急性离心运动后恢复期大鼠骨骼肌微管形态及自噬体降解特征研究

Study on the morphology of microtubules and the characteristics of autophagosome degradation in rats skeletal muscle during the recovery period after acute eccentric exercise

目的:观察急性离心运动后恢复期大鼠骨骼肌微管形态变化及自噬体降解特征。方法:SD大鼠随机分为安静组、急性离心运动组,于运动后0h、12h、24h、48h、72h取材。分离单根肌纤维检测微管形态,Western Blot检测LC3、p62蛋白表达,腹腔注射秋水仙碱检测恢复前期(12h内)骨骼肌自噬流。结果:①急性离心运动后恢复期内骨骼肌微管形态改变,恢复12h大量横向微管丢失且纵向微管束密度降低;②恢复12h肌纤维中多见自噬体,24h与48h自噬溶酶体增多;③微管相关蛋白1轻链3(microtubule-associated protein1 light chain 3,LC3)Ⅱ型蛋白表达在恢复12h、24h、48h显著增加(P<0.05),0h、72h显著降低(P<0.05),p62蛋白表达呈先增加后降低趋势,于恢复12h最高(P<0.05);④急性离心运动组运动后恢复前期(12h内)骨骼肌自噬流显著低于安静组(P<0.05)。结论:急性离心运动后恢复期内骨骼肌自噬体降解存在“瓶颈”,恢复前期(12h内)自噬流降低,可能与骨骼肌横向、纵向微管丢失有关。

Objective:To observe the morphological changes of microtubules and the characteristics of autophagosome degradation in rat skeletal muscle following eccentric exercise.Method:SD rats were randomly divided into control group and eccentric exercise group.The muscles were taken at 0h,12h,24h,48h,72h postexercise.Single muscle fiber was isolated to detect the morphology of microtubules.Western Blot was used to detect the expression of LC3 and p62.Moreover,autophagy flux in the first 12h after exercise was detected via intraperitoneal injection colchicine.Result:The results showed that eccentric exercise changed the morphology of muscle microtubules.Transverse microtubules were lost at 12h postexercise,and the density of longitudinal microtubule bundles also decreased at this time point.The results of transmission electron microscope showed that there were more autophagosomes at 12h postexercise,while autophagolysosomes were more at 24h and 48h.The results of Western Blot showed that the level of autophagy marker protein LC3-Ⅱincreased significantly at 12h,24h,and 48h postexercise(P<0.05),and significantly decreased at 0h,72h(P<0.05),and p62 protein level increased at first then decreased,the expression was the highest at 12h postexercise(P<0.05).Furthermore,autophagy flux of skeletal muscle in the first 12h after exercise was significantly lower than that in control group(P<0.05).Conclusion:These results indicate that there is a"bottleneck"in autophagosome degradation of muscle after eccentric exercise,the autophagy flux was decreased in the early stage of recovery(first 12h),which may be related to the loss of muscle transverse and longitudinal microtubules.