Omi/HtrA2介导的细胞自噬在运动性骨骼肌损伤中的作用

Role of Omi/HtrA2-mediated Autophagy in Motor Skeletal Muscle Injury

目的以Omi/HtrA2为切入点,探讨大负荷运动诱导骨骼肌细胞自噬的可能机制。方法将168只SD大鼠随机分为对照组(C)、安慰剂组(D)、抑制剂组(U)、运动组(E)和抑制剂运动组(EU)。E组进行一次大负荷下坡跑,U组注射Omi/HtrA2特异性抑制剂ucf-101,分别于干预后0 h、12 h、24 h、48 h和72 h取材。透射电镜观测比目鱼肌细胞内自噬体超微结构变化;Western Blot检测比目鱼肌Omi/HtrA2、Hax-1和Beclin1蛋白表达;免疫共沉淀技术测定Hax-1与Beclin1蛋白结合水平。结果D组与对照组蛋白表达无显著性差异;U组干预后电镜下线粒体稍有肿胀,未见明显自噬现象,细胞内Omi/HtrA2和Beclin1蛋白一过性降低(P<0.01或P<0.05),同时Hax-1一过性升高(P<0.01或P<0.05),Hax-1与Beclin1蛋白结合增强;E组比目鱼肌出现损伤,肌原纤维间出现不同成熟阶段的自噬小泡,细胞内Omi/HtrA2和Beclin1蛋白一过性升高(P<0.01或P<0.05),Hax-1一过性降低(P<0.01或P<0.05),Hax-1与Beclin1蛋白结合减弱;EU组比目鱼肌细胞内自噬体不明显,蛋白变化趋势与U组基本一致,但变化幅度低于U组。结论一次大负荷离心运动可诱导骨骼肌细胞自噬现象;Omi/HtrA2通过下游Hax-1-Beclin1途径参与自噬,抑制Omi/HtrA2可降低运动诱导骨骼肌细胞的自噬水平。

Objective This study explored the molecular mechanism of skeletal muscle autophagy induced by high-intensity exercise with Omi/HtrA2 as the entry point.Methods 168 SD rats were randomly divided into control group,dummy control group,ucf-101 group,exercise group and exercise+ucf-101 group.The exercise group was given a high-intensity downhill running intervention,and the ucf-101 group was injected with ucf-101 intervention.Samples were taken at 0 h,12 h,24 h,48 h and 72 h after intervention.Ultrastructural changes of auto phagosomes in soleus muscle cells were observed by transmission electron microscopy.Western Blot analysis of protein expression of Omi/HtrA2,hax-1 and Beclin1.The binding levels of Hax-1 and Beclin1 weredetermined by immunoprecipitation.Results There was no significant difference in protein expression between group D and control group.After intervention,the mitochondria in the U group were slightly swollen under electron microscope,no obvious autophagy was observed,and the intracellular Omi/HtrA2 and Beclin1 proteins were reduced temporarily(P<0.01 or P<0.05),and the transient increase of hax-1(P<0.01 or P<0.05),the binding of hax-1 to Beclin1 was enhanced;In group E,soles muscle was injured,autophagic vesicles at different stages of maturation appeared between myofibrils,and intracellular Omi/HtrA2 and Beclin1 proteins were transient elevated(P<0.01 or P<0.05),hax-1 transient decreased(P<0.01 or P<0.05),the binding of hax-1 to Beclin1 was weakened;The auto phagosomes in EU group were not obvious,and the protein changes were basically the same as those in group U,but the changes were lower than those in group U.Conclusion A single high-intensity centrifugal exercise can induce autophagy enhancement in skeletal muscle cells;Omi/HtrA2 participates in autophagy through the downstream hax-1-beclin1 pathway,and inhibition of Omi/HtrA2 can reduce the autophagy level of motor induced skeletal muscle cells.