The Effect And Mechanism Of Formononetin On CKD Muscle Atrophy Based On Myostatin Mediated Satellite Cell Function And PI3K/Akt/FoxO3a Pathway

BackgroundMuscle atrophy is a common complication of chronic kidney disease(CKD).The pathogenesis of CKD muscle atrophy is complex,and the current treatment is very limited.To explore the molecular mechanism of CKD muscle atrophy and actively seek effective treatment drugs have become a major problem to be solved in the field of kidney disease.Mounting evidence has shown that myostatin plays an important role in CKD muscle atrophy.Our previous study found that Shenshuai nutrition capsule(SSYYJN)significantly improved CKD muscle atrophy.Through transcriptome sequencing analysis,we found that myostatin may be one of the important targets of SSYYJN in improving CKD muscle atrophy.Formononetin is the main active component of myostatin in Astragalus membranaceus,which is the main prescription of SSYYJN.Studies have shown that formononetin has many pharmacological effects,such as anti-inflammatory,antioxidant and so on.However,the role of formononetin in CKD muscle atrophy has not been reported.ObjectivesThis study was conducted to investigate the therapeutic effect of formononetin on CKD muscle atrophy and explore its molecular mechanism based on myostatin mediated satellite cell function and PI3K/Akt/FoxO3a pathway.Methods5/6 nephrectomized rats and tumor necrosis factor-α(TNF-α)-induced C2C12 myoblasts were used for in vitro and in vivo models of muscle atrophy.First,the effective component formononetin binding with myostatin was screened from SSYYJN by molecular docking technology.The renal protective effects of formononetin on CKD rats were evaluated by serum creatinine(Scr),blood urea nitrogen(BUN)and Cystatin C.Values for body weight,muscle volume,wet weight to body weight ratio,cross-sectional area of muscle fibers,hemoglobin(Hb),albumin(ALB),transferrin(TF),prealbumin(PA),the expression levels of Muscle-specific RING finger protein 1(MuRF-1)and Muscle atrophy F-box(MAFbx/Atrogin-1)were measured to investigate the therapeutic effect of formononetin on CKD muscle atrophy.Second,the expression of myostatin and the levels of inflammatory related factors such as serum C-reactive protein(GRP),tumor necrosis factor-α(TNF-α),interleukin 6(IL-6)and IL-1β in vivo and vitro was determined to clarify the regulatory effect of formononetin on inflammation and myostatin.We also detected the nuclear factor-κB(NF-κB)pathway related proteins to elucidate the possible upstream mechanism of formononetin inhibiting the expression of myostatin in vitro.Then,the expressions of myogenic determination(MyoD)and myogenin in muscles of CKD were detected to clarify the improvement effect of formononetin on muscle satellite function of CKD rats.To further investigate the effects of formononetin on the proliferation and differentiation of C2C12 cells,the cell activity of C2C12 cells and the expression of MyoD,myogenin and myosin heavy chain(MyHC)were detected.The phosphorylation levels of phosphatidylinositol 3-kinase(PI3K),Akt and forkhead box containing protein O subfamily-3 a(FoxO3a)in vitro and in vivo were detected to investigate the effect of formononetin on PI3K/Akt/FoxO3a pathway.Finally,overexpression of myostatin in vitro was used to verify the target of formononetin and elucidate the regulatory mechanism of formononetin on CKD muscle atrophy in vitro and in vivo.ResultsThe results of qRT-PCR and Western blot showed that SSYYJN significantly inhibited the expression of myostatin in muscle of CKD rats.The results of molecular docking showed that when SSYYJN combined with myostatin active region,the active component with the least binding energy was formononetin.The results showed that formononetin significantly reduced the levels of Scr,BUN and Cystatin C in CKD rats.Values for body weight,muscle weight,cross-sectional area of muscle fibers,muscle volume,and wet weight to body weight ratio were significantly larger and the levels of Hb,ALB,TF and PA were significantly increased in the formononetin treatment rats.Furthermore,formononetin significantly suppressed the expression of MAFbx and MuRF-1 in the muscles of CKD rats and TNF-α-induced C2C12 myoblasts.In addition,formononetin significantly inhibited the expressions of myostatin,CRP,TNF-α,IL-6,IL-1β and the activation of NF-κB pathway in vivo and vitro.The mechanism of formononetin improving skeletal muscle atrophy in CKD rats was studied and the results showed that formononetin significantly increased the levels of MyoD,myogenin,p-PI3k,p-Akt and p-FoxO3a in CKD rats.At the same time,formononetin significantly increased the cell activity and the levels of MyoD,myogenin,MyHC,p-PI3k,p-Akt and p-FoxO3a in C2C12 cells.Notably,Overexpression of myostatin significantly decreased the expression of MyoD,myogenin,MyHC,p-PI3k,p-Akt and p-FoxO3a in C2C12 cells and reversed the improvement effect of formononetin on the muscle satellite cells function and PI3K/Akt/FoxO3a pathway.ConclusionsIn conclusion,myostatin might be an important target gene of SSYYJN to improve CKD muscle atrophy.Formononetin,a small molecule active component combined with myostatin in SSYYJN,improved CKD muscle atrophy by inhibiting myostatin mediated muscle satellite cell function and PI3K/Akt/FOXO3a pathway.