The Effect And Melocular Mechanism Of Caylcosin In Chronic Kidney Disease Induced Skeletal Muscle Atrophy Based On AMPK/SKP2/CARM1 Signaling

Objective:Skeletal muscle atrophy is a common serious complication of chronic kidney disease(CKD).More and more studies have shown that oxidative stress and autophagy are very important molecular mechanisms of skeletal muscle atrophy.Calycosin is the main active ingredient of Astragalus.It exerts anti-inflammatory,anti-oxidation,and anti-autophagy effects,but its role in skeletal muscle atrophy of CKD is unclear currently.The purpose of this article is to explore the role and mechanism of calycosin in skeletal muscle atrophy of CKD.Methods:5/6 nephrectomy(5/6 Nx)was subjected to make a CKD rat model.After Shenshuai Nutritional Capsules(SSYYJN)treatment,the mRNA and protein were isolated and extracted from skeletal muscle in rats and RNA sequencing was performed to identify the differentially expressed genes.qRT-PCR and western blot were used to detect the level of co-activator associated arginine methyltransferase 1(CARM1)in muscles.The siRNA-CARM1 interference plasmid were constructed by siRNA interference technology and transfected into C2C12 myotube.In addition,Tumor necrosis factor-α(TNF-α)was used to induced C2C12 myotube atrophy.Cell activity,apoptosis ratio,antioxidant enzyme activity,autophagy gene expression,and skeletal muscle atrophy gene levels were performed to clarify the effect of CARM1 on the phenotype of C2C12 cells.Then the machine learning molecular docking method was used to screen and predict small molecule drugs that could bind to the CARM1 active region from the active ingredients of SSYYJN,and calycosin was selected as best Chinese medicine monomer that could work with CARM1.Then,the serum creatinine(Scr),blood urea nitrogen(BUN),serum albumin(ALB),Hemoglobin(Hb),renal histopathological morphology,rat weight,skeletal muscle mass,muscle cross-sectional area,Muscle-specific RING finger protein 1(MuRF1),Muscle atrophy F-Box(MAFbx)and other indicators were used to evaluate the effect of calycosin and Compound α keto acid(KA,Positive control group)on renal and skeletal muscle atrophy in CKD rats.The level of Superoxide Dismutase(SOD),Catalase(CAT),Glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)was used to assess the regulation of calycosin on oxidative stress in CKD skeletal muscle.The number of autophagosomes and mitochondrial morphology were observed by transmission electron microscope.The levels of LC3A/B and Atg7 were detected to explore the effect of calycosin on autophagy in CKD skeletal muscle by immunohistochemistry and Western blot.Effects of calycosin on oxidative stress and autophagy in TNF-αinduced C2C12 myoblasts.Finally,the key protein levels of AMPK/SKP2/CARM1 signal transduction pathway in CKD skeletal muscle and C2C12 cells were detected by western blot.The protective role of calycosin in skeletal muscle atrophy was reversed by AMPK activator,and its target was clarified.Results:38 differentially expressed gene were found between CKD model and SSYYJN group by RNA sequencing.CARM1 was found to be one of these genes and was highly expressed in skeletal muscle tissues through gene information platform analysis.This study confirmed that CARM1 mRNA expression and protein levels were significantly up-regulated in CKD skeletal muscles and down-regulated with SSYYJN treatment by qRT-PCR and western blot.Compared with the control plasmid,after siRNA CARM1 interfering in TNF-α induced C2C12,cell apoptosis,level of oxidative stress and autophagy related genes expressions were reduced.Importantly,myotube atrophy was mitigated.The molecular docking screened out the calycosin was the best one in those could combine with CARM1 active area.In vivo experiments,we have found that calycosin and KA significantly improved body weight,reduced the levels of Scr and BUN,increased the levels of ALB and Hb,inhibited the expression of TGF-β.Calycosin and KA improved renal function and alleviated renal fibrosis in CKD rats.Calycosin also increased the masses of gastrocnemius muscles,tibialis anterior muscles and soleus muscles,improved the cross-sectional area of muscle fibers and reduced the levels of muscle atrophy molecules MuRF1 and MAFbx.In addition,calycosin inhibited cell apoptosis,increased the antioxidant enzymes SOD,CAT and GSH-Px activity and reduce the level of MDA in skeletal muscle.Furthermore,the formation of autophagosomes and the level of LC3 A/B and Atg7 were reduced in skeletal muscle with calycosin administration.In vitro experiments,the results showed that calycosin inhibited myotube apoptosis,reduced ROS production and autophagy activation,and alleviated myotube atrophy in TNF-α induced C2C12.Mechanically,calycosin inhibited AMPK and FOXO3a phosphorylation,increased SKP2 levels which promotes CARM1 degradation,resulted in down-regulation of CARM1 and H3R17me2a and decreasing the expression of LC3A/B and Atg7 autophagy genes both in CKD skeletal muscle and TNF-α induced C2C12 myotube.Interestingly,AMPK activator(AICAR)partially reversed the protective effect of calycosin in TNF-αinduced C2C12 myotube atrophy.Conclusion:CARM1 might be an important target gene for skeletal muscle atrophy.CARM 1 plasmid interfering inhibited cell apoptosis,oxidative stress and autophagy activation,and reduce myotube atrophy in TNF-α induced C2C12.Calycosin and KA improved kidney injury and reduce skeletal muscle atrophy in CKD rats.Importantly,calycosin extenuated oxidative stress and autophagy in CKD skeletal muscles and in TNF-α induced C2C12 myotube atrophy partially by regulating AMPK/SKP2/CARM1 signaling pathway.