| Objective : With aging,skeletal muscle mass and strength decline progressively,affecting the body's ability to exercise and quality of life,and increasing the incidence of neurodegenerative diseases,diabetes,tumors.Studies have shown that age-related skeletal muscle atrophy is closely related to mitochondrial dysfunction.As an important mechanism to maintain mitochondrial homeostasis,the mitochondrial quality control system mainly includes the mitochondrial unfolded protein response(UPRmt),mitophagy and mitochondrial biogenesis.Among them,UPRmt is to sense mitochondrial stress and activate the transcription process of a series of nuclear coding genes,so as to improve mitochondrial protein homeostasis and repair damaged mitochondria.Exercise can effectively improve the aging related skeletal muscle atrophy,and the mechanism is related to the mitochondrial appropriate stress.We speculate that exercise may ameliorate age-related mitochondrial degeneration through the UPRmt pathway.Currently,studies on UPRmt and aging are mainly focused on nematodes,drosophila and other model animals.Age and exercise intervention in this mouse model,to observe the process of increasing age and exercise intervention in skeletal muscle in mice,the change rule of mitochondrial force can learn and UPRmt observed UPRmt and mitophagy and mitochondrial biogenesis of temporal relations,mitochondrial quality control system of each component in the exercise intervention age related biological roles in skeletal muscle atrophy.Method : In this study,20 C57BL/6 mice aged 2 months,9 months and 16 months were bred respectively,and then the mice of the three age groups were randomly divided into the normal feeding group(C)and the aerobic exercise group(T).Mice in the aerobic exercise group were trained on the treadmill with moderate intensity exercise load(~ 54% VO2 Max).They exercised continuously for 1h every day for 5days a week for 12 weeks.Based on mice,JC-1 probe method is used to detect the mitochondrial membrane potential ? ?;H2O2 was measured by DCFH-DA probe method to reflect the ROS production level of mitochondria.Western-blotting assay was used to detect UPRmt-related proteins c-jun,CHOP,HSP60,AKT,Htr A2,SIRT3,FOXO3a and SOD2.Mitochondrial protein involved in biosynthesis of PGC-1 ?,NRF1,COX ?;Mitophagy related protein PINK1,Parkin,LC3 ? / ?,P62 expression quantity.Results:I.Effects of exercise and aging on mitochondrial membrane potential and ROS production in skeletal muscle of mice In the aging process of mice,compared with the group of 5 months old,the mitochondrial membrane potential of skeletal muscle at 12 months and 19 months was significantly reduced(P < 0.05),and the mitochondrial ROS level was significantly increased at 19 months old(P < 0.05).Compared with the quiet group,the mitochondrial membrane potential level in the training group increased significantly at 5 months,12 months and 19 months(P < 0.05),and the mitochondrial ROS level decreased significantly at 12 months and 19 months(P < 0.05).Ii.Effects of exercise and aging on UPRmt related proteins in skeletal muscle of mice In mitochondrial matrix : In the aging process of mice,the expression levels of chaperone HSP60 protein at 12 and 19 months of age were significantly reduced compared with those in the 5 month group(P < 0.05).Compared with 12 months old,the expression level of C-Jun protein at 19 months old was significantly reduced(P <0.05).There was no significant change in the expression of CHOP protein.Compared with the quiet group,the expression levels of transcription factor C-Jun and molecular chaperone HSP60 were significantly increased at 5,12 and 19 months,and the expression levels of transcription factor CHOP were significantly increased at 12 and19 months(P < 0.05).In the intermembrane space:In the aging process of mice,the expression level of protease Htr A2 protein at 12 months of age was significantly lower than that of the group at 5 months of age(P < 0.05),and the expression level of AKT protein at 19 months of age was significantly lower than that of the group at 5 months of age(P <0.05).Compared with the 12-month group,the expression level of Htr A2 protein at 19 months was significantly increased(P < 0.05),and the expression level of AKT protein at 19 months was significantly decreased(P < 0.05).Compared with the quiet group,the expression level of Htr A2 increased significantly at 12 months of age(P <0.05),while the expression level of AKT did not change significantly at 5 months,12 months or 19 months of age.In mitochondrial matrix : In the aging process of mice,the expression levels of SIRT3,FOXO3 a and SOD2 proteins at the age of 19 months were significantly reduced compared with those in the group of 5 months(P < 0.05).Compared with the quiet group,SIRT3 protein expression was significantly increased at 5,12 and 19 months,FOXOa protein expression was significantly increased at 19 months,and SOD2 protein expression was significantly increased at 12 and 19 months(P < 0.05).Iii.Effects of exercise and aging on mitochondrial biosynthesis related proteins in skeletal muscle of mice In the process of mice age,compared with five months of age group,19 months PGC-1 ? and COX ? protein expression was significantly decreased(P <0.05);There was no significant change in NRF1 protein expression.Compared with quiet group,training group PGC-1?,NRF1 and COX ? protein expression in the12 months were significantly increased(P < 0.05).Iv.Effects of exercise and aging on mitophagy related proteins in skeletal muscle of mice In the aging process of mice,compared with the group of 5 months,the expression level of PINK1 protein at 12 months was significantly increased(P < 0.05).Compared with the group of 5 months old,Parkin protein expression was significantly increased at 19 months old(P < 0.05).LC3 ? / ? P62 protein expression and no obvious change.Compared with quiet group,training group LC3 ?/? protein expression in five months,12 months and 19 months were significantly elevated,amount of PINK1 protein expression in 5 months increased significantly(P < 0.05),the amount of Parkin protein expression in a significant rise in five months and 12 months of age(P< 0.05),the amount of P62 protein expression was not significantly change.Conclusions:In this study,skeletal muscle of mice was taken as the research object to observe the effects of aging(Age of 5 months represents youth,age of 12 months represents middle age,and age of 19 months represents old age)and exercise intervention on UPRmt,mitochondrial autophagy and mitochondrial biosynthesis of skeletal muscle.Preliminary conclusions are obtained as follows:1)With aging,the activation degree of three pathways of skeletal muscle UPRmt(mitochondrial matrix non-folding protein pathway,membrane cavity pathway and matrix oxidative stress pathway)decreases progressively in middle and old age,while mitochondrial biogenesis only decreases in the old age.It is suggested that UPRmt abnormality is involved in age-related mitochondrial dysfunction earlier.2)Aerobic exercise can significantly activate three pathways of skeletal muscle UPRmt in middle-aged mice(up-regulation of molecular chaperone to restore correct protein conformation,up-regulation of protease to clear misfolded protein,up-regulation of antioxidant enzyme to inhibit oxidative stress),and up-regulation of mitophagy and mitochondrial biogenesis.It is suggested that exercise in middle age can improve the mitochondrial function of skeletal muscle by regulating multiple links of mitochondrial quality control system.3)Aerobic exercise can significantly activate skeletal muscle UPRmt in aged mice,but has no significant improvement effect on mitochondrial autophagy and mitochondrial biosynthesis.It is suggested that the improvement effect of exercise on mitochondria in old age depends on UPRmt activation.In summary,in the mitochondrial quality control system,UPRmt is more sensitive to aging and exercise intervention than mitochondrial biosynthesis and mitochondrial autophagy,and it can be used as a biological marker for early senescence muscular atrophy and a potential target for exercise simulation... |
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