大负荷运动后时钟基因Bmal1调控骨骼肌线粒体结构功能的作用机制

Effects of clock gene Bmal1 on the the structure and function of skeletal muscle mitochondria after heavy load exercise

目的:探讨大负荷运动后大鼠骨骼肌时钟基因Bmal1节律振荡变化及其对线粒体结构功能的调控作用。方法:将156只成年SD大鼠随机分为对照组和运动组,运动组予以一次大负荷运动训练。每6 h取各组大鼠腓肠肌,使用RT-qPCR检测各时相时钟基因Bmal1的mRNA水平,并用余弦分析软件CircaCompare(R Packages)获取拟合余弦曲线参数,分析时钟基因Bmal1的mRNA表达节律性振荡情况,透射电镜下观察每周期始末(ZT0、ZT24、ZT48和ZT72)骨骼肌线粒体的形态学变化,Western blot检测Bmal1和过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator activated receptorγcoactivator-1α,PGC-1α)的蛋白表达,ELISA测定ATP和ADP含量,以及线粒体氧化呼吸链酶细胞色素C氧化酶复合物亚单位Ⅱ(subunitsⅡof cytochrome C oxidase complex,COXⅡ)和COXⅣ的活性。结果:运动组Bmal1的mRNA表达在ZT0~ZT24时节律出现紊乱(P>0.05),ZT24~ZT48和ZT48~ZT72时节律恢复(P<0.05)。大负荷运动后运动组线粒体形态于ZT0出现肿胀、嵴结构损伤等异常,于ZT24和ZT48时有所恢复,ZT72时损伤基本消失。与对照组相比,运动组Bmal1、PGC-1α的蛋白表达于ZT0时显著升高(P<0.05),ATP和ADP含量分别于ZT0时显著下降和升高(P<0.05),COXⅡ和COXⅣ活性于ZT0时显著升高和下降(P<0.05),在ZT24时二者活性下降至最低(P<0.05)。结论:大负荷运动可诱发骨骼肌时钟基因Bmal1的节律紊乱,可能参与调控了线粒体的结构功能异常。

AIM:To investigate the effection of skeletal muscle clock gene Bmal1 on mitochondrial structure and function after heavy load exercise in rats.METHODS:156 adult SD rats were randomly divided into control group and exercise group. The gastrocnemius muscle of each group were isolated in every 6 h,and the Bmal1 mRNA levels were detected by RT-qPCR. The parameters of fitting cosine curve were obtained by using the cosine analysis software CircaCompare(R Packages),and the rhythmic oscillation of clock gene Bmal1 mRNA expression was analyzed. Morphological changes of skeletal muscle mitochondria at zeitgeber time(ZT)0,ZT24,ZT48 and ZT72 were observed by transmission electron microscopy. Protein levels of Bmal1 and peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α)were detected by Western bolt,contents of ATP and ADP and the activity of enzymes involved in the mitochondrial respiratory chain such as subunit Ⅱ of cytochrome C oxidase complex(COX Ⅱ)and COX Ⅳ were determined by ELISA.RESULTS:(1)The expression of Bmal1 mRNA was disturbed in ZT0~ZT24(P>0. 05),and recovered in ZT24~ZT48 and ZT48~ZT72(P<0. 05).(2)After heavy load exercise,the morphology of mitochondria in exercise group showed swelling,crest damage at ZT0~ ZT24,recovered at ZT24~ ZT48 and ZT48~ ZT72.(3)Compared with control group,the protein levels of Bmal1 and PGC-1α in exercise group were significantly increased at ZT0(P<0. 05).(4)Decreased ATP and increased ADP in exercise group were observed at ZT0(P<0. 05),the stimulated COXⅡ and decreased COXⅣ were measured at ZT0,and both decreased to the lowest at ZT24(P<0. 05).CONCLUSION:Heavy load exercise induce the rhythm disorder of skeletal muscle clock gene Bmal1,which may be linked to the abnormal mitochondrial structure and function.