针刺对运动性骨骼肌损伤大鼠骨骼肌线粒体自噬的影响

摘要

目的:通过观察针刺对大负荷运动大鼠骨骼肌线粒体自噬相关蛋白表达的影响,探讨针刺在运动性骨骼肌损伤修复中的作用及其机制.方法:将128只成年雄性Sprague-Dawley大鼠随机分为4组:空白对照(control,C;n=8)组、单纯运动(exercise,E;n=40)组、单纯针刺(acupuncture,A;n=40)组和运动针刺(exercise and acupuncture,EA;n=40)组.其中,E和EA组进行1次下坡跑运动,A组和EA组(运动后即刻)施加针刺处理.后3组根据干预后不同时相又分为0h、12h、24h、48h和72h亚组(n=8),分别于对应时点分离比目鱼肌进行检测,使用透射电子显微镜观察骨骼肌线粒体超微结构变化;采用ELISA法检测比目鱼肌线粒体定量酶柠檬酸合成酶(CS)的含量变化;应用Western blot法检测骨骼肌PTEN诱导假定激酶1(PINK1)、parkin和微管相关蛋白1轻链3(LC3)的蛋白表达变化.结果:1次大负荷运动后大鼠比目鱼肌线粒体出现明显肿胀和肌膜下积聚等超微结构异常变化,伴有大量自噬体形成;同时CS的含量明显减少(P<0.05);线粒体自噬蛋白PINK1、parkin和LC3均出现一过性的表达升高(P<0.05).运动后针刺明显改善了线粒体超微结构的异常变化,减少自噬溶酶体的出现,同时抑制CS的含量减少,下调PINK1、parkin和LC3在线粒体上的表达(P<0.05).结论:1次大负荷运动后骨骼肌线粒体结构和数量受损,通过激活PINK1/parkin途径诱发线粒体自噬的过度发生.大负荷运动后针刺可以缓解骨骼肌线粒体的损伤,其作用机制可能是通过下调线粒体外膜蛋白PINK1表达,抑制其对下游胞浆蛋白par-kin的招募,进而影响LC3与线粒体的结合以抑制线粒体自噬过度激活.%AIM:The effect of acupuncture on mitophagy-related protein expression in skeletal muscle of rats after heavy-load exercise was investigated to explore the role of acupuncture in the repairment of exercise-induced skeletal muscle damage. METHODS:Male adult Sprague-Dawley rats (n=128) were randomly divided into 4 groups:control (C,n=8) group, exercise (E, n=40) group, acupuncture (A, n=40) group, and exercise and acupuncture (EA, n=40) group. The rats in E group and EA group performed an eccentric exercise,and the rats in A group and EA group immediately after exercise received acupuncture treatment. The rats in the latter 3 groups were further divided into 0 h,12 h,24 h,48 h and 72 h sub-groups(n=8),and soleus muscle was collected at each time point. The transmission electron microscopy was used to observe the ultrastructural changes of the mitochondria in skeletal muscle. The content of citrate synthase (CS) was measured by ELISA. The protein expression of skeletal muscle PTEN-induced putative kinase 1 (PINK1),parkin and microtubule-associated protein 1 light chain 3 (LC3) was determined by Western blot. RESULTS:After the heavy-load exercise,the mitochondria swelled and accumulated under cell membrane. The number of mitophago-somes was increased,and the content of CS was significantly decreased(P<0.05). The expression of PINK1,parkin and LC3 was significantly elevated (P<0.05). However,the acupuncture intervention after exercise promoted the recovery of mitochondrial ultrastructure, attenuated mitophagolysosome formation, maintained CS content and down-regulated the ex-pression of PINK1,parkin and LC3 (P<0.05). CONCLUSION:Heavy-load exercise causes the damages of mitochon-drial structure and function in the skeletal muscle and activates PINK1/parkin pathway to induce excessive occurrence of mitophagy. Acupuncture intervention after exercise is able to alleviate the damage of mitochondria in the skeletal muscle through decreasing the expression of mitochondrial outer membrane protein PINK1,reducing the recruitment of downstream cytoplasmic protein parkin,thereby affecting the combination of LC3 and mitochondria to inhibit the overactivation of mito-phagy.