Mechanism Of Heavy Load Exercise-induced Mitophagy In Rat Skeletal Muscle And Intervention Study Of Acupuncture

Objective:To observe the effect of a heavy load exercise on mitochondrial structure and function in skeletal muscle of rats and analyze the change of mitophagy protein and related proteins, and to explore the molecular mechanism of mitophagy induced by the eccentric exercise; to observe the effect of acupuncture intervention on skeletal muscle mitochondria after heavy load exercise and clarify its mechanism.Methods:The male SD rats which were used for the study were divided into two groups:control group (C group) and exercise group (E group) in the experiment one and two, and the rats in E group were got a heavy load running. In experiment three the rats were divided into blank control group (C group), exercise group (E group), acupuncture group (A group), exercise and acupuncture group (EA group). The rats in E group and EA group received an eccentric exercise, and the others in A group and EA group applied acupuncture treatment. Each group was further divided into Oh、12h、24h、48h and 72h group, and in the corresponding point of time the soleus was isolated. The mitochondrial ultrastructural changes in skeletal muscle was observed by transmission electron microscope; the content of quantitative enzyme CS and the activities of mitochondrial respiratory chain complex Hand IVwere measured by ELISA; the protein expressions of skeletal muscle COX I、PINK1 and mitochondrial Parkin、 LC3 were detected by western blot; the amount of co-localization of mitochondria respectively with Parkin、Ub、p62、LC3 was measured by immunofluorescence double labeling technique.Results:(1) After heavy load exercise, the mitochondrial structure appeared the typical abnormal changes such as swelling and accumulating under cell membrane, and formed a large number of mitophagosomes which were in different stages of maturity. The content of CS and the activities of mitochondrial respiratory chain complex Ⅱ and Ⅳ and the protein expression of COX I were significantly decreased. (2) After heavy load exercise, the expression of PINKl、Parkin、Ub、p62、LC3 were significantly elevated. The immunofluorescence results were same as the results of protein expression. (3) Acupuncture intervention after exercise, the ultrastructure of mitochondria recovery was promoted, and complete mitophagolysosome formation was induced. The reduction of CS content was suppressed, and the activities of mitochondrial respiratory chain complex Hand IV and the protein expression of COX I were significantly increased. The expression of PINK1、Parkin、Ub、p62、LC3 in mitochondria were decreased. The immunofluorescence results were same as the results of protein expression.Conclusion:(1) As a heavy load exercise could cause mitochondrial structure and function damage on skeletal muscle and form a lot of mitophagosomes, it may induce the mitophagy occurring. (2) A heavy load exercise activated PINK1/Parkin pathway, then made the Ub and p62 express in the mitochondria and promoted LC3 combining with mitochondria eventually, so they coordinated regulation of mitophagy. (3) Acupuncture intervention after exercise could decrease the expression of mitochondrial outer membrane protein PINK1, reducing the recruitment of Parkin in the cytoplasm, thereby affecting its downstream Ub and p62 mitochondria translocation and the combination of LC3 and mitochondria, regulating the autophagy-lysosome pathway and inhibitting excessive activation of mitophagy and alleviating the damage of mitochondrial structure and function.