大负荷运动诱导大鼠骨骼肌细胞凋亡的作用及线粒体凋亡机制

Effects of heavy-load exercise on skeletal muscle cells apoptosis and mechanisms of mitochondrial apoptosis in rats

目的:以丝氨酸蛋白酶Omi为切入点,探究大负荷运动诱导大鼠骨骼肌细胞凋亡的可能机制。方法:126只健康雄性SD大鼠随机分为安静对照组(C),离心运动组(E),单纯阻断组(U),二甲基亚砜(DMSO)组(D),运动阻断组(EU)。除C组外,其余4组随机分为干预后0 h组、12 h组、24 h组、48 h组、72 h组,每组6只。E组与EU组大鼠在跑台上进行坡度为-16°,速度为16 m/min,90 min的一次性大负荷运动。U组、D组与EU组进行一次性药物干预,给予U组和EU组大鼠腹腔注射1.5μmoL/kg Omi特异性抑制剂Ucf-101,同样给予D组大鼠腹腔注射1.5μmoL/kg的0.5%DMSO,于实验后不同时间点分批取比目鱼肌,检测其Caspase-3,-8,-9,-12的活性以及Omi和X染色体连锁的凋亡抑制蛋白(XIAP)的表达。结果:与C组相比,E组骨骼肌线粒体形态结构发生典型的病理改变,骨骼肌线粒体膜通透性转换孔(MPTP)开放程度明显增加(P<0.01)或(P<0.05),同时骨骼肌Omi、XIAP蛋白表达均明显增加(P<0.01或P<0.05),Caspase-9,-3活性也均明显增加(P<0.01或P<0.05)。与C组相比,U组和D组XIAP蛋白以及Caspase-9,-3活性均无显著差异。EU组XIAP蛋白以及Caspase-9,-3活性变化趋势均与E组基本一致,但XIAP蛋白变化幅度明显高于E组(P<0.01),Caspase-9,-3活性变化幅度明显低于E组(P<0.01)。结论:大负荷运动可以诱导大鼠骨骼肌线粒体形态结构改变,使MPTP高通透性开放,Omi蛋白表达上调,进而通过其下游的XIAP-Caspase途径,启动依赖Caspase-9介导线粒体凋亡途径,最终导致肌细胞发生凋亡,而抑制Omi可降低运动诱导骨骼肌细胞的凋亡水平。

Objective:To analyze the molecular mechanisms of skeletal muscle cells apoptosis induced by heavy-load exercise with Omi as the entry point.Methods:One hundred and twenty-six adult SD rats were randomly divided into five groups:control group(C),eccentric exercise group(E),simple blocking group(U),DMSO group(D)and exercise block group(EU).In addition to the C group,the other four groups were randomly divided into 0 h after experiment,12 h after experiment,24 h after experiment,48 h after experiment and 72 h after experiment with 6 rats in each group.E and EU group were submitted to a heavy-load exercise on a treadmill down a 16°decline,16 m/min for 90 minutes.U,D and EU group were one-time intervened with drugs.U and EU groups were intraperitoneally injected with 1.5μmol/kg ucf-101,D group were intraperitoneally injected with 1.5μmoL/kg 0.5%DMSO.The rats were sacrificed in batches at different time points after experiment,then the soleus were saved to detect the Caspase-3,-8,-9,-12 activities and protein expressions of Omi and XIAP.Results:Compared with group C,the mitochondrial distribution and morphology appeared the typical ultrastructure pathological changes,the opening degree of MPTP was increased significantly(P<0.01)or(P<0.05),protein expressions of Omi and XIAP were increased significantly(P<0.01 or P<0.05),the activities of Caspase-9 and Caspase-3 were increased significantly(P<0.01 or P<0.05)in group E.Compared with group C,there was no significant difference in XIAP protein and caspase-9,-3 activities in group U and Group D.The change trend of XIAP protein and Caspase-9,-3 activities was the same as those between EU group and E group,but the change range of XIAP protein in EU group was significantly higher than that in E group(P<0.01),and the change ranges of caspase-9,-3 activities in EU group were significantly lower than those in E group(P<0.01).Conclusion:A single heavy-load exercise can induce changes in the mitochondria morphology and structure in rats,open the high permeability of MPTP,and improve the expression of Omi protein,then through its downstream XIAP-Caspase pathway,start the mitochondrial apoptosis pathway mediated by caspase-9,and finally lead to myocyte apoptosis.The inhibition of Omi can reduce the cell apoptosis level of motor induced skeletal muscle cells.