The Effects And Mechanisms Of Saikosaponin A And D On Muscle Atrophy In Chronic Kidney Disease Based On PI3K/AKT/Nrf2 Pathway

BackgroundSkeletal muscle atrophy,a common complication of chronic kidney disease(CKD),leads to a decline in the quality of life in CKD patients.Oxidative stress is essential in the progression of CKD-related muscle atrophy.Modern pharmacological research reveals that Saikosaponin A(SSA)and Saikosaponin D(SSD)exert diverse pharmacological functions such as antioxidant and anti-inflammatory.ObjectivesThe purpose of this study was to investigate the effects of SSA and SSD on CKD complicated with muscle atrophy and explored their underlying mechanisms based on PI3K/AKT/Nrf2 pathway.MethodsIn part one,skeletal muscle atrophy model was established using 5/6 nephrectomized mice in vivo.C57BL/6 mice were divided into four groups:Sham operation group,CKD model group,SSA group and SSD group.Serum creatinine(Scr),Blood urea nitrogen(BUN)and kidney histomorphology were used to evaluate the effects of SSA and SSD on the renal function of CKD mice.Aspartate aminotransferase(AST)and Alaninetransaminase(ALT)were measured to evaluate the effects of SSA and SSD on the liver function of CKD mice.Value for wet weight/body weight ratio,cross-sectional area(CSA)of muscle fibers,the type of muscle fiber,Dystrophin,Myogenic determination(MyoD)and Muscle-specific RING finger protein 1(MuRF-1)were performed to explore the therapeutic effects of SSA and SSD on CKD-induced myotube atrophy.Tumor necrosis factor α(TNF-α),Interleukin 6(IL-6),Interleukin 1β(IL-1β)and Interleukin 10(IL-10)were used to assess the effects of SSA and SSD on the inflammatory level of CKD mice.The generation of Reactive oxygen species(ROS)and the activities of Superoxide dismutase(SOD),Catalase(CAT)and total glutathione(T-GSH)were used to investigate the effects of SSA and SSD on th e oxidative stress level of CKD mice.The key protein levels of PI3K/AKT/Nrf2 pathway were detected to explore the targets of SSA and SSD in improving CKD-induced skeletal muscle atrophy.In the second part,myotube atrophy model was adopted with Dexamethasone(Dex)-managed C2C12 myotube in vitro.Cells were divided into four groups:Control group,Dex model group,SSA group and SSD group.The inner diameter of C2C12 myotube was performed to explore the therapeutic effects of SSA and SSD on Dex-induced myotube atrophy.The generation of ROS and malondialdehyde(MDA),and the level of CAT were used to investigate the effects of SSA and SSD on the oxidative stress level of C2C12 cells.The key protein levels of PI3K/AKT/Nrf2 pathway were detected to explored the targets of SSA and SSD in Dex-induced myoatrophy.In the last part,PI3K/AKT inhibitor LY294002 and Nrf2-siRNA were used to verify the target of SSA in Dex-induced myoatrophy.ResultsIn vivo,treatment with SSA and SSD significantly lowered the levels of Scr and BUN and against kidney damage in CKD mice.There were no significant differences in the distribution of serum AST and ALT among the four groups.SSA and SSD increased the wet weight/body weight ratio,CSA of the muscle fibers and the proportion of slow muscle fibers in CKD mice.These two components enhanced the expression of Dystrophin and MyoD,and suppressed the expression of MuRF-1.SSA and SSD exerted great effects on the down-regulation of proinflammatory cytokines(e.g.TNF-α,IL-6 and IL-1β)and the up-regulation of anti-inflammatory cytokine IL-10 in CKD mice.These two agents inhibited the excessive accumulation of ROS and increased the activities of antioxidant enzymes.SSA and SSD enhanced the expression of PI3K,p-AKT，p-mTOR,p70S6K,Nrf2,p62,NQO1 and HO-1 in the muscles of CKD mice.In vitro,SSA and SSD increased the inner diameter of C2C12 myotube.These two antioxidants inhibited the generation of ROS and MDA,and enhanced the expression of CAT.SSA and SSD enhanced the expression of p-AKT,p-mTOR,p70S6K,Nrf2 and HO-1 in Dex-induced C2C12 myotube.Finally,LY294002 reversed the effect of SSA on the upregulation of p-AKT,p-mTOR,Nrf2 and HO-1,as well as the downregulation of ROS and MDA.Knocking out Nrf2 reversed the effect of SSA on the upregulation of p62,NQO1,HO-1 and MyoD,as well as the downregulation of MDA and MuRF-1.ConclusionSSA and SSD improved CKD-related muscle atrophy and Dex-induced myotube atrophy by reducing oxidative stress through the PI3K/AKT/Nrf2 signaling pathway.