Changes in expression of autophagy-related factors during acute contusion repair of skeletal muscle

doi:10.12047/j.cjap.5647.2018.024

Abstract

Abstract: Objective: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. Methods: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, n=6). On the 3^rd, 5^th, 7^th and 14^th 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. Results:The results of HE staining showed that compared with the control group, the inflammation reached its peak on the 5^th 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 (P<0.01). Similarly, compared with the control group, the expression of P62 reached its peak on the 3^rd day after injury (P<0. 01), and returned to normal level on the 14^th 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 (P<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 (P<0.01, P<0.05, P<0.01). Conclusion: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.

Key words: skeletal muscle, acute blunt trauma, damage repair, autophagy, rat

LUO Ao, TANG Cheng-lin, HUANG Si-qin, ZHAO Dan-dan, ZHANG An-ning, GUO Quan-hu, GAO Rui-qi, CAO Jing. Changes in expression of autophagy-related factors during acute contusion repair of skeletal muscle[J]. CJAP, 2018, 34(2): 97-101.

References

[1] Crisco JJ, Jokl P, Heinen GT, et al. A muscle contusion injury model. Biomechanics physiology histology[J]. AM J Sports Med, 1994, 22(5):702-710.
[2] Järvinen TA, Järvinen TL, Kääriäinen M, et al. Muscle injuries:biology and treatment[J]. Am J Sports Med, 2005, 33(5):745-764.
[3] 寇现娟, 陈宁. 自噬在骨骼肌质量维持中的作用与调控[J]. 武汉体育学院学报, 2012, 46(11):66-71.
[4] 朱焕勉, 陈然, 薛峰, 等. 自噬抑制剂氯喹对低氧诱导肺动脉平滑肌细胞增殖的影响[J]. 中国应用生理学杂志, 2014, 30(1):8-12.
[5] 胡中慧, 马慧萍, 樊鹏程, 等. PC12细胞在缺氧条件下的自噬现象[J]. 中国应用生理学杂志, 2017, 33(1):65-68.
[6] Pfeifer U. Morphological and functional aspects of cellular autophagy[J]. Acta Morphol Acad Sci Hung, 1972, 20(3):247-267.
[7] 田源, 唐成林, 黄思琴, 等. 按摩对大鼠骨骼肌急性钝挫伤后膜修复蛋白dysferlin和annexinA1表达的影响[J]. 中国康复医学杂志, 2016, 31(8):841-846.
[8] 杨辉, 常青, 唐成林, 等. 跑台运动训练与按摩联合作用对大鼠骨骼肌急性损伤修复过程中炎症的发展及肌卫星细胞增殖的影响[J]. 体育科学, 2015, 35(3):51-58.
[9] 杨辉, 唐成林, 常青, 等. 在大鼠骨骼肌急性损伤修复过程中按摩对神经型一氧化氮合成酶、肝细胞生长因子mRNA表达的影响[J]. 中国康复医学杂志, 2015, 30(3):224-230.
[10] Kami K, Masuhara M, Kashiba H, et al. Changes of vinculin and extracellular matrix components following blunt trauma to rat skeletal muscle[J]. Med Sci Sport Exer, 1993, 25(7):832-840.
[11] Suzuki K, Ohsumi Y. Molecular machinery of autophagosome fo rmation in yeast, Saccharomyces cerevisiae[J]. FEBS Lett, 2007, 581(11):2156-2161.
[12] Ferraro E, Cecconi F. Autophagic and apoptotic response to stress signals in mammalian cells[J]. Arch Biochem Biophys, 2007, 462(2):210-219.
[13] Levine B, Yuan J. Autophagy in cell death:an innocent convict[J]? J Clin Invest, 2005, 115(10):2679-2688.
[14] Ohsumi Y. Molecular dissection of autophagy:two ubiquitinlike systems[J]. Nat Rev Mol Cell Biol, 2001, 2(3):211216.
[15] Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[J]. EMBO J, 2000, 19(21):5720-5728.
[16] Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting[J]. Autophagy, 1900, 3(6):542-545.
[17] Hayes JD, Mcmahon M. NRF2 and KEAP1 mutations:permanent activation of an adaptive response in cancer[J]. Trends Biochem Sci, 2009, 34(4):176-188.
[18] Su Y, Qian H, Zhang J, et al. The diversity expression of p62 in digestive system cancers[J]. Clin Immunol, 2005, 116(2):118-123.