Alcohol Acts Via P38 MAPK To Induce Expression Of Atrogin-1 In The Differentiation Process Of C2C12 Myoblasts

Alcohol-related myopathy is characterized by severe biochemical and structural changes in skeletal muscle,muscle atrophy is one of the main clinical manifestations.Ethanol produces large amounts of reactive oxygen species and free radicals in the body's metabolism,causing oxidative stress and lipid peroxidation in cells.Ubiquitin ligase is a key enzyme in skeletal muscle atrophy.Atrogin-l,a novel E3 ubiquitin ligase,is thought to be the early biomarker of muscle atrophy.In this study,we establish the model of acute alcoholism by exposuring C2C12 myoblasts to alcohol.With the use of ROS detection and Western Blotting technique,the change of ROS,atrogin-1,protein P38 after exposure of C2C12 myoblasts to alcohol and the expression change of atrogin-1 followed by treating C2C12 myoblasts with specific inhibitors of each MAPK pathway were detected,then the oxidative damage of alcohol on C2C12 myoblasts and the mechanism of its effect on atrogin-1 expression were analysed.The results showed that ROS levels were rised after exposure of C2C12 myoblasts to alcohol(100mM)for 2 hours,and there was a significant difference compared to the control group(P<0.05);In addition,between 2-12 hours of exposure of alcohol,the expression levels of atrogin-1 were significantly increased compared to control group(P<0.05),and at 6 hours its expression reached the maximal.After exposure of C2C12 myoblasts to alcohol(100mM)for 2 hours,the levels of phospho-P38 were significantly increased compared to control group(P<0.05).However after adding P38 pathway inhibitor(SB203580),the levels of atrogin-1 were significantly reduced compared to the alcohol group(P<0.05).In summary,alcohol can cause the increase of ROS level,then induce oxidative stress in C2C12 cells.The elevated ROS will result in the expression increase of atrogin-1 via P38 MAPK signaling pathway.In this study,we observe the oxidative damage and the expression change of atrogin-1 of alcohol on skeletal muscle cells and investigate its intracellular signal transduction pathways by cultivating C2C12 mouse skeletal muscle cells in vitro,which provides new ideas for exploring the mechanism of skeletal muscle atrophy in acute alcoholic myopathy.Objectives:To investigate the oxidative damage and the expression of atrogin-1 protein of alcohol on skeletal muscle,we establish the model of acute alcoholism by exposuring C2C12 myoblasts to alcohol in vitro,observe the change of atrogin-1 protein expression and explore its intracellular signal transduction pathways of the effect of alcohol on C2C12 cells,which may offer new therapeutic approaches to suppress skeletal muscle atrophy.Methods:1.Induce the differentiation of C2C12 myoblasts by replacing growth medium with differentiation medium.2.Detection of cell activity by CCK-8 method to determine the optimal concentration of alcohol causing cell damage.3.After exposing alcohol(100mM)to C2C12 cells:0h,2h,4h,6h,8h,observe ROS levels within the cell among different groups using DCF method and the inverted microscope.4.Alcohol(100mM)were acting on C2C12 cells at different time points,namely:0h,2h,4h,6h,8h,10h,12h,then detect Atrogin-1,P-P38,P-JNK protein expression levels among different groups,in order to observe the relationship between different exposing times of alcohol(100mM)on C2C12 cells with the expression of atrogin-1 protein and the phosphorylation of various MAPK signal transduction pathways.5.Previously added(without)each MAPK signal transduction pathway inhibitors:SB203580(P38 inhibitor),SP600125(JNK inhibitor),then add alcohol(100mM)to C2C12 cells for 6 hours,with Western Blotting technique to detect the expression of atrogin-1 protein among different groups to explore the effect of alcohol on atrogin-1 protein and its relationship with various MAPK signal transduction pathway.Results:1.The C2C12 myoblast differentiation results.2.CCK-8 method shows:alcohol(0?200mM)has no effect on C2C12 cell activity.3.ROS levels were rised after exposure of C2C12 myoblasts to alcohol(100mM)for 2 hours,and peaked at 4 hours,there was a significant difference compared to the control group(P<0.05).4.Between 2-12 hours of exposing alcohol on C2C12 cells,Atrogin-1 protein levels were significantly increased and peaked at 6 hours,there was a significant difference compared to the control group(P<0.05).5.The phosphorylation levels of P38 protein were significantly increased after exposure of C2C12 myoblasts to alcohol(100mM)for 2 hours,and there was a significant difference with the control group(P<0.05);However after adding its inhibitor SB203580,the level of atrogin-1 protein was significantly lower compared with the alcohol group.Conclusions:1.Alcohol(100mM)could cause an increase in intracellular ROS levels and induce the oxidative stress reaction in the skeletal muscle,causing the increased expression of atrogin-1 protein in C2C12 cells.2.Alcohol induce the increased expression of atrogin-1 protein in C2C12 cells via P38 MAPK signaling pathway.