Effects Of Alcohol On Expression Of E3 Ligase Atrogin-1 In C2C12 Myoblasts

Excessive alcohol could lead to skeletal muscle myopathy. It is characterized by biochemical, structural, and physiological changes. Alcohol-induced chronic myopathy is characterized by muscle soreness and muscle atrophy. Reactive oxygen species(ROS) and free radicals are generated during alcohol metabolism, causing oxidative stress and lipid peroxidation. Ubiquitin ligase plays important roles in muscle atrophy. Atrogin-1,a novel E3 ubiquitin ligase, is thought to be the early biochemical maker of muscle atrophy. In humans, during some diseases associated with muscle atrophy, the expression of Atrogin-1 is induced by oxidative stress via P-38 MAPK. However, there is few research to explore the relationship between alcohol and muscle atrophy, and most of them focus on animal modle. Our study is aimed at investgating the related mechanism and signal pathway of alcohol-induced muscle atrophy with C2C12 cells.Objectives: To investigate the oxidative damage and the expression of Atrogin-1 Protein, explore the effects of alcohol(100m M) on Atrogin-1 protein, alcohol expourse in the mouse C2C12 cells. A further object is to provide a new insight into the effects of alcohol and ROS on the expression of Atrogin-1 protein.Methods:(1)alcohol was expoursed 24 h, NAC(2u M) was pretreated 30 min, then intracellular ROS level was detected;(2)The C2C12 cells was treated with different time: 0h, 8h, 16 h, 24 h. Then the level of Atrogin-1 protein was detected by Western Blot;(3)The C2C12 cells was treated 8h with alcohol(100 m M), NAC(2u M) was pretreated 30 min, then the expression of Atrogin-1 was detected by Western Blot;(4)The C2C12 cells was treated 8h with alcohol(100 m M),NAC(2u M) was pretreated 30 min, then the expression of P-38 and P-P38 was detected by Western Blot;The C2C12 cells was treated with SB203580(50u M), a specific p38 inhibitor. Then the expression of Atrogin-1 was detected by Western Blot.Results:(1) 100 m M alcohol caused an significant increase in intracellular ROS levels compared with the control group(P<0.05); The ROS level of NAC(2u M) pretreatment decreased compared with alcohol group;(2)The expression of Atrogin-1 protein was time-dependent with alcohol(100m M) expourse 8h and 24 h and reach peak at 8h;(3)After alcohol(100m M) expourse 8h, the expression of Atrogin-1 increased compared with the control group(P<0.05); However, the increased Atrogin-1 induced by alcohol was decreased by NAC(2u M) pretreatment;(4)The level of P-P38 protein was increased after alcohol(100m M) exposure compared the control group(P < 0.05); The P-P38 protein level of NAC(2u M) pretreatment decreased compared with alcohol group(P<0.05); After treatment of SB202580(50u M), the expression of Atrogin-1 decreased compared with alcohol group(P<0.05).Conclusions:(1)100m M Alcohol could cause an increase in intracellular ROS levels and induce the oxidative stress reaction in the skeletal muscle. 2u M NAC could decrease the ROS level induced by alcohol(2) ROS could induce the expression of Atrogin-1 protein via P-38 MAPK pathway.