Luo Ao, Tang Cheng-Lin, Huang Si-Qin, Zhao Dan-Dan, Zhang An-Ning, Guo Quan-Hu, Gao Rui-Qi, Cao Jing
College of Traditional Chinese Medicine, Chongqing Medical University, Chongqing 400016, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2018 Apr 8;34(2):97-101. doi: 10.12047/j.cjap.5647.2018.024.
To explore the possible biological mechanism of skeletal muscle contusion repair through researching the changes in expression of autophagy-related genes and proteins in SD rats with acute skeletal muscle contusion.
Six rats were randomly selected as the control group from 30 male SD rats, acute skeletal muscle contusion model were established in the remaining 24 rats with self-made hitter, then the model rats were randomly divided into 4 groups (3 d, 5 d, 7 d, 14 d groups, =6). On the 3, 5, 7 and 14 day after injury, injured gastrocnemius of each group was harvested. The morphological and the ultra-microstructure changes of gastrocnemius after injury were observed by HE staining and transmission electron microscope (TEM) respectively. The relative protein levels of (LC3-Ⅱ) and P62 of each group were observed by Western blot. The relative mRNA levels of atg7, atg10, atg12, atg16L1 of each group were observed by RTPCR.
The results of HE staining showed that compared with the control group, the inflammation reached its peak on the 5 day after injury, new muscle fibers were clearly observed in 7 d group. The results of TEM showed that, compared with the control group, oncotic mitochondria could be clearly seen in the 3 d, 5 d, 7 d groups. Also, the Z line changed from disappearing to drift thickening, sarcoplasmic reticulum dilatation gradually improved, there was no evident difference between the 14 d group and the control group, suggesting that the damage has preliminarily healed. The results of Western blot showed that the expressions of LC3-Ⅱand P62 were increased at first and then decreased. The expression of LC3-Ⅱwas markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (<0.01). Similarly, compared with the control group, the expression of P62 reached its peak on the 3 day after injury (<0. 01), and returned to normal level on the 14 day. The results of RT-PCR showed that the expression of atg10 mRNA in the natural recovery group of 3 d, 5 d, 7 d, 14 d was firstly decreased and then increased, the atg10 mRNA was markedly down-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (<0. 01). The expression of atg7, atg12, atg16L1 mRNA was generally increased at first and then decreased, it was markedly up-regulated in the 3 d, 5 d, 7 d groups compared with the control group and the 14 d group (<0.01, <0.05, <0.01).
The above results indicate that the autophagy is involved in repair of skeletal muscle injury by its autophagyrelated factors,regularly changes after contusion, and the rate of damage repair may be related to the level of autophagy.
通过研究急性骨骼肌挫伤的SD大鼠自噬相关基因及蛋白表达变化，探讨骨骼肌挫伤修复可能的生物学机制。
从30只雄性SD大鼠中随机选取6只作为对照组，其余24只大鼠用自制击打器建立急性骨骼肌挫伤模型，然后将模型大鼠随机分为4组（3 d组、5 d组、7 d组、14 d组，每组n = 6）。于伤后3、5、7、14天，分别取各组大鼠的损伤腓肠肌。分别采用苏木精-伊红（HE）染色及透射电子显微镜（TEM）观察伤后腓肠肌的形态及超微结构变化。采用蛋白质免疫印迹法观察各组微管相关蛋白1轻链3（LC3-Ⅱ）及P62的相对蛋白水平。采用逆转录聚合酶链反应（RT-PCR）观察各组自噬相关基因7（atg7）、自噬相关基因10（atg10）、自噬相关基因12（atg12）、自噬相关基因16样蛋白1（atg16L1）的相对mRNA水平。
HE染色结果显示，与对照组相比，损伤后第5天炎症达到高峰，7 d组可见明显的新生肌纤维。TEM结果显示，与对照组相比，3 d组、5 d组、7 d组可见肿胀的线粒体；同时，Z线由消失变为漂移增粗，肌浆网扩张逐渐改善，14 d组与对照组无明显差异，提示损伤已初步愈合。蛋白质免疫印迹法结果显示，LC3-Ⅱ及P62表达先升高后降低。与对照组及14 d组相比，3 d组、5 d组、7 d组LC3-Ⅱ表达明显上调（P < 0.01）。同样，与对照组相比，P62表达于伤后第3天达到高峰（P < 0.01），并于第14天恢复至正常水平。RT-PCR结果显示，3 d、5 d、7 d、14 d自然恢复组atg10 mRNA表达先降低后升高，与对照组及14 d组相比，3 d组、5 d组、7 d组atg10 mRNA表达明显下调（P < 0.01）。atg7、atg12、atg16L1 mRNA表达总体先升高后降低，与对照组及14 d组相比，3 d组、5 d组、7 d组表达明显上调（P < 0.01、P < 0.05、P < 0.01）。
上述结果表明，自噬通过其自噬相关因子参与骨骼肌损伤修复，挫伤后呈规律性变化，损伤修复速率可能与自噬水平有关。
