急性运动诱导大鼠骨骼肌线粒体生物合成：H2O2参与介导PGC-1α转录

Acute Exercise Induces Mitochondrial Biogenesis in Skeletal Muscle of Rat:Involvement of H_2O_2 in Contractile Activity-induced PGC-1α Transcription

目的:线粒体产生的活性氧(ROS)是多种重要细胞信号转导通路的调节中心,本文拟探讨运动源性的过氧化氢(H2O2)是否作为信号分子参与了运动诱导的骨骼肌线粒体生物合成过程。方法:以大鼠急性递增负荷跑台运动为模型,连续观察和分别测定对照组(C),运动中45min、90min1、20min 150min(分别以E45、E90、E120和E150组表示)及运动后恢复3h、6h、12h、18h和24h(分别以R3h、R6h、R12h、R18h和R24h组表示)各时间点骨骼肌组织中H2O2浓度,PGC-1α、NRF-1、Tfam、COX IV mRNA表达,PGC-1α、Tfam、COX IV和p38MAPK蛋白含量以及p38MAPK活性的变化。结果:1)与对照组比较,E45组骨骼肌H2O2浓度即开始呈现显著性增加,并持续到R6h组(均P<0.001);在R12h组出现短暂降低后,又持续显著性增加至R24h组(均P<0.001);2)E150和R3h组PGC-1αmRNA表达与对照组相比显著增加(P<0.01和P<0.001);随后,R6h^R18h组PGC-1αmRNA表达降低(P>0.05),R24h组PGC-1α基因表达又呈现显著性增加(P<0.05)。PGC-1α蛋白含量滞后于其基因表达,在R6h和R12h组中显著增加(均P<0.001);3)R3h^R12h组骨骼肌NRF-1mRNA表达较对照组显著增加(P<0.01和P<0.001)。随后,NRF-1基因转录开始下降(P>0.05);4)除E150组外,从E90~R24h组的骨骼肌Tfam mRNA均较对照组显著增加(P<0.01和P<0.001)。E45和E90组的Tfam蛋白含量较对照组呈现短暂的显著增加(P<0.001),在E120组降低后又呈现显著性增加,直至R24h组(均P<0.001);5)E120组骨骼肌COX IV基因表达较对照组显著增加(P<0.05),恢复期R3h^R18h组呈显著性增加(均P<0.001)。运动中各组COX IV蛋白含量较对照组未呈现显著增加,但恢复期各时间段COX IV蛋白水平均呈显著性增加(均P<0.001);6)骨骼肌p38 MAPK活性(磷酸化p38 MAPK)在E90~R6h组,R18h^R24h组呈显著性增加(分别P<0.05、P<0.01和P<0.001)。仅R3h组p38 MAPK蛋白含量较对照组呈显著性增加(P<0.05)。结论:一次急性运动即可影响COX IV基因和蛋白表达,诱导骨骼肌线粒体生物合成;运动源性的H2O2可能通过先行激活p38MAPK磷酸化和继发诱导p38MAPK信号通路两个途径影响PGC-1α基因转录和线粒体生物合成的下游信号级联反应。

Objective The purpose of present study was to investigate the role of H2O2 involved in exercise-induced mitochondrial biogenesis in skeletal muscle. Methods Sprague-Dawley rats were divided into 10 groups （6 rats in each group） ：control group （C） and exercise groups including running for 45min （E45）, 90min （E90）, 120 （E120）, 150min （E150）, and post-exercise groups including 3h （R3h）, 6h （R6h）, 12h （R12h）, 18h （R18h） and 24h （R24h） after exercise. Rats in exercise groups were forced to perform an acute bout of incremental treadmill running. Rats were sacrificed at rest and immediately at each time point. The soleus and gastrocnemius muscle were harvested for analyzing the H2O2 concentration, the expressions of PGC-1α, NRF-1, Tfam and COX IV mRNA, and the protein contents of PGC-1α, Tfam, COX IV, and p38MAPK, and the activity of p38MAPK. Results It was found that： 1） Except R12h, muscular H2O2 concentration increased significantly in group E45 to group R24h as compared with that in group C （P〈0. 001） ; 2） PGC-1α mRNA expression increased significantly in group El50 and R3h as compared with that in group C （P〈0.01 and P〈0. 001）. The expression increased significantly once again in group R24h （P〈0.05）. The PGC-1α protein level increased significantly in group R6h and R12h （P〈0. 001） ; 3） The expression of NRF-1 mRNA was up-regulated in group R3h, R6h and R12h （P〈0. 01 and P〈0. 001, respectively） ; 4） The expression of Tram mRNA was remarkably increased in most groups （P〈0.05, and P〈0. 001, respectively） as compared with that in group C, except in group E150. The Tfam protein content was also up-regulated significantly in most groups （P〈0. 001, respectively）, except in group E120; 5） The expression of COX IV mRNA increased significantly in group E120 （P〈0.05） and in group R3h to group R18h （P〈0. 001） as compared with that in group C. COX IV protein level was significantly higher in group R3h to group R24h （P〈0. 001, respectively）; 6） p38MAPK （p38MAPK phosphorylated protein） was activated significantly in group Eg0 and in group R6h, R18h and R24h （P〈0.05, P〈0.01 and P〈0. 001, respectively）. However, p38MAPK protein content was significantly increased only in group R3h （P〈0.05）. Conclusion Our data suggested that an acute bout of exercise could affect the expression of COX IV mRNA and its protein, and induce mitochondrial biogenesis in skeletal muscle. We hypothesized that exerciseinduced H2O2 might up-regulate PGC-1α mRNA transcription and its downstream cascade in the contractile activity-induced mitochondrial biogenesis through initial activation of p38MAPK phosphorylation and secondary induction of p38MAPK signaling pathway.