Cycloastragenol Prevents The Oxidative Damage Induced By T-BHP In C2C12 Myoblasts Through Keap1/Nrf2 Pathway

Objective:Sarcopenia is a chronic degenerative disease characterized by loss of skeletal muscle mass and strength as the body ages.Studies have shown that aging is closely related to the continuous production of reactive oxygen species(ROS)and sarcopenia.In this experiment,the mice C2C12 myoblasts were induced by tert-butyl hydroperoxide(t-BHP)to investigate whether cycloastragenol(CAG),the main component of Astragalus membranaceus,could protect C2C12 myoblasts from oxidative stress.To explore the regulation mechanism of cycloastragenol on Keap1/Nrf2 antioxidant pathway,and provide treatment ideas for sarcopenia related diseases.Method:1.C2C12 myoblasts were cultured in vitro and t-BHP was used to treat C2C12 myoblasts to establish a cellular oxidative stress model.C2C12 myoblasts were treated with 0,1,10,25,50,75,100,150,200 μmol/L of t-BHP for 6 h.CCK-8 was used to detect cell viability and determine the intervention concentration of t-BHP.C2C12 myoblasts were treated with 0,0.1,0.5,1,5,10,50,100,500 μmol/L of cycloastragenol for 24 h.CCK-8 was used to detect cell viability and determine the intervention concentration of cycloastragenol.2.C2C12 myoblasts were divided into five groups: Control group,t-BHP group(100 μmol/L t-BHP),cycloastragenol low-dose group(1 μmol/L CAG+100 μmol/L t-BHP),cycloastragenol medium-dose group(10 μmol/L CAG+100 μmol/L t BHP),cycloastragenol high-dose group(20 μmol/L CAG+100 μmol/L t-BHP),and CCK-8was used to detect the effect of different concentrations of cycloastragenol on t-BHP-induced oxidative damage in C2C12 myoblasts.β-galactosidase staining was used to detect cellular senescence.EDU-488 assay was used to detect cell proliferation.The intracellular ROS levels were measured by DCFH-DA.The levels of SOD and MDA were detected by biochemical method.The relative expres sion levels of Pax7,Myo D,Keap1,Cyto-Nrf2,Nuclear-Nrf2 and HO-1 protein were detected by Western blot analysis.Nuclear translocation of Nrf2 was characterized by immunofluorescence.3.Knockdown of Nrf2,C2C12 myoblasts were divided into 6 groups(t-BHP using a concentration of 100 μmo/L and cycloastragenol using a concentration of 20μmo/L): Control group,t-BHP group,t-BHP+si-NC group,t-BHP+si-Nrf2 group,t-BHP+si-Nrf2+CAG group,DCFH-DA fluorescent probe assay was used to determine the ROS level in each group.Western blot was used to detect the expression of Pax7,Myo D and HO-1 proteins.Results:1.CCK-8 results showed that t-BHP less than 50 μmol/L had no significant effect on the viability of C2C12 myoblasts,while t-BHP greater than 75 μmol/L had obvious cytotoxic effect in C2C12 myoblasts.Low concentration of 1-100 μmol/L cycloastragenol(CAG)significantly increased cell viability,while 500 μmol/L cycloastragenol(CAG)significantly inhibited cell viability.Therefore,C2C12 myoblasts were treated with 1,10 and 20 μmol/L cycloastragenol for 24 h and 100μmol/L t-BHP for 6 h as subsequent experimental concentrations.2.After C2C12 myoblasts were induced by t-BHP,the expression of ROS and MDA increased,the expression of SOD decreased,the percentage of β-galactosidase staining positive cells increased,Ed U positive proliferating cells decreased,and the expression of proliferation-related protein Pax7 and differentiation-related protein Myo D decreased.Compared with the t-BHP group,cycloastragenol improved the viability of C2C12 myoblasts,reduced the content of ROS and MDA,increased SOD activity,and increase Ed U positive proliferating cells.Cycloastragenol could also promote the expression of cell proliferation and differentiation-related proteins Pax7 and Myo D.Cycloastragenol can promote the expression of HO-1 protein,a key downstream molecule,by inhibiting the expression of Keap1 protein and promoting the expression of Nrf2 after uncoupling with Keap1 into the nucleus.3.After Nrf2 knockdown,intracellular ROS content increased,HO-1 protein level was down-regulated,and Nrf2/HO-1 signaling pathway was inhibited,the expression of Pax7 and Myo D protein decreased,and the proliferation and differentiation ability of skeletal muscle decreased.Conclusion:The effect of cycloastragenol on oxidative damage of C2C12 myoblasts may be related to inhibition of Keap1 expression,promotion of Nrf2 nuclear translocation and up-regulation of downstream target molecule HO-1 expression.Cycloastragenol can also promote the expression of proteins related to skeletal muscle proliferation and differentiation,providing a new idea for the prevention and treatment of sarcopenia.