【摘要】 目的:探讨骨骼肌钝挫伤后线粒体自噬相关基因与自噬体变化的分子机制。方法:Wistar雄性大鼠64只,随机分为对照组、钝挫伤组(按照取材时间分为12 h组、2 d组、5 d组、7 d组、10 d组、15 d组、30 d组)。采用重物下落装置导致右小腿内侧面(其皮下为腓肠肌中段)一次打击伤,建立急性骨骼肌钝挫伤模型,通过Western-Blotting和qRT-PCR技术分别检测钝挫伤后腓肠肌缺氧及自噬相关因子,包括低氧诱导因子-1α(HIF-1α)、腺苷酸活化蛋白激酶α2亚基(AMPKα2)、Bcl-2腺病毒E1B19ku相关蛋白3(BNIP3)和Bcl-2家族的类NIP3蛋白(NIX)的蛋白和mRNA表达变化情况;透射电镜观察受损部位不同恢复时间的超微结构及自噬体形成情况。结果:(1)钝挫伤后,HIF-1α、AMPKα2蛋白表达在12 h、2 d达到峰值,至伤后10 d,HIF-1α表达均明显高于对照组(P<0.05);AMPKα2表达在伤后5 d仍显著高于对照组(P<0.05),至10 d回落至正常水平;BNIP3持续高表达至5 d后开始回落,但至10 d仍高于对照组;NIX表达峰值出现在伤后12 h、2 d,持续高表达至7 d。(2)钝挫伤后,相关基因表达水平,Hif-1α与Ampkα2mRNA在2 d显著高于对照组(P<0.01),至5 d表达下降但仍高于对照组(P<0.05),随后回落至正常水平;Bnip3、Nix的mRNA变化情况与其蛋白表现基本一致。损伤后12 h在单个网孔视野中即可见到多个自噬体,至2～7 d自噬体数量逐渐增多,10 d后自噬体数量比钝挫伤后12 h～7 d组相比呈现减少趋势,至15 d后自噬体数量逐渐减少。结论:骨骼肌钝挫伤发生后,损伤早期代谢调控因子AMPKα2、缺氧敏感因子HIF-1α表达变化情况表明损伤后受损骨骼肌内部出现能量危机并形成缺氧环境;线粒体自噬、细胞凋亡相关因子BNIP3、NIX表达变化表明损伤后线粒体自噬被启动,缺氧诱导了骨骼肌钝挫伤早期的线粒体自噬;缺氧诱导的线粒体自噬可及时清除受损线粒体,维持线粒体质量、为新生线粒体提供原料,从而减小损伤程度,以利于受损骨骼肌的快速恢复,这可能是损伤发生后机体的一种代偿性机制。更多还原

【Abstract】 Objective To investigate the molecular mechanism of mitochondrial autophagy-related genes and autophagosome changes after the blunt trauma of skeletal muscles. Methods Sixty-four male Wistar rats were randomly divided into a control and a blunt trauma group,each of 32. The acute skeletal muscle contusion was induced by a single blow on the middle segment of the gastrocnemius muscle,using the heavy objects dropping device to rats of the blunt trauma group,while no trauma was induced in the control group. The expression of hypoxia and autophagy-related factors in the skeletal muscle of the blunt trauma,including the hypoxia inducible factor-1α(HIF-1α),α2 subunit of AMP-activated protein kinase(AMPKα2),BCL2 interacting protein 3(BNIP3) and NIP3-like protein X(NIX) were measured using the Western blotting and qRT-PCR 12 h,2 d,5 d,7 d,10 d,15 d and 30 d after the modeling for both groups. The ultrastructure and autophagosomes at different time points were observed using a transmission electron microscopy(TEM). Results The expression of HIF-1α and AMPKα2 reached the peak at 12 h and 2 d after the trauma,and the expression of HIF-1αwas significantly higher than that of the control group 10 days after the modeling(P<0.05). The expression of AMPKα2 was significantly higher than the control group 5 days after the injury(P<0.05),but reached the normal level 5 days later. The expression of BNIP3 remained at a high level at the beginning,and began to decline till 5 days after the modeling,but was still higher than the control group till 10 days after the trauma(P<0.05). The NIX expression peaked at 12 h and 2 d after injury,keeping high-level expression till 7 days after injury. The expression of Hif-1α and Ampkα2 mRNA was significantly higher than that of the control group 2 days after the modeling,and began to decrease 3 days later,but still significantly higher than the control group. The mRNA expression of Bnip3 and Nix was basically the same as their proteins. A number of autophagosomes were observed at 12 h after injury,and the number of autophagosomes increased gradually from 2 to 7 days after the modeling,followed by a decrease from 10 days after the modeling. Moreover,15 days after the modeling,the number of autophagosomes decreased gradually. Conclusion The changes of the early-stage metabolic regulator AMPKα2 and hypoxia-sensitive factor HIF-1α after the blunt trauma of skeletal muscles indicate that an energy crisis occurs in the skeletal muscle after injury,and the hypoxic environment was formed. The mitochondrial autophagy and the expression of BNIP3 and NIX show that the mitochondrial autophagy is activated by the hypoxia. Hypoxia-induced mitochondrial autophagy can remove the damaged mitochondria,maintain mitochondrial quality and provide raw materials for the new generation of mitochondrias and facilitate the rapid recovery of damaged skeletal muscles,which may be a compensatory mechanism of the body response to injury.更多还原