Mechanism Of SIRT3 Mediating Mitophagy In Rat Skeletal Muscle To Regulate Sepsis-Induced Muscle Atrophy

Objective: To establish a rat model of sepsis-induced muscle atrophy,and explore the mechanism of SIRT3 mediating mitophagy in skeletal muscle to regulate sepsis induced muscular atrophy.Methods: Sixty-six adult male SD rats(about 200-300 g in body weight)were randomly divided into two groups.The first group of 30 rats were randomly divided into 5 groups(6 rats in each group):(1)Normal group(Normal);(2)Intraperitoneal injection of LPS group(S);(3)Immobilization group(I);(4)Intraperitoneal injection of LPS + immobilization group(S+I,the model group);(5)Intraperitoneal injection of LPS+ immobilization + SIRT3 agonist Honokiol group(H).The second group of 36 rats were randomly divided into 6 groups(6 rats in each group):(1)Intraperitoneal injection of LPS + immobilization group(S+I);(2)Intraperitoneal injection of LPS +immobilization + SIRT3 agonist Honokiol group(H);(3)Intraperitoneal injection of LPS+immobilization+SIRT3 agonist Honokiol + mitophagy inhibitor Mdivi-1 group(H+M);(4)Intraperitoneal injection of LPS + immobilization + mitophagy agonist Berberine group(BBR);(5)Intraperitoneal injection of LPS + immobilization+Dimethyl sulfoxide group(DMSO);(6)Intraperitoneal injection of LPS +immobilization + Normal saline group(NS).The levels of serum inflammatory factors TNF-α and IL-6 were detected by ELISA,and the changes of transverse diameter of tibialis anterior muscle were observed by HE staining.The success of modeling was evaluated by combining the two methods.Mitochondrial membrane potential(MMP),ATP and ROS levels of tibialis anterior muscle were detected to evaluate mitochondrial function.The protein expression levels of SIRT3,FOXO3 a,PINK1,Parkin and LC3-Ⅱ/Ⅰ were detected by Western Blot.Results: 1.Compared with Normal group,the levels of serum inflammatory factors TNF-α and IL-6 in S+I group were significantly increased,and body weight was decreased(P<0.01);Appear listless,stand unsteadily,limb tremors,canthus secretions increase,pyuria and loose stool.Compared with Normal group,S group and I group,the transverse diameter of tibialis anterior muscle fiber in S+I group was significantly decreased(P<0.01),suggesting the successful establishment of a sepsis-induced muscle atrophy model2.Compared with Normal group,S group and I group,mitochondrial membrane potential(MMP)level and ATP content in S+I group were significantly decreased,and ROS level was significantly increased(P<0.01),indicating mitochondrial damage in skeletal muscle of S+I group.3.Compared with Normal group,S group and I group,the protein expression levels of SIRT3,FOXO3 a,PINK1,Parkin and LC3-Ⅱ/Ⅰ in S+I group were significantly decreased(P<0.01),suggesting that the level of mitophagy in skeletal muscle of septic muscular atrophy rats decreased.4.Compared with S+I group,protein expression levels of SIRT3,FOXO3 a,PINK1,Parkin and LC3-Ⅱ/Ⅰ in tibialis anterior muscle of H group after SIRT3 agonist were significantly increased(P<0.01),and MMP level and ATP content were increased,while ROS production was significantly decreased(P<0.01).It indicates that mitochondrial function has been restored.Compared with the S+I group,the BBR group treated with Berberine enhanced mitophagy increased MMP and ATP content,and decreased ROS production(P<0.01),suggesting that promoting mitophagy can reverse sepsis-induced muscle atrophy.Compared with the H group,the MMP and ATP content of tibialis anterior muscle in the H+M group decreased(P<0.01)after the treatment of mitophagy inhibitor Mdivi-1,and ROS production increased(P<0.01),and the expressions of FOXO3 a,PINK1,Parkin and LC3-Ⅱ/Ⅰ decreased significantly(P<0.01),suggesting that inhibition of mitophagy can inhibit mitochondrial function in skeletal muscle of sepsis-induced muscle atrophy rats.Conclusion: In the process of sepsis-induced muscle atrophy,the morphology of skeletal muscle fibers in rats was changed,mitochondrial function was impaired,and the levels of SIRT3 and mitophagy decreased.Intervention of SIRT3 and mitophagy can reverse the development of sepsis-induced muscle atrophy.