运动对衰老骨骼肌卫星细胞成肌分化中线粒体活性氧生成的影响及调控机制

目的:观察耐力运动训练对衰老骨骼肌卫星细胞成肌分化中线粒体活性氧(ROS)生成的影响,并探讨过氧化物酶体增殖物激活受体γ辅激活因子1(PGC-1α)对ROS生成调控的潜在机制。方法:C57BL/6小鼠分为青年对照组(YN组,2月龄,12只)、老年对照组(ON组,12月龄,12只)和老年运动训练组(OT组,12月龄,12只),其中OT组进行中等强度跑台训练(0°,17 m/min,25 min/天,5天/周)。12周后,HE染色鉴定骨骼肌萎缩程度。两步酶消化法分离骨骼肌卫星细胞,原代培养并体外诱导成肌分化24 h。倒置显微镜观察卫星细胞成肌分化程度,测定线粒体呼吸功能、细胞ROS水平、线粒体ROS生成速率、肌球蛋白重链(MyHC)各亚基mRNA表达,及MyHC、PGC-1α、Tfam、COXⅣ、MnSOD蛋白表达量。结果:与YN组比较,ON组肌纤维横截面积、肌管形成数量、My-HC蛋白表达、MyHCⅠ、MyHCⅡa、MyHCⅡx mRNA表达、态3呼吸速率(ST3)、呼吸控制比(RCR)和PGC-1α、Tfam、COXⅣ、MnSOD蛋白表达均显著降低(P<0.05～0.01),ON组态4呼吸速率(ST4)、细胞ROS水平、线粒体ROS生成速率及MyHCⅡb mRNA表达显著升高(P<0.05～0.01)。与ON组比较,OT组湿重/体重比值、肌纤维横截面积、肌管形成数量、MyHC蛋白表达、MyHCⅠ、MyHCⅡa、My-HCⅡx mRNA表达、ST3、RCR和PGC-1α、Tfam、COXⅣ、MnSOD蛋白表达均显著升高(P<0.05～0.01),OT组细胞ROS水平、线粒体ROS生成速率及MyHCⅡb mRNA表达显著降低(P<0.05～0.01)。结论:耐力运动训练提高衰老骨骼肌卫星细胞成肌分化中PGC-1α表达,继而通过上调Tfam和MnSOD提高线粒体能量代谢,减少线粒体ROS产生,以促进成肌分化。

Endurance Exercise Affects the Mitochondrial ROS Generation during Aged Muscle Satellite Cells Differentiation

Objective To observe the effect of endurance exercise on the mitochondrial reactive oxygen species(ROS) generation,and the role of perxisome proliferator-activated receptor γ coactivator 1α(PGC-1α) in regulation of ROS generation during aged muscle satellite cells differentiation.Methods C57 BL/6 mice were randomly divided into three groups:young normal group(YN),aged normal group(ON) and aged exercise training group(OT).The moderate endurance training on a treadmill(0°,17 m/min,25 min/day,5 day/week) was imposed on group OT.After 12 weeks,muscle atrophy was observed through HE staining.Purified satellite cells were obtained by two step enzymatic digestion method and primary culture technique.Differentiation was induced by moving the cells into the differential medium composed of 2% horse serum,and was observed by inverted microscopy.At 24h of differentiation,cytoplasmic ROS,mitochondrial ROS generation,mitochondrial respiration parameters,MyHC isoforms mRNA expression,MyHC,PGC-1α,Tfam,COXⅣ and MnSOD protein expression were measured.Results Comparing with group YN,the formation of myotubes,ST3,RCR,MyHCⅠ,MyHCⅡa and MyHCⅡx mRNA expression,MyHC,PGC-1α,Tfam,COXⅣ,and MnSOD protein expression in group ON significantly decreased(P < 0.05~0.01).ST4,cytoplasmic ROS,mitochondrial ROS generation and MyHCⅡb mRNA in group ON significantly enhanced(P < 0.05~0.01).Comparing with group ON,the formation of myotubes,ST3,RCR,MyHCⅠ,MyHCⅡa and MyHCⅡx mRNA expression,MyHC,PGC-1α,Tfam,COXⅣ and MnSOD protein expression in group OT significantly increased(P < 0.05~0.01).Cytoplasmic ROS,mitochondrial ROS generation and MyHCⅡb mRNA expression in group OT significantly depressed(P < 0.05~0.01).Conclusion Endurance exercise markedly promoted PGC-1α expression in aged muscle satellite cells and attenuated ROS generation through upregulation of Tfam and MnSOD,which in turn enhanced the myogenic differentiation.