基于Nrf2调控Pink1/Parkin介导的线粒体自噬在老年肌肉减少症骨骼肌纤维化中的作用及机制研究

Study on the effect and mechanism of mitochondrial autophagy on sarcopenia and skeletal muscle fibrosis that mediated by Pink1/Parkin based on regulation of Nrf2

目的探讨线粒体自噬在老年肌肉减少症模型小鼠中的作用,并进一步探讨其作用机制。方法选取13.5个月龄SPF级雄性C57BL/6J小鼠13只分为老龄鼠组(O组,n=6)、老龄鼠+核转录因子红系2相关因子2(Nrf2)激动剂组(O+SFN组,n=7),另3月龄SPF级雄性C57BL/6小鼠8只作为青年鼠组(Y组)。O组按体重0.1 mL/10 g给予0.1%DMSO溶液,每周3次,O+SFN组按体重0.1 mL/10 g给予1%SFN溶液,每周3次,Y组给予等体积的0.1%DMSO溶液,各组持续10周。干预10周后,检测各组小鼠的体重、腓肠肌/体重比、抓力、腓肠肌细胞活性氧水平、腓肠肌细胞线粒体膜电位和腓肠肌细胞线粒体DNA(mtDNA)拷贝数;天狼星红染色观察各组小鼠腓肠肌组织纤维化情况;蛋白质印迹法检测腓肠肌组织中纤维化相关蛋白collagen 1、collagen 3和fibronectin及自噬相关蛋白Nrf2、PINK1、Parkin、LC3、BNIP3和FUNDC1的相对表达。结果干预10周后,O组和O+SFN组小鼠体重均高于Y组,腓肠肌/体重比均显著低于Y组,差异均有统计学意义(P<0.05);O+SFN组小鼠的体重及腓肠肌/体重比值均显著高于O组,差异均有统计学意义(P<0.05)。干预10周后,O组和O+SFN组小鼠抓力均低于Y组,而O+SFN组小鼠抓力显著高于O组,差异均有统计学意义(P<0.05)。干预10周后,O组和O+SFN组小鼠的活性氧含量和线粒体膜电位均显著高于Y组,mtDNA拷贝数显著低于Y组,差异均有统计学意义(P<0.05);O+SFN组小鼠的活性氧含量和线粒体膜电位低于O组,mtDNA拷贝数高于O组,差异均有统计学意义(P<0.05)。天狼星红染色观察可见,O组和O+SFN组小鼠的胶原纤维的比例显著高于Y组,O+SFN组的胶原纤维比例低于O组。干预10周后,O组和O+SFN组小鼠腓肠肌组织中collagen 1、collagen 3和fibronectin蛋白的相对表达均显著高于Y组,而Nrf2、BNIP3、FUNDC1、PINK1和Parkin蛋白的相对表达均显著低于Y组,差异均有统计学意义(P<0.05);O+SFN组小鼠腓肠肌组织中collagen 1、collagen 3和fibronectin蛋白的相对表达均低于O组,而Nrf2、BNIP3、FUNDC1、PINK1和Parkin蛋白的相对表达均高于O组,差异均有统计学意义(P<0.05)。结论骨骼肌纤维化是老年肌肉减少症发生发展的重要机制之一,激活Nrf2可上调PINK1/Parkin信号通路而对线粒体自噬发挥保护作用,进而抑制骨骼肌纤维化,缓解肌肉减少症,可作为临床防治老年肌肉减少症的新思路和新切入点。

Objective To investigate the effect of mitochondrial autophagy on sarcopenia mice,and ulteriorly explore its potential mechanism.Methods Thirteen 13.5 month old SPF grade male C57BL/6J mice were selected and divided into an elderly mouse group(O group,n=6),an elderly mouse+nuclear factor-erythroid 2-related factor2(Nrf2)agonist group(O+SFN group,n=7),and eight 3-month old SPF grade male C57BL/6 mice were selected as the young mouse group(Y group).O group was given 0.1%DMSO solution at weight of 0.1 mL/10 g,three times a week.O+SFN group was given 1%SFN solution at weight of 0.1 mL/10 g,three times a week.Y group was given an equal volume of 0.1%DMSO solution,and each group lasted for 10 weeks.After 10 weeks of the intervention,body mass,gastrocnemius muscle/body mass ratio,grip,reactive oxygen species level of gastrocnemius cells,mitochondrial membrane potential and mitochondrial DNA(mtDNA)copy number of gastrocnemius cells were examined in each group of mice.The fibrosis of gastrocnemius tissue was observed by Sirius red staining.The relative expression of fibrosis-related proteins collagen 1,collagen 3 and fibronectin,and autophagy-related proteins,Nrf 2,PINK 1,Parkin,LC3,BNIP 3,and FUNDC1 in gastrocnemius muscle tissue were detected by Western blotting.Results After 10 weeks of intervention,the body weight in the O group and the O+SFN group mice was higher than that in the Y group,and the gastrocnemius muscle to body weight ratio was significantly lower than that in the Y group,the differences were statistically significant(P<0.05);The weight and gastrocnemius muscle weight ratio of the O+SFN group mice were significantly higher than those of the O group,and the differences were statistically significant(P<0.05).After 10 weeks of intervention,the grip of O group and the O+SFN group mice was lower than that of the Y group,while the grip of O+SFN group mice was higher than that of the O group,and the differences were statistically significant(P<0.05).After 10 weeks of intervention,the reactive oxygen species content and mitochondrial membrane potential of the O group and O+SFN group mice were significantly higher than those of the Y group,and the mtDNA copy number was significantly lower than that of the Y group,and the differences were statistically significant(P<0.05);the reactive oxygen species content and mitochondrial membrane potential of the O+SFN group mice were lower than those of the O group,and the mtDNA copy number was higher than that of the O group,and the differences were statistically significant(P<0.05).Sirius red staining showed that the proportion of collagenous fiber of O group and O+SFN group was more than group Y,while O+SFN group was less than O group.After 10 weeks of intervention,the relative expression of collagen 1,collagen 3 and fibronectin of O group and O+SFN group were higher than those of group Y,while Nrf2,BNIP3,FUNDC1,PINK1 and Parkin were lower than those of group Y,and the differences were statistically significant(P<0.05);the relative expression of collagen 1,collagen 3 and fibronectin of O+SFN group were lower than those of O group,while Nrf2,BNIP3,FUNDC1,PINK1 and Parkin were higher than those of O group,and the differences were statistically significant(P<0.05).Conclusion Skeletal muscle fibrosis is one of the important mechanism of sarcopenia,activation of Nrf2 can up-regulate PINK1/Parkin signal pathway and exert protection effect on mitochondrial autophagy,which can inhibit skeletal muscle fibrosis and relief sarcopenia,which can regarded as new thought and breakthrough point for clinical prevention and treatment of sarcopenia.