【作者】 孟思进；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 衰老性肌萎缩是指骨骼肌质量和力量随着增龄逐渐下降,会增大摔倒、骨折的风险,危害老年人的健康。已有研究表明,衰老性肌萎缩与氧化应激增强、蛋白质代谢失衡以及线粒体机能下降等诸多方面密切相关。随着老年人口增多,衰老性肌萎缩已成为关注热点,研究开发行之有效的治疗、干预甚至逆转衰老性肌萎缩的方法显得尤为迫切。本文在预实验确定运动是增强骨骼肌结构和机能的关键因素以及延缓衰老的重要手段基础上,重点研究了衰老性肌萎缩的运动干预及其作用机理,以期寻找最佳运动方法并提供相应的科学依据。本文系统地比较研究了耐力运动、抗阻运动或组合运动等不同运动方法对衰老性肌萎缩的干预效果,并从氧化应激、蛋白质合成信号激酶、线粒体机能等方面研究分析了不同运动方法的干预作用机制。本文成功构建了耐力训练、抗阻训练、组合训练的老年小鼠运动模型：分别训练18月龄昆明雌性小鼠3个月,训练结束时达21月龄。训练结束后,在安静状态下取小鼠后肢股直肌、腓肠肌,采用生物化学方法测量肌肉内MDA含量,以及SOD、CAT、GPX酶活性；采用Western blotting测量蛋白激酶mTOR、p70S6K和ERK1／2磷酸化水平；采用PCR技术测量线粒体DNA缺失突变,采用生物化学方法测定线粒体复合物Ⅰ、Ⅲ、Ⅳ酶活性。应用SPSS11.5统计软件包,进行多因素方差分析和多重比较,显著性水平为P<0.05。实验研究取得了以下创新性实验结果：(1)研究发现,成年小鼠随着增龄进入老年时,股直肌和腓肠肌质量显著下降；骨骼肌组织中MDA含量和GPX活性升高,而SOD活性和CAT活性明显下降；老年小鼠肌肉内mTOR、p70s6k和ERK1／2磷酸化水平下降；在衰老模型小鼠肌肉内,线粒体缺失突变率(mtDNA%)较高,线粒体复合物Ⅰ、Ⅲ、Ⅳ酶活性下降。结果表明,随着增龄,小鼠进入老年期会发生衰老性肌萎缩,并伴随着氧化应激水平升高,蛋白质合成信号激酶活性下降,线粒体机能下降。(2)研究发现,抗阻运动及组合运动后小鼠腓肠肌重量显著增加,而耐力运动对小鼠腓肠肌重量无显著影响；耐力运动可使小鼠腓肠肌MDA含量明显下降,且SOD、CAT和GPX活性显著升高；抗阻运动可使小鼠腓肠肌GPX活性显著升高,MDA含量、SOD和CAT活性无显著性变化；组合运动可使小鼠腓肠肌MDA含量明显下降,且SOD、CAT和GPX活性极显著升高。结果表明,抗阻运动并没有对老年肌肉内氧化应激产生显著干预作用；耐力运动无明显增加老年小鼠骨骼肌内mTOR、p70s6k磷酸化水平和股直肌线粒体呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ活性；抗阻运动与组合运动能显著增加老年小鼠骨骼肌内mTOR、p70s6k和ERK1／2活性和股直肌线粒体呼吸链酶复合体Ⅰ、Ⅲ、Ⅳ活性。因此,耐力训练、抗阻训练、组合训练等不同类型运动训练对衰老性肌萎缩的干预效果及其机制存在明显差异：耐力训练增强了老年肌肉抗氧化能力和线粒体机能,但没有诱导肌肉质量增大；抗阻训练能诱导肌肉肥大,增强蛋白质合成信号分子活性,逆转了衰老性肌质量下降,还能增强线粒体机能；组合运动亦能增强老年肌肉蛋白质合成,诱导肌肉肥大,同时引起肌肉质量增长,组合运动对衰老性肌萎缩能起到较全面的干预作用,是较优的干预策略。更多还原

【Abstract】 Age-related muscle atrophy refers to the decline in muscle mass and strength with advancing age, which results in the increased risk of fall and fracture in the elderly, and the decreased ability of daily activities. With the increasing elderly population, research on age-related muscle atrophy has become a hot issue in recent years, more understanding of its pathogenesis and intervention strategies should be of significance. Due to the complication of the mechanisms, there was still lack of highly targeted and effective method of treatment, intervention or even reversal to this type of muscle atrophy up to date. Exercise has been suggested as a key factor to enhance the structure and function of skeletal muscle and an effective method delay aging. But endurance exercise or resistance exercise may interfere with age-related muscle atrophy by distinct mechanisms, and its interventional effects may also be different. We hypothesisd that the combination of endurance exercise and resistance exercise might play a more prominent role in intervention to aged muscle.The article focused primarily on the intervention effects and its mechanisms of different types of exercise such as endurance exercise, resistance exercise, or combined exercise in age-related muscle atrophy, including the level of oxidative stress, the phosphorylated level of signaling kinases and mitochondrial function, to explore the best exercise intervention.Eighteen-month-old female KM mice were assigned to undergo endurance training, resistance training, or combined training for 10-12 weeks, respectively. Within 1-2 days at end of training, mice were sacrificed in rest state, then rectus femoris and gastrocnemius were collected quikly, weighed, frozen in liquid nitrogen at -80℃. The content of MDA, the activity of anti-oxidant enzyme SOD, CAT, and GPX were measured by biochemical assays. The phosphorylation levels of protein kinases mTOR, p70S6K, ERK1/2 were analysized by Western blotting. The deletion mutant rates of mtDNA were determined by PCR, the activity of mitochondrial complexⅠ,Ⅲ,Ⅳwere measured by biochemical methods. SPSS11.5 statistical software package was used for multi-factor analysis of variance and multi-comparation, significance level was set at P<0.05.The results in the present study showed that with advancing age, the muscle mass of the rectus femoris and gastrocnemius decreased significantly. The MDA content and GPX activity increased in aged skeletal muscle tissue, while SOD activity and CAT activity decreased significantly. In addition, The phosphorylation levels of protein kinases mTOR, p70S6K, ERK1/2 decreased in aged mice. The deletion mutation rate of mitochondrial (mtDNA%) was higher in aged mice compared with controls, while the activity of mitochondrial complexⅠ,Ⅲ,Ⅳdecreased. Therefore, elevated levels of oxidative stress, and decreased signaling kinase activity of protein synthesis, and mitochondrial dyfunction may be cellular and molecular mechanism of age-related muscle atrophy.Moreover, there was an significant increase in muscle mass of the rectus femoris and gastrocnemius from aged mice after resistance exercise and combined exercise, but no marked effect from aged mice after endurance exercise.After endurance exercise, there were significant decrease in MDA content, and increase in SOD, CAT and GPX activity in the gastrocnemius muscle of aged mouse. After resistance exercise, GPX activity significantly increased, while MDA content, SOD, and CAT activity didn’t change. By combined exercise, MDA content in the gastrocnemius muscle of aged mice significantly decreased, SOD, CAT and GPX activity were significantly higher. The phosphyorylation levels of mTOR, p70S6K and ERK1/2 kinases in aged mice were enhanced significantly by resistance exercise and combined exercise. However, there was no significant increase in phosphorylated mTOR, p70s6k after endurance exercise in aged mice compared with controls.Through different types of exercise training interventions, the rate of mitochondrial deletion and oxidative damage were reduced significantly in muscles of aging model. Resistance exercise significantly increased the activity of complexⅠ,Ⅲ,Ⅳin the rectus femoris muscle of aged mice. Combined exercise markedly improved the activity of complexⅠ,Ⅳin aging mice rectus femoris muscle. However, endurance exercise did not significantly improve the complexⅠ,Ⅲ,Ⅳactivity in aged muscle.It had been shown in the present study, that the intervention effects and related mechanisms of different types of exercise training including endurance training, resistance training, combined training on age-related muscle atrophy were obviously different. Endurance training enhanced the antioxidant capacity of aged muscle and mitochondrial function, but did not induce increased muscle mass. Resistance exercise not only induced muscle hypertrophy, increased protein synthesis activity of signaling molecules, reversd age-related muscle mass decline, but also enhanced mitochondrial function. Combination of exercise also improved the status of oxidative stress, enhanced the ability of protein synthesis, and induced muscle mass growth in aged mice. Combined exercise has more comprehensive effects of intervention to age-related muscle atrophy.Further research on the mechanisms of age-related muscle atrophy needs to be performed. Based on these researches, the best strategy for the future is to develop effective combined interventions to age-related muscle atrophy.更多还原