Research On The Theory Of Mitochondrial Autophagy Involved In Energy Compensation Of Rat Skeletal Muscle Contusion

Research purposesAfter the blunt trauma of skeletal muscle happens,in addition to the classic vascular response and inflammatory response the damaged skeletal muscle within can also form ischemia and hypoxia environments.The formation of this environment may cause mitochondrial dysfunction,resulting in insufficient ATP production capacity.The results will inevitably have prolonged duration,incomplete recovery of postoperative function and other more serious adverse effects of rehabilitation of the injury.Mitochondrial autophagy is an important force to maintain the quality of mitochondria.It has been confirmed that mitochondrial autophagy is an important compensatory response to metabolic changes in the body.The process can promptly remove the damaged mitochondria in order to reduce the production of ROS,thereby reducing excessive ROS‘s attack on the healthy mitochondria to prevent the damage range from expanding.According to the above-mentioned basis,the hypothesis was put forward: 1.Ultrastructural changes of skeletal muscle after injury: mitochondrial morphological changes,loss of integrity,presence of mitochondrial autophagosome;2.ROS level in skeletal muscle recovery after blunt injury,Mitochondrial function decreased;3.mitochondrial autophagy was induced during the recovery period of skeletal muscle contusion,and it was most significant at early stage of injury,and mitochondrial dysfunction also occurred during this period.In this study,animal models of animal skeletal muscle were injured by weighting,the contusion of skeletal muscle during exercise was simulated,the mitochondrial autophagosome and the amount of mitochondrial autophagosome were observed by transmission electron microscope,the amount of ROS in the damaged tissue was detected and compared with mitochondrial respiratory chain enzyme complex Activity and mitochondrial autophagy-related indicators of time-course changes,changes in these indicators to observe and evaluate mitochondrial autophagy after blunt trauma,mitochondrial function changes,from a new perspective to observe,explain skeletal muscle contusion occurred,The possible mechanism of mitochondrial autophagy induction in this process was speculated.It is hoped that it will provide more theoretical support for clinical treatment,rehabilitation and prevention of blunt contusion of skeletal muscle in future.Research methodsIn 64 male Wistar rats,eight rats were randomly selected as normal control group.Another 56 rats were divided into 7 groups after the injury to the right hind leg triceps to establish blunt contusion model,8 in each group.The rats in each group were drawn from 12 h,2d,5d,7d,10 d,15d and 30 d respectively after the injury to detect the changes of AMPK?2,HIF-1?,NIX,BNIP3 Protein and m RNA Expression in Triceps Triceps.Transmission electron microscopy was used to observe ultrastructural changes of skeletal muscle and mitochondrial autophagosome.And ROS,ATP,mitochondrial transmembrane potential(? ? m),electron transport chain complex(C?-?),ATP synthase and other mitochondrial function indicators were measured.The change of mitochondrial function and whether mitochondrial autophagy occurred in the process was analyzed by detecting the changes of the above indexes during the recovery process after injury.The study deduced the mechanism of mitochondrial autophagy,the possible compensation consequences and whether mitochondrial function was damaged and explored the possible mechanisms and possible consequences of mitochondrial function in blunt trauma.Results1 The skeletal muscle ultrastructure was observed under transmission electron microscope(TEM)at different time points after skeletal muscle contusion: The presence of autophagy was observed from injury up to 30 days after injury.The amount of autophagosome in the group was more 10 days after injury.Since then the number of autophagosomes began to decline.2 There was a significant increase of metabolism-related protein AMPK?2,hypoxia-associated factor HIF-1? in the early stage of injury.Over time,the expression of the two decreased to the normal level from 5-7 days after injury until 30 days after injury.On the two m RNA expression,HIF-1? protein and its m RNA expression is not consistent.This is presumably because changes in HIF-1? expression occur mainly at the protein level with changes in endogenous oxygen levels.3 The expression of mitochondrial autophagy-related protein BNIP3 and NIX also increased at the early stage of injury.It suggested that Mitochondrial autophagy induced by both was mainly induced by hypoxia causing the occurrence of apoptosis.Both of them were directly regulated by HIF-1?.So the performance of the two was basically close to the changes of the HIF-1? level.In addition,In the two groups,a greater number of mitochondria coated with lysosomes was observed by electron microscopy.4 Mitochondrial transmembrane potential(??m)is currently considered as one of the important indicators of mitochondrial function,mitochondrial autophagy,and apoptosis.Through the observation of the index during the recovery process,it was found that ??m had obvious temporal change.At the early stage of injury(12h,2d,5d),??m decreased significantly.The change of this index was consistent with that of BNIP3 and NIX.5 Compared with the control group,ROS were increased in different degrees from 12 hours after injury up to 5 days after injury and the difference was significant.It is hypothesized that the indirect cause of the energy crisis after injury was local ischemia and hypoxia.But the direct damage to the mitochondria was caused by excessive ROS in the environment of ischemia and hypoxia.6 And 12 h,2d and 5d after injury,the activities of mitochondrial electron transport chain complexes C1 and CIII were significantly increased.This also occurs with the rise of the ROS level in the same time period.While the other two enzyme complex activity did not change significantly.Conclusion1 High concentration of ROS can directly attack the mitochondrial respiratory chain,resulting in mitochondrial damage.While the site of endogenous ROS happens to be on the mitochondrial electron transport chain because the stage of ROS rise is basically the same to the time period of C?,C? activity.This shows that mitochondrial electron transport chain reacts directly with the hypoxia by C?,C? activity rise.C?,C? is the main site of ROS production,so the damaged mitochondria at this stage produce large amounts of ROS and more than ROS removal system scavenging capacity.2 The expression of AMPK?2 and hypoxia-associated factor HIF-1? were all up-regulated during mitochondrial function changes.The number of autophagosomes in this stage was also significantly increased.It suggests that both of them may make compensatory adjustment through the synthesis of catabolic regulation,angiogenesis,mitochondrial quality control,mitochondrial generation.3 The expression of mitochondrial autophagy-related protein BNIP3 and NIX was also significantly increased during mitochondrial dysfunction.At the same time,mitochondria coated with lysosomes were also found in the damaged skeletal muscle at this stage by transmission electron microscopy.This suggested that mitochondrial autophagy was involved in the process of mitochondrial dysfunction after injury.Through mitochondrial autophagy,impaired mitochondria were removed,the production of ROS was reduced,and the expansion of the scale of injury was prevented.Although excessive mitochondrial autophagy could cause cell apoptosis,for this experiment,bilateral limb function was gradually restored and did not appear skeletal muscle necrosis indicating that the degree of mitochondrial autophagy is not excessive,the role is positive.4 Mitochondrial transmembrane potential(??m)is currently considered as one of the important indicators of mitochondrial function,mitochondrial autophagy,and apoptosis.During the recovery of skeletal muscle during blunt trauma,??m also showed a significant temporal change.At the early stage of injury(12h,2d,5d),??m decreased significantly.Combined with the BNIP3,NIX expression,we infered that mitochondrial autophagy was induced after injury and was involved in the compensatory mechanism after injury.But the change of ??m could also cause apoptosis,mitochondrial autophagy could also cause apoptosis.