Effect And Mechanism Of Matrine On The Rugulation Of Cancer Cachexia Induced Skeletal Muscle Atrophy By BDK

Cancer cachexia(CC)is a metabolic,multi-factorial and irreversible syndrome.Its main feature is weight loss,especially the consumption of skeletal muscle and fat.Loss of skeletal muscle causes mobility disorders in patients with cancer cachexia,reduced tolerance to cancer treatment,and increased complications and mortality.In contrast,previous studies have reported that prolonging the survival rate of patients with cancer cachexia by preserving skeletal muscle mass.In addition,in 2011,multinational experts formed a consensus,"the definition and classification of cancer cachexia-international consensus ",redefining the cancer cachexia,also stressed the importance of skeletal muscle atrophy on the incidence and develop of cancer cachexia.However,there has been no effective drug developed for the clinical treatment of skeletal muscle atrophy caused by cancer cachexia so far.The clinical study of cancer cachexia biomarkers was carried out in our previous research.Results showed that metabolic changes were not only an important feature of cancer cachexia,but also suggested that key limiting metabolic enzymes expression or activity abnormalities of metabolite biomarkers were important characteristics of cancer cachexia.It could be used as the target of drug intervention in cancer cachexia.Further studies have shown that cancer cachexia skeletal muscle atrophy was closely related to the metabolic process of branched amino acid(BCAA).The restricted irreversible metabolic enzyme of BCAA is the Branched-chain alpha-keto acid dehydrogenase complex(BCKDC).Our preliminary experimental studies showed that BCKDC protein activity increased significantly in the gastrocnemius of cancer cachexia mice and dexamethasone-treated C2C12 myotube.BCKDC is inactivated by phosphorylation of BDKDC kinase(BDK)and actived by dephosphorylation BCKDC phosphatase(BDP).Previous studies found that interfering with BDK expression or specifically inhibiting BDK activity caused an increase in BCKDC activity accompanied with decreased expression of Myosin heavy chain(MHC),MyoD,and MyoG.Therefore,this study suggests that inhibition of BDK is a new mechanism of cancer cachexia induced skeletal muscle atrophy.Matrine is one of the main components of Sophora flavescens.It is reported that matrine has a wide range of effects in anti-hepatitis,myocardial injury,fibrotic diseases and tumors.In recent years,studies have shown that matrine also has a certain effect on anti-cancer cachexia.Concurrently,the China Food and Drug Administration(CFDA)has approved matrine injection for clinical prevention and treatment of cancer cachexia.However,the mechanism of matrine on cancer cachexia is still unclear.Especially,its effect on skeletal muscle still remains further studies.Preliminary experimental studies showed that matrine increased the expression of BDK mRNA while improving dexamethasone-induced C2C12 myotube atrophy,increasing MyoD and MHC expression.However,after knocking down the BDK gene in C2C12 myotubes,the reversal effect of matrine on the down-regulation of dexamethasone-induced MyoD and MHC expression diminished.Therefore,we proposed the hypothesis that matrine regulates muscle protein synthesis and degradation through BDK,thereby improving cancer cachexia skeletal muscle atrophy.Purpose: 1.To clarify the effect of matrine on the improvement of cancer cachexia skeletal muscle atrophy.2.To elucidate the regulation of BDK on skeletal muscle atrophy and its effects on muscle protein synthesis and degradation.3.To explore the relationship between matrine ameliorates cachexia skeletal muscle atrophy and BDK regulation.Methods: 1.To establish a cancer cachexia skeletal muscle atrophy model by using CT26 mice colon cancer cells.The effects of matrine on CT26 tumor growth,body weight,organ quality,food intake and serum inflammatory factors were measured.HE staining,qPCR,Western blot and immunofluorescence staining were used to detect the effects of matrine on skeletal muscle fibers and the regulation of E3 ubiquitin ligase and MHC expression.2.Dexamethasone(Dex),tumor necrosis factor alpha(TNF?)and CT26 cell culture supernatant were used for establishing C2C12 cell atrophy models in vitro,respectively,followed by matrine treatement.Immunofluorescence staining was used for observing C2C12 myoblast differentiation and C2C12 myotube atrophy.The effect of matrine on the expression of E3 ubiquitin ligase and MyoD were analyzed by Western blot,qPCR and immunofluorescence staining to reveal its function on the synthesis and degradation of muscle protein.Finally,Western blot was used to analyze the effect of matrine on the signaling pathways involved in muscle protein synthesis and degradation.3.BDK knockdown(AdshBDK)and overexpression(AdBDK)of adenovirus were constructed for infecting of C2C12 myoblast or myotube.MTT was used to detect the effect of BDK knockdown on myotubes activity.The effects of BDK knockdown/overexpression on myoblast differentiation and on the size of the myotubes was examined by immunofluorescence staining.qPCR and Western blot were used to detect the effects of BDK on muscle protein synthesis and degradation.Secondly,the effects of BT2,a specificity BDK activity inhibitor,on C2C12 myotube atrophy and muscle protein synthesis and degradation were detected by immunofluorescence staining,qPCR and Western blot.Immunofluorescence staining and Western blot were also used to detect the protective effects of BDK overexpression on Dex-induced myotube atrophy.Finally,Western blot was used to detect the effects of BDK on the signal pathways involved in muscle protein synthesis and degradation.4.The AdshBDK/AdBDK were used to infect the mice gastrocnemius muscle to study the effects of BDK knockdown/overexpression on skeletal muscle mass,muscle fiber size and muscle protein synthesis and degradation in normal mice.Meanwhile,the protective effects of BDK overexpression on skeletal muscle atrophy induced by CT26 cancer cachexia were investigated.Finally,Western blot was used to detect the effects of BDK knockdown or overexpression on the signal pathways involved in muscle protein synthesis and degradation in the gastrocnemius muscle.5.The CT26 mice cancer cachexia model and Dex-induced C2C12 myotube atrophy model were estabilished as previous.With knocking down of the BDK gene,HE-staining and Western blot were used to determind the myofiber size,the expression of muscle proteins and the signaling pathway involved in the synthesis and degradation of muscle proteins.Results: 1.The intraperitoneal injection of matrine to mice had no significant inhibitory effect on CT26 tumor growth,while it alleviated the weight loss of cachexia mice,improved the performance of mice organs and reduced the concentration of serum inflammatory factors,such as TNF?,IL-6 and IL-1?.Meanwhile,the mechanism studies showed that matrine preserved the skeletal muscle mass and reduced the expression level of E3 ubiquitin ligase in skeletal muscle of cancer cachexia mice.2.In vitro studies,matrine promoted the differentiation of C2C12 myoblast,improved the inhibitory effect of Dex on the differentiation of C2C12 myoblast,and also counteracted Dex-induced C2C12 myotube atrophy.Mechanism studies showed that matrine reduced degradation of skeletal muscle protein by inhibiting the expression of E3 ubiquitin ligase and upregulating the expression of MyoD.Signal pathway studies showed that matrine up-regulated the phosphorylation levels of Akt,mTOR and FoxO3? in C2C12 myotubes,while wortmanin,a specific PI3 K inhibitor,partly blocked the regulation of above signal protein by matrine.This indicates that the effect of matrine on muscle atrophy has relationship with Akt/mTOR/FoxO3? signaling pathway.3.Dex,TNF? and CT26 culture medium supernatant all lead to lower expression of BDK in C2C12 myotube.Knockdown of BDK also inhibited the differentiation of C2C12 myoblast and induced atrophy of myotube.It was associated with the decreased expression of MyoD and MyoG,and upregulation of E3 ubiquitin ligase.However,the effect of BDK overexpression on C2C12 myotube was contrary to BDK knockdown,and BDK overexpression partially reversed Dex-induced C2C12 myotube atrophy.Mechanism syudies suggested that BDK gene regulated Akt/mTOR/FoxO3? signaling pathway in C2C12 myotube.4.Animal studies showed that the expression of BDK in mice skeletal muscle with CT26 cancer cachexia was also significantly reduced.Knockdown of BDK reduced the mass of the gastrocnemius muscle and decreased the size of muscle fibers.Concurrently,the expression of MuRF1 was up-regulated and the expression of MyoD was down-regulated.BDK overexpression had the opposite effect.In addition,BDK overexpression also improved skeletal muscle atrophy in CT26 tumor cachexia mice.Signaling pathway analysis indicated that BDK up-regulates phosphorylation of Akt and mTOR.5.Matrine up-regulated the expression of BDK mRNA in cachexia mice gastrocnemius muscle and atrophied C2C12 myotube.The preservation effect of matrine on cachexia muscle fiber size and muscle protein decreased after knockdowning of BDK in gastrocnemius muscle.In addition,the reversal effect of matrine on the Dex-induced myotube atrophy and muscle protein synthesis almost diminished,and decreased phosphorylation level of Akt and mTOR were detected after knockdowning of BDK in C2C12 myotube.Conclusion:Matrine improves the symptoms of CT26 cancer cachexia,and its effect is mainly relate to the preservation of skeletal muscle mass.The analysis of action characteristics showes that matrine inhibit the expression of E3 ubiquitin ligase,therefore,reduces the degradation of skeletal muscle protein.On the other hand,it promotes the differentiation of C2C12 myoblast,improves of myotube atrophy,and up-regulates the phosphorylate level of key proteins Akt,mTOR and FoxO3? in the PI3K/Akt signaling pathway which improves skeletal muscle protein synthesis.Further mechanism studies indicates that the BDK gene plays an important role in skeletal muscle atrophy.Muscle atrophy leads to down-regulation of BDK expression,while knockdown of BDK also reduces skeletal muscle mass and causes skeletal muscle atrophy.Overexpression of BDK improves skeletal muscle atrophy due to cachexia factor.Matrine increases skeletal muscle mass and muscle fiber size by regulating BDK expression,and its mechanism is relate to the increasing phosphorylation of Akt and mTOR,promoting skeletal muscle protein synthesis,and inhibiting E3 ubiquitination ligase expression to improve muscle protein degradation.