【作者】 崔迪； 王海燕； 张喆； 张坦； 孙易； 丁树哲；

【Author】 CUI Di;WANG Haiyan;ZHANG Zhe;ZHANG Tan;SUN Yi;DING Shuzhe;The Key Laboratory of Adolescent Health Assessment and Exercise Intervention, Ministry of Education of China;School of PE and Health, East China Normal University;

【机构】 青少年健康评价与运动干预教育部重点实验室； 华东师范大学体育与健康学院；

【摘要】 研究目的探讨自主跑轮运动对生长期小鼠骨骼肌细胞自噬、线粒体质量(包括线粒体生物发生及线粒体自噬相关基因表达)的影响,旨在揭示自主运动介导的健康促进作用的分子机制,并为代谢性疾病的运动防治提供理论及数据支持。方法:20只4周龄SPF(Specific Pathogen Free)级C57BL/6雄性小鼠随机分为安静对照组(C组)、自主跑轮运动组(E组),每组10只,各组小鼠均单笼饲养且在每只饲养笼相同位置安装跑轮装置,C组小鼠饲养笼中跑轮处于"制动"状态,E组小鼠饲养笼中跑轮与遥感计数器相连;E组小鼠进行为期8周的自主跑轮运动,每日定时监测并记录跑轮圈数、及时清零,根据跑轮内径、圆周率π及每日跑轮圈数计算小鼠日均跑程(单位为Km/day),最后一周实验结束24h后处死小鼠并取材,分别提取骨骼肌DNA、RNA及蛋白裂解物,制备骨骼肌总DNA、c DNA及总蛋白样品,Real-time PCR技术检测小鼠骨骼肌线粒体DNA(mt DNA)相对含量,Real-time PCR、Western Blot技术检测细胞自噬、线粒体质量、线粒体生物发生、线粒体自噬相关基因mR NA及蛋白表达。结果:1)实验开始后,E组小鼠日均跑程持续增加,在第三周达到峰值后基本稳定在12Km/day;2)C组、E组小鼠经过8周实验干预后体重均显著增加(p<0.05),但E组小鼠体重增加幅度普遍低于C组;3)E组小鼠骨骼肌细胞自噬相关基因ULK1(Unc-51-like kinase 1)、LC3(Microtubule associated protein 1 light chain 3)、LAMP2(Lysosome-associated membrane protein 2)mR NA含量显著高于C组(p<0.05),p62(Ubiquitin binding protein p62/SQSMT1)蛋白含量下降,而用以指征细胞自噬的黄金指标LC3Ⅱ/Ⅰ比值显著增加(p<0.05);4)8周自主跑轮运动后,E组小鼠骨骼肌mt DNA含量较C组有所增加,指征线粒体质量的分子COX2(Cytochrome c oxidase subunit 2)、COX4(Cytochrome c oxidase subunit 4)mR NA及蛋白水平均显著高于C组(p<0.05);5)E组小鼠骨骼肌线粒体生物发生相关基因PGC-1α(Peroxisome proliferator-activated receptor gamma coactivator 1-alpha)、PPARβ(Peroxisome proliferator-activated receptor beta or delta)、P53(Tumor suppressor p53)、NRF(Nuclear Respiratory Factor)1/2 mR NA水平较C组显著上调(p<0.05),线粒体生物发生关键分子PGC-1α蛋白水平显著增加(p<0.05);6)与C组比,E组小鼠骨骼肌线粒体自噬相关基因PINK1(PTEN-induced putative kinase 1)、BNIP3(BCL2/adenovirus E1B 19 k Da protein-interacting protein 3)、NIX(BCL2/adenovirus E1B 19 k Da proteininteracting protein 3-like)、Mieap(Mitochondrial eating protein)mR NA表达明显增加(p<0.05),同时NIX、PINK1、Parkin(Parkinson disease protein 2)蛋白表达亦显著上调(p<0.05)。结论:自主跑轮运动可有效增加LC3Ⅱ/Ⅰ的比值促进骨骼肌细胞自噬水平及活性,这可能从逆向生物学角度揭示了运动对健康促进的分子机制,即自主运动可通过增加细胞自噬的活性参与代谢性疾病的防控机制;自主跑轮运动可有效促进骨骼肌mt DNA含量及COX2/4的表达增加线粒体质量,从而参与机体能量代谢的改善,且在该过程中不仅线粒体生物发生参与其中,线粒体自噬这一分子事件也参与其中并发挥至关重要的作用。更多还原

【Abstract】 Objective To study the effects of volunteer wheel running in growth period on the autophagy and mitochondrial quality in skeletal muscle of mice and to ascertain the molecular mechanism of health promotion effect regulated by volunteer exercise especially in the excise induced metabolic diseases’ prevention. Method: The 20 male mice as the research object, after feed to 4 weeks just in growth period, were assigned as control group C and volunteer wheel running group E, 10 mice in each group; all the mice were fed in separately and the volunteer wheels were equipped in each cage, and the wheels stayed braked in group C and intact and mating counters in group E; the mice in group E executed volunteer wheel running and the running distance was calculated through the inner diameter of the wheel, PI and the laps which was collected in a fixed time everyday and the counters were also needed to reset each day; after experiment, all the mice were executed and the total DNA, c DNA and total protein sample of skeletal muscle were prepared to detected the autophagy and mitochondrial quality related genes mR NA and protein expression using real-time PCR and Western Blot techniques. Results: 1)The running distance of mice each day in group E increased steadily and reach a peak in the third week, then the data sustained about 12 Km/day; 2) All the body weight of mice augmented gradually after experiment(p<0.05), while the amplification of the body weight in group E was lower than it in group C; 3) In skeletal muscle, compared to C, the mR NA expression of autophagy related genes ULK1(Unc-51-like kinase 1), LC3(Microtubule associated protein 1 light chain 3), LAMP2(Lysosome-associated membrane protein 2)unregulated significantly in skeletal muscle of mice in group E(p<0.05), whereas the p62(Ubiquitin binding protein p62/SQSMT1) protein expression decreased with the ratio of LC3 Ⅱ / Ⅰ accelerated obviously(p<0.05); 4) After 8 weeks exercise intervention, the relative content of mt DNA was improved in group E, as both the mR NA and protein expression of the mitochondrial quality index genes COX2(Cytochrome c oxidase subunit 2), COX4(Cytochrome c oxidase subunit 4) increased significantly compared with group C(p<0.05); 5) At the side of group C, the mR NA expression of PGC-1α(Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), PPARβ(Peroxisome proliferator-activated receptor beta or delta), P53(Tumor suppressor p53) and NRF(Nuclear Respiratory Factor)1/2 mR NA went up evidently(p<0.05) while the protein relative content of PGC-1αincreased clearly in group E(p<0.05); 6) The mitophagy related genes PINK1(PTEN-induced putative kinase 1), BNIP3(BCL2/adenovirus E1 B 19 k Da protein-interacting protein 3), NIX(BCL2/adenovirus E1 B 19 k Da protein-interacting protein 3-like), Mieap(Mitochondrial eating protein) mR NA expression and Nix, PINK1, Parkin(Parkinson disease protein 2) protein expression unregulated distinctly in group E while compared with them in group C(p<0.05). Conclusion: The volunteer wheel running can raise the ratio of LC3Ⅱ/Ⅰand increase the autophagy activity effectively, which may explain the molecular mechanism of health promotion induced by volunteer exercise; The mitochondrial quality in skeletal muscle has been enhanced to ameliorate the body energy metabolism state, and in this course not only mitochondrial biogenesis but also mitophagy involves in it and plays a critical role.更多还原