运动激活自噬对小鼠骨骼肌抗氧化防御功能的影响

摘要

目的:探讨一次性力竭运动能否激活骨骼肌自噬及运动性自噬对骨骼肌抗氧化防御功能的影响,为运动性自噬与骨骼肌抗氧化防御功能之间的发生机制提供实验依据.方法:30只健康雄性4周龄C57BL/6小鼠随机分为安静对照组(n=6)和运动组(n=24),运动组进行一次性力竭跑台运动,分别于运动完成后的0 h(n=6)、6h(n=6)、12 h(n=6)和24h(n=6)取小鼠两侧腓肠肌,采用黄嘌呤氧化酶法测定腓肠肌锰超氧化物歧化酶(MnSOD)、总超氧化物歧化酶(T-SOD)和铜锌超氧化物歧化酶(CuznSOD)的活性;比色法测定腓肠肌总抗氧化能力(T-AOC)的含量;荧光定量PCR和Western Blot分别检测腓肠肌自噬相关因子Beclin1、P62和B淋巴细胞瘤-2(Bc12)的mRNA和蛋白表达.结果:与安静对照组相比,(1)小鼠腓肠肌Beclin1 mRNA表达在运动后0h、6h均极显著升高(P＜0.01),P62 mRNA表达在运动后24 h极显著升高(P＜0.01);P62蛋白表达在运动后0h、12 h和24h均显著下降(P＜0.05或P＜0.01),Bcl2蛋白表达在运动后Oh、6h和12 h均显著升高(P＜0.05或P＜0.01);(2)MnSOD和CuznSOD活性在0h均显著升高(P＜0.05),T-SOD活性在6h极显著下降(P＜0.01),T-AOC含量在运动后6h、12 h、24 h均显著升高(P＜0.05或P＜0.01);P62蛋白表达与T-AOC含量呈负相关.结论:一次性力竭跑台运动可在一定程度上激活骨骼肌自噬相关因子Beclin1 mRNA和P62蛋白的表达,可适度提高自噬;且自噬相关因子P62蛋白表达与骨骼肌抗氧化防御功能具有相关性,这可能是运动增加骨骼肌抗氧化防御功能的分子机理之一.%Objective To explore whether a single bout of exhaustive exercise can activate autophagy in mice skeletal muscles and to investigate the effect and possible mechanisms of exercise-induced autophagy on antioxidant defense function in vivo.Methods Thirty healthy 4-week-old male C57BL/6 mice were randomly divided into a control group(n=6) and an exercise group(n=24).The latter was subjected to a single bout of exhaustive treadmill exercise.Gastrocnemius muscles of 6 mice on both sides were then isolated right after,as well as 6h,12h and 24h after the running.The manganese super oxide dismutase(MnSOD),total superoxide dismutase(T-SOD) and Copper-Zinc superoxide dismutase (CuznSOD) activities were determined using the xanthine oxidase method and the content of the total anti-oxygen capability(T-AOC) using the colorimetric method.The expression of Beclin1,P62 and Bcl2 mRNAs,as well as their proteins were determined using the fluorescent quantitative PCR and Western blotting.Results The expression of Beclin1 mRNA in gastrocnemius muscles of the exercise group increased more significantly than that of the control group right after the exercise,as well as 6 hours later(P＜0.01).P62 mRNA expression also increased significantly(P＜0.01) 24h after the exercise.In contrast,the expression of P62 protein at 0h,12h and 24h after the exercise decreased more significantly(P＜0.05 or P＜0.01) than that of the control group.Similarly,the expression of Bcl2 protein at 0h,6h and 12h increased significantly(P＜0.05 or P＜0.01).The MnSOD and CuznSOD activity increased significantly right after the exercise(P＜0.05).The T-SOD activity decreased significantly(P＜ 0.01) 6h after the exercise.T-AOC content increased significantly 6h,12h and 24h after the exercise (P＜0.05 or P＜0.01).The P62 protein expression was negatively correlated with T-AOC content.Conclusion The single bout of exhaustive treadmill exercise can activate the expression of such autophagyrelated factors as Beclin1 mRNA and P62 protein and increase the autophagy to a certain extent to maintain antioxidant defensive function of the skeletal muscle.