AMPK调控不同强度电刺激引起的C2C12肌管线粒体氧化应激  

AMPK regulates mitochondrial oxidative stress in C2C12 myotubes induced by electrical stimulations of different intensities

作　　者：董合玲 吴洪渊 唐钰 黄银伟 林锐章 赵军 徐晓阳[3] DONG Heling;WU Hongyuan;TANG Yu;HUANG Yinwei;LIN Ruizhang;ZHAO Jun;XU Xiaoyang(College of Sports Science, Jinan University, Guangzhou 510632, China;Department of Health Management, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;College of Sports Science and Physical Education, South China Normal University, Guangzhou 510631, China)

机构地区：[1]暨南大学体育学院,广东广州510632 [2]南方医科大学南方医院健康管理科,广东广州510515 [3]华南师范大学体育科学学院,广东广州510631

出　　处：《南方医科大学学报》2018年第6期742-747,共6页Journal of Southern Medical University

基　　金：广东省自然科学基金(2017A030310178);中央高校教育教学改革专项(82617058)

摘　　要：目的研究不同时间电刺激模拟不同强度运动对C2C12肌管线粒体氧化应激水平的影响,并进一步探讨5′-磷酸腺苷活化蛋白激酶(AMPK)信号通路在不同运动强度过程中所发挥的作用。方法 C2C12肌管分化7 d后给予1次电刺激,刺激强度为15 V,30 ms,3 Hz,刺激时间分别为0、60、120和180 min。实验共4组,对照组(Con);电刺激60 min组(E60),120 min(E120)和180 min(E180)。细胞电刺激处理后于倒置显微镜观察肌管形态;试剂盒法检测脂质过氧化产物(MDA)和活性氧自由基(ROS);流式细胞技术检测线粒体ROS和膜电位;免疫蛋白印迹法检测PGC1、AMPK-Ser485、AMPK-Thr172、AMPK的蛋白表达。结果不同时间的电刺激后,C2C12肌管的形态无显著差异;电刺激60 min,MDA、ROS、AMPK-Ser485和AMPK-Thr172显著上升(P<0.05);电刺激120 min和180 min,MDA、ROS、线粒体ROS、AMPK-Ser485、PGC1显著上升(P<0.05),线粒体膜电位显著下降(P<0.05)。结论电刺激导致肌管氧化应激,且随时间延长,线粒体氧化应激程度越强烈,线粒体损伤越严重;AMPKThr172调控中长时间电刺激引起的氧化应激,而AMPK-Ser485及PGC1调控超长时间电刺激引起的氧化应激。Objective To study effect of electrical stimulations of different intensities on mitochondrial oxidative stress in C2 C12 myotubes and explore the molecular mechanisms. Methods After 7 days of differentiation, C2 C12 myotubes were subjected to electrical stimulations（15 V, 3 Hz, 30 ms） for 60, 120, or 180 min, and the morphological changes of muscular tubes were observed under inverted microscope. The levels of MDA and SOD activity of the cells were detected, and flow cytometry was used to detect mitochondrial reactive oxygen species（ROS） and membrane potential. Western blotting was used to detect the expression of PGC1, AMPK-Ser485, AMPK-Thr172, and AMPK in the cells. Results No significant changes occurred in the morphology of C2 C12 myotubes in response to electrical stimulations. Electrical stimulation for 60 min resulted in significantly increased levels of MDA, AMPK-Ser485 and AMPK-Thr172 in the cells（P〈0.05）; simulations of the cells for 120 and 180 min caused significantly increased MDA, ROS, mitochondrial ROS, AMPK-Ser485 and PGC1 along with marked reduction of mitochondrial membrane potential（P〈0.05）. Conclusion Electrical stimulation significantly activates oxidative stress, and a longer stimulation time causes stronger mitochondrial oxidation. AMPK-Thr172 regulates oxidative stress induced by stimulations for a moderate time length, while AMPK-Ser485 and PGC1 function to modulate oxidative stress following prolonged stimulations.

关 键 词：电刺激 线粒体 氧化应激 5′-磷酸腺苷活化蛋白激酶 

分 类 号：R363[医药卫生—病理学]