)分为3组(n=12):对照组(C组)、缺氧复氧损伤组(H/R组)和七氟醚预处理(SP组);采用随机数字表法,将AMPK基因敲除(AMPK siRNA)的H9C2细胞分为3组(n=12):对照组(A-C组)、缺氧复氧损伤组(A-H/R组)和七氟醚预处理(A-SP组).H9C2细胞置于95％N2+ 5％CO2培养箱孵育14 h,之后置于95％O2+ 5％CO2培养箱中孵育3h,建立缺氧复氧模型.SP组H9C2细胞培养液中加入七氟醚,终浓度为2.0 mmol/L,每次孵育15 min,共3次,间隔15 min进行七氟醚预处理.七氟醚预处理结束后15min建立缺氧复氧模型.复氧3h时,采用lucigenin增强化学发光法和DHE染色测定超氧阴离子水平,并测定LDH释放量和caspase-3活性.结果 与C组比较,H/R组和SP组H9C2细胞LDH释放量增多,caspase-3活性增强,超氧阴离子水平升高(P＜0.01);与H/R组比较,SP组H9C2细胞LDH释放量减少,caspase-3活性减弱,超氧阴离子水平降低,A-H/R组H9C2细胞LDH释放量增多,caspase-3活性增强,超氧阴离子水平升高(P＜0.01);与SP组比较,A-SP组H9C2细胞LDH释放量增多,caspase-3活性增强,超氧阴离子水平升高(P＜0.01);与A-H/R组比较,A-SP组H9C2细胞LDH释放量减少,caspase-3活性减弱,超氧阴离子水平降低(P＜0.01).结论 AMPK信号通路参与了七氟醚预处理增强大鼠心肌细胞缺氧复氧时的抗氧化能力,但并未起主要作用.%Objective To investigate the role of 5'-AMP-activated protein (AMPK) signaling pathway in sevoflurane preconditioning-induced enhancement of antioxidant capacity during hypoxia-reoxygenation (H/R) in the cardiomyocytes of the rats.Methods The rat embryonic cardiomyocytes (H9C2 cells) were randomly divided into 3 groups (n =12 each) using a random number table:control group (group C),H/R group,and sevoflurane preconditioning group (group SP).AMPK gene knockout (AMPK siRNA) H9C2 cells were randomly divided into 3 groups (n =12 each) using a random number table:control group (group A-C),A-H/R group,and sevoflurane preconditioning group (group A-SP).The cells were exposed to 95％ N2 + 5％ CO2 in an incubator for 14 h followed by reoxygenation with 95％ O2 + 5％ CO2 for 3 h.In SP group,the cells were preconditioned with 15 min sevoflurane (final concentration 2.0 mmol/L) three times at 15 min interval and H/R model was established at 15 min after the end of preconditioning.At 3 h of reoxygenation,the level of superoxide anion was detected using lucigenin-enhanced chemiluminescence and DHE staining,and the release of dehydrogenase (LDH) and caspase3 activity were measured.Results Compared with group C,the release of LDH and level of superoxide anion were significantly increased,and caspase-3 activity was enhanced in group H/R.Compared with group H/R,the release of LDH and level of superoxide anion were significantly decreased,and caspase-3 activity was weakened in group SP,and the release of LDH and level of superoxide anion were significantly increased,and caspase-3 activity was enhanced in group A-H/R.Compared with group SP,the release of LDH and level of superoxide anion were significantly increased,and caspase-3 activity was enhanced in group A-SP.Compared with group A-H/R,the release of LDH and level of superoxide anion were significantly decreased,and caspase-3 activity was weakened in group A-SP.Conclusion AMPK signaling pathway is involved in the mechanism by which sevoflurane preconditioning enhances the antioxidant capacity during H/R in the cardiomyocytes of the rats,but it does not play a major role.