一次性力竭运动致大鼠骨骼肌氧化应激的机制

Mechanism of oxidative stress in skeletal muscle of rats induced by acute exhaustive exercise

目的:观察一次性力竭运动对大鼠骨骼肌氧化应激相关酶表达的影响。方法:雄性SD大鼠40只,分为4组(n=10),分别为对照组(C组)、力竭运动组(E组)、运动+PKC抑制剂组(EC组)、运动+NOX抑制剂组(EA组)。三组运动大鼠进行3 d的跑台适应性运动(5 m/min,1次/日,无坡度),然后休息1 d;EC组于运动前1 d和运动前1 h注射PKC抑制剂白屈菜红碱(5 mg/kg),EA组同期注射NADPH氧化酶抑制剂Apocynin(10 mg/kg),C组和E组注射同等剂量生理盐水;三组运动大鼠进行一次性跑台力竭运动,力竭后取大鼠的跖肌,DCF荧光探针检测活性氧(ROS),Western blot分析NOX2、NOX4、3-NT,免疫沉淀分析PKC、NOX2、NOX4。结果:与C组相比,E组的ROS水平、NOX2和NOX4蛋白表达、PKC-NOX2和PKC-NOX4复合物水平、3-NT生成均显著增加(P<0.01,P<0.05),EC组、EA组ROS无显著差异(P>0.05),EC组NOX4蛋白表达显著增加(P<0.05);与E组相比,EC组和EA组的ROS水平、NOX2和NOX4蛋白表达、PKC-NOX2和PKC-NOX4复合物水平、3-NT生成均显著降低(P<0.01,P<0.05)。结论:力竭运动诱导骨骼肌NOX2、NOX4蛋白表达增加,PKC通过调控NOX2介导ROS的生成。

Objective:To observe the effects of acute exhaustive exercise on the expressions of oxidative stress related enzymes in skeletal muscle of rats.Methods:Forty male SD rats were divided into 4 groups,10 rats in each group,which were the control group(C group),exhausted exercise group(E group),exercise+PKC inhibitor group(EC group),exercise+NOX inhibitor group(EA group).Three groups of exercise rats were familiarized with treadmill running for 3 days(5 m/min,once/d,no incline),then rested for one day.EC group was injected with PKC inhibitor chelerythrine(5 mg/kg)one day before and one hour before exercise,EA group was injected with NADPH oxidase inhibitor apocynin(10 mg/kg)at the same time,group C and group E were injected with the same dose of normal saline.Three groups of exercise rats were subjected to a one-time treadmill exhaustion exercise,and the plantaris were taken after exhaustion.Reactive oxygen species(ROS)were detected by DCF fluorescent probe,NOX2,NOX4,3-NT were analyzed by Western blot,and PKC,NOX2,NOX4 were analyzed by immunoprecipitation.Results:Compared with group C,ROS level,NOX2 and NOX4 protein expressions,PKC-NOX2 and PKC-NOX4 complex levels,and 3-NT production in group E were significantly increased(P<0.01,P<0.05),and ROS level was no significant difference in group EC and group EA(P>0.05),and NOX4 protein expression in group EC was significantly increased(P<0.05).Compared with group E,ROS level,NOX2 and NOX4 protein expressions,PKC-NOX2 and PKC-NOX4 complex levels,3-NT production were decreased significantly(P<0.01,P<0.05).Conclusion:Exhausted exercise induces increased expressions of NOX2 and NOX4 proteins in skeletal muscle,and PKC mediates the production of ROS by regulating NOX2.