肌少症对大鼠骨骼肌线粒体自噬的影响

Influence of sarcopenia on skeletal muscle mitochondrial autophagy

目的探讨肌少症对大鼠骨骼肌线粒体自噬相关蛋白表达的影响及相关机制。方法20只雌性SD大鼠随机分为对照组和模型组各10只,模型组采用卵巢摘除+地塞米松给药法制备肌少症模型(卵巢摘除7 d后腹部皮下注射地塞米松5 mg/kg,连续给药7 d);对照组不予任何操作,正常喂养。2组造模前及造模第1、4、7、10、13、16、19、22天测量体质量;造模第22天应用抓力测定仪测量抓力,并测量大鼠双侧腓肠肌质量,计算腓肠肌指数;采用HE染色观察腓肠肌组织形态;采用Western blot法检测腓肠肌组织微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)、Beclin1、磷酸化腺苷酸活化蛋白激酶(p-AMPK)、磷酸化失调51样激酶-1(p-ULK1)、Atg5蛋白相对表达量。结果模型组造模前及造模第1、4、7、10、13天体质量逐渐升高,第16、19、22天体质量逐渐降低;对照组造模前及造模第1、4、7、10、13、16、19、22天体质量逐渐升高;造模前及造模第1、4、7天,2组体质量比较差异均无统计学意义(P>0.05);造模第10、13、16、19、22天,模型组体质量均低于对照组(P<0.05)。模型组造模第22天抓力低于造模前(P<0.05);对照组造模第22天抓力与造模前比较差异无统计学意义(P>0.05);造模前,2组抓力比较差异无统计学意义(P>0.05);造模第22天,模型组抓力、左侧和右侧腓肠肌质量、腓肠肌指数[(234.52±52.21)g、(1.62±0.01)g、(1.67±0.01)g、(1.32±0.01)%]均低于对照组[(506.93±42.81)g、(2.45±0.10)g、(2.34±0.05)g、(1.84±0.05)%](P<0.05)。造模第22天,对照组腓肠肌肌纤维结构完整,排列紧密,肌纤维间无炎症细胞浸润;模型组腓肠肌肌纤维排列紊乱,肌纤维轻度萎缩、少量自溶,纤维束膜轻度破裂,肌纤维间少量淋巴细胞浸润。模型组腓肠肌组织LC3-Ⅱ、Beclin1、p-AMPK、p-ULK1、Atg5蛋白相对表达量(0.70±0.07、0.65±0.02、0.60±0.01、0.38±0.01、0.76±0.02)均低于对照组(0.92±0.03、0.80±0.01、0.73±0.03、0.51±0.02、1.15±0.04)(P<0.05)。结论肌少症大鼠骨骼肌线粒体自噬信号通路AMPK/ULK1下调,自噬水平降低。

Objective To investigate the influence of sarcopenia on the expression of autophagy related proteins in skeletal muscle mitochondria and the related mechanisms.Methods Twenty female SD rats were randomly divided into control group and model group,with 10 rats in each group.Sarcopenia rat models were prepared in model group by ovarian extraction plus 5 mg/kg dexamethasone administration subcutaneously 7 days later continuously for 7 days.Control group was fed normally without any treatment.The body mass was measured before modeling and by day 1,4,7,10,13,16,19 and 22 after modeling in two groups.By day 22 after modeling,the gripping force was measured with a gripping force meter,and the bilateral gastrocnemius mass was measured to calculate the gastrocnemius index.The morphology of gastrocnemius was observed by HE staining.The relative expressions of microtubule-associated protein 1light chain 3-Ⅱ(LC3-Ⅱ),Beclinl,phosphorylated AMP-activated protein kinase(p-AMPK),phosphorylated uncoordinated 51 like kinase-1(p-ULK1)and Atg5 proteins in gastrocnemius were detected by Western blot assay.Results In model group,the body mass increased gradually before and by day 1,4,7,10 and 13 after modeling,and decreased gradually by day 16,19 and 22 after modeling.In control group,the body mass increased gradually before and by day 1,4,7,10,13,16,19 and 22 after modeling.The body mass showed no significant differences before and by day 1,4 and 7 after modeling between two groups(P>0.05),and was lower in model group than that in control group by day 10,13,16,19 and 22 after modeling(P<0.05).The gripping force was lower by day 22 after modeling than that before modeling in model group(P<0.05),and showed no significant difference in control group(P>0.05).The gripping force showed no significant difference before modeling between two groups(P>0.05).The gripping force,left and right gastrocnemius masses,and gastrocnemius index were lower in model group[(234.52±52.21)g,(1.62±0.01)g,(1.67±0.01)g,1.32±0.01]than those in control group[(506.93±42.81)g,(2.42±0.10)g,(2.34±0.05)g,1.84±0.05]by day 22 after modeling(P<0.05).By day 22 after modeling,the muscle fibers of gastrocnemius were intact and tightly arranged,and there was no inflammatory cell infiltration between the muscle fibers in control group.In model group,the muscle fibers of gastrocnemius were disordered,the muscle fibers were slightly atrophied and a little autolysis,the fiber bundle membrane was slightly broken,and there was a little lymphocyte infiltration between the muscle fibers.The relative expressions of LC3-Ⅱ,Beclin1,p-AMPK,p-ULK1 and Atg5proteins were lower in model group(0.70±0.07,0.65±0.02,0.60±0.01,0.38±0.01,0.76±0.02)than those in control group(0.92±0.03,0.80±0.01,0.73±0.03,0.51±0.02,1.15±0.04)(P<0.05).Conclusion The autophagy signaling pathway AMPK/ULK1 is down-regulated in skeletal muscle mitochondria of rats with sarcopenia,and the level of autophagy decreases.