Study On Orotective Effect And Mechanism Of Angelica Sinensis Polysaccharide On TNF-?-induced Injury In Mouse C2C12 Myoblasts

Background:Skeletal muscle,composed of muscle fibers and connective tissue,is the main store of human protein,containing 50%-75%of the total body protein and accounting for about 40%of the total body weight.Skeletal muscle atrophy is a decline in muscle mass and function that can be seen in a variety of physiological or pathological conditions,including aging,prolonged bed rest,severe burns,and a variety of serious chronic diseases,such as chronic heart failure,chronic obstructive pulmonary disease,chronic kidney failure,AIDS,cancer,and malnutrition.From the microscopic point of view,the essence of skeletal muscle atrophy is the reduction in the volume and quantity of muscle fibers that make up skeletal muscle.A large number of studies have confirmed that ATP-dependent Ubiquitin-proteasome system(UPS)is involved in the occurrence of skeletal muscle atrophy by Ubiquitin-mediated protein degradation.In recent years,more and more studies have shown that Oxidative stress(OS)is widely involved in the occurrence and development of skeletal muscle atrophy.Reactive oxygen species(ROS)can damage skeletal muscle satellite cell(MSC)and affect the repair and regeneration of skeletal muscle tissue.In addition,apoptosis of skeletal muscle cells leads to a decrease in the number of muscle fibers that make up skeletal muscle,which is also one of the causes of skeletal muscle atrophy.Angelica sinensis polysaccharide(ASP)is the main water-soluble component of Angelica sinensis.It has the functions of promoting hematopoiesis,regulating immunity,anti-tumor and anti-oxidation,but its role in skeletal muscle atrophy has not been reported.Objective:C2C12 myoblasts induced by tumor necrosis factor-alpha(TNF-?)were used as research objects to investigate the effect of Angelica sinensis polysaccharide(ASP)on skeletal muscle atrophy and its mechanism.Methods:C2C12 myoblasts were induced by TNF-? to establish skeletal muscle atrophy model,and CCK-8 method was used to detect the effects of different concentrations of ASP on cell viability to determine the appropriate drug intervention concentration.The experiment was divided into 4 groups,including control group,TNF-? model group,ASP 20?g/ml group and ASP 60 ?g/ml group.HE staining was used to detect the transverse diameter of muscle tubes in each group,and the ROS level in cells in each group was detected by the ROS assay kit.Corresponding detection kits were used to detect the activity of antioxidant enzymes superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-Px),and Malondiald-ehyde(MDA)level.Hoechst 33342 staining was used to evaluate the rate of apoptosis.Expression of MAFbx and MuRF-mRNA was detected by RT-PCR.Western blot(WB)was used to detect the level of Akt,mTOR,p70S6K,Fox03a,MAFbx,and MuRF-1,key proteins in protein synthesis and catabolic pathways.The expression level of apoptosis-related proteins Bcl-2,Bax,and cleaved caspase-3 was also detected by WB.Result:1.CCK-8 test results showed that 80?g/ml and above concentration of ASP had significant inhibitory effect on C2C12 myoblasts activity(P<0.01),while 60 ?g/ml and below concentration of ASP had no significant effect on C2C12 cell activity(P>0.05);2.HE staining results showed that TNF-? significantly reduced the transverse diameter of C2C12 muscle tubes(P<0.01).After ASP intervention,compared with TNF-? model group,the transverse diameter of C2C12 muscle tubes was significantly increased(P<0.01);3.RT-PCR showed that ASP could down-regulate the expression of MAFbx and MuRF-1 mRNA.WB showed that ASP could up-regulate the expression of Akt,mTOR and p70S6K and down-regulate the expression of FoxO3a,MAFbx and MuRF-1;4.ASP could increase the activity of SOD,CAT and GSH-Px,and reduce the level of MDA and the production of ROS;5.ASP could inhibit the apoptosis of C2C12 myoblasts induced by TNF-?,up-regulate the expression of Bcl-2,and down-regulate the expression of Bax and cleaved caspase-3.Conclusion:Angelica sinensis polysaccharide(ASP)can improve the skeletal muscle atrophy induced by TNF-?,and its mechanism is related to regulating protein synthesis and degradation,reducing oxidative stress and inhibiting apoptosis.