Study On The Mechanism Of Excercise And Acupuncture Intervention Affecting Skeletal Muscle Endoplasmic Reticulum Stress-autophagy Response

Objective: The molecular effects of CRT,FAM134 B and LC3 were used as the starting point,to observe the occurrence of stress – autophagy reaction in skeletal muscle endoplasmic reticulum after heavy load exercise,and to analyze the changes of endoplasmic reticulum function;to observe the effect of acupuncture on stress and autophagy reaction in rats skeletal muscle after exercise,and to analyze the improved effect of acupuncture on the damage of endoplasmic reticulum;to study the molecular mechanism of stress and autophagy reaction in skeletal muscle cells.Methods: The male SD rats and isolated rat skeletal muscle cells?L6 cells?were selected as the research object.Study One was divided into a quiet control group and a motor group,the exercise group performed a heavy load downhill running.Study 2 was divided into the rats into quiet control group,simple exercise group,simple acupuncture group and exercise acupuncture group,which exercise group and exercise acupuncture group carried out a heavy load downhill running,acupuncture group and acupuncture group were treated with acupuncture.Rats in each group were divided into 0h,12 h,24h,48 h and 72 h groups according to different time,took the soleus muscle to conduct index test in the corresponding time.The concentration of Ca²⁺([Ca²⁺] ER)was determined by fluorescence probe method,the content of Ca²⁺-ATPase?SERCA?and protein disulfide isomerase?PDI?were determined by ELISA method;the expression changes of the stress protein GRP78,CRT,endoplasmic reticulum autophagy receptor protein FAM134 B and autophagy marker protein LC3 were detected by Western Blot method,the co-localization of FAM134 B and CRT,LC3 and CRT was observed by immunofluorescence double staining combined with laser confocal microscopy;the stress protein CRT located in the endoplasmic reticulum were labeled by the immunogold gold technique to label,the formation of endoplasmic reticulum autophagy in CRT were observed by Electron microscopy.Study 3 was divided into normal control group,endoplasmic reticulum stress induction agent carotene?TG?treatment group and endoplasmic reticulum stress inhibitor 4-phenylbutyric acid?4-PBA?treatment group,collected the corresponding indicators of cell testing at the same time,and used immunoprecipitation method,to confirm the interaction between endoplasmic reticulum stress protein CRT in L6 cells and autophagy receptor protein FAM134 B,FAM134B and autophagy marker protein LC3.Results:?1?The rats skeletal muscle [Ca²⁺] ER decreased sharply after heavy load exercise,SERCA and PDI dysfunction;endoplasmic reticulum stress protein GRP78,CRT expression increased,the peak appeared immediately after exercise;the expression of autophagy receptor protein FAM134 B and autophagy marker protein LC3 increased in the endoplasmic reticulum and co-localization with CRT increased,the peak appeared 12 hours after exercise;a large number of aggregation of CRT immune markers in autophagy at different stages can be seen under Electron microscopy,12 hours after exercise is the most obvious.?2?Acupuncture intervention could increase the content of [Ca²⁺] ER,SERCA and PDI in skeletal muscle of exercise rats;GRP78 and CRT protein expression decreased accordingly;FAM134B and LC3 translocation and co-localization with CRT decreased.?3?After treatment with endoplasmic reticulum stress inducer,rat skeletal muscle cell endoplasmic reticulum Ca²⁺ depleted,CRT expression up;FAM134B and LC3 were transcribed to the endoplasmic reticulum and co-localization with CRT increased;The results of immunoprecipitation showed that the interaction between CRT and FAM134 B,FAM134B and LC3 was enhanced.Conclusion:?1?Large-load exercise could induce skeletal muscle endoplasmic reticulum dysfunction,the endoplasmic reticulum stress-autophagy was then initiated by CRT,FAM134 B and LC3.?2?Acupuncture could down-regulate CRT protein expression after exercise,inhibited FAM134 B and LC3 translocation,and then effectively regulated the endoplasmic reticulum stress-autophagy to relieve skeletal muscle endoplasmic reticulum dysfunction.?3?The mechanism of endoplasmic reticulum stress and autophagy in skeletal muscle cells was: CRT protein expression was up-regulated after endoplasmic reticulum steady state imbalance,FAM134 B selectively anchors the CRT and recruits LC3,these three interacts to coordinate the occurrence of skeletal muscle cell endoplasmic reticulum stress – autophagy reaction.