Effect Of Exercise On IL-6/NOX Oxidative Stress Pathway In Alcoholic Liver Disease

Objective:Through the inhibition of NOX activity and aerobic exercise intervention during the formation and recovery of alcoholic liver disease（ALD）,it is revealed that exercise regulation of IL-6/p47^phoxoxidative stress pathway can improve liver cell injury and dyslipidemia.In order to provide theoretical and experimental basis for exercise to improve the function of immune system,enhance the body’s anti-inflammatory ability,prevent and cure immune suppression,and promote rehabilitation of alcoholic liver disease.Methods:Sixty-four clean-grade C57BL/6J male rats aged 6 weeks were divided into 3groups.In experiment 1,mice were randomly divided into CC group,CN group,S group,L group and LN group.CC and CN group were fed with ordinary solid diet.mice in CN group were intraperitoneally injected by 10 mg/Kg Apocynin for 4consecutive days,while CC group was injected with the same dose of solvent.ALD model was constructed by Lieber-De Carli liquid diet with TP4030C in S group as well as TP4030B in L group and LN group.Mice in LN group was intraperitoneally injected with 10 mg/Kg Apocynin 6 days a week for 6 weeks,while mice in other two groups were injected with the same dose of solvent.In experiment 2,mice were randomly divided into C group,A group,AN group,AE group and AEN group.All mice was constructed by Lieber-De Carli liquid diet with TP4030C in C group as well as TP4030B in other groups.At the end of modeling,they were fed with ordinary solid diet.The AE group and AEN group swam for 1h/d and 6d/week for consecutive6 weeks.6h before exercise,mice in the AN group and AEN group were intraperitoneally injected with Apocynin（10mg/kg）,and other groups were injected with the same dose of solvent.In experiment 3,mice were randomly divided into S group,L group,LE group and LEN group.The S group was given TP4030C control diet.The remaining groups were fed with TP4030B alcohol diet and exercise intervention was started synchronously combined with or without Apocynin intraperitoneal injection on every exercise training day for 5 weeks.During the experimental intervention,the mice in each group were observed by general behavior,and their body weight was recorded weekly.After the experiment,All mice were weighed and anesthetized for 16 hours after fasting and were sacrificed.Epididymis fat,triceps calf muscle,intact liver.Fasting blood glucose was measured or glucose tolerance test was performed to detect ALT and AST levels in serum,TG,LDL-C and HDL-C concentrations and MDA and SOD activities in tissues.HE or oil-red O staining in liver tissues was observed.Western Blot was used to detect the expression of NOX4,gp91^phox,p47^phoxand IL-6 proteins in liver tissue or muscle tissue,and the correlation analysis of IL-6 and p47^phoxwas conducted.Results:Experiment 1:1)Compared with CC group,the content of TG in liver tissue and the levels of serum ALT and AST increased significantly in CN group;the expressions of NOX4and gp91^phoxdecreased evidently in liver tissue（P<0.05,P<0.01）;2)Compared with S group,the relative content of epididymal fat and the content of HDL-C in serum were strikingly decreased in L group（P<0.05,P<0.01）,and the level of serum ALT increased;the levels of TG and the protein expression of NOX4 were particularly increased in liver tissue（P<0.01）;3)Compared with L group,the levels of HDL-C in serum increased notably in ALD in LN group（P<0.05,P<0.01）;4)The structure of liver cells in S group.In L group,the boundaries of hepatocyte were blurred and the hepatic cords were disordered.After Apocynin,the lobule structure of liver tissue was intact and the infiltration degree of inflammatory cells was alleviated.Experiment 2:1)Compared with S group,the levels of TG in serum and IL-6 protein content in liver tissue in L group were increased significantly（P<0.05）;2)p47^phoxexpression in muscle tissue in AN group was increased notably compared with A group（P<0.05）.The liver index and the level of HDL-C in AEN group was decreased notably compared with AE group（P<0.05）;3)Compared with A group,the level of TG in serum was decreased notably in AE group（P<0.05）;the level of TG in serum,the ratio of AST/ALT,the expression of IL-6 protein in liver and muscle tissue were decreased notably（P<0.05）,and the level of MDA in muscle tissue was increased significantly（P<0.01）.Compared with AN group,the ratio of AST/ALT,the level of TG in serum,SOD activity in liver tissue,the expression of p47^phoxprotein in liver and muscle tissue were decreased significantly（P<0.01）;4)Compared with C group,mice had hepatic cell swelling,loose cytoplasm and fat vacuole,accompanied by inflammatory cell infiltration in A group.After exercise intervention,the degree of hepatocyte steatosis and inflammatory infiltration was reversed;5)There was a high correlation between p47^phoxand IL-6 protein level in liver tissue（R²=0.7913）,and a low correlation between p47^phoxand IL-6 protein level in muscle tissue（R²=0.3001）.Experiment 3:1)Compared with L group,the level of TG in serum,MDA activity in liver tissue,the expression of NOX4 and p47^phoxprotein were increased significantly,ALT level and weight in LE group were decreased significantly（P<0.05,P<0.01）;the level of TG in liver tissue,MDA activity in muscle tissue,the expression of gp91phox protein,ALT level and weight in LEN group were decreased significantly（P<0.05,P<0.01）;2)Compared with LE group,the levels of ALT and MDA activity in muscle tissue,the expression of gp91^phoxand p47^phoxprotein in liver tissue were decreased significantly in LEN group;3)There was a high correlation between p47^phoxand IL-6 protein level in liver tissue（R²=0.6333）.Conclusion:1)Apocynin had a more significant effect on NOX4 protein in liver tissue.During ALD model establishment,inhibition of NOX alleviates alcoholic liver cell injury and lipid metabolism disorder.Apocynin has different effects on liver physiology and pathological state of alcoholic liver injury;2)During ALD recovery,exercise intervention may reduce IL-6/p47^phoxoxidative stress pathway to improve hepatocyte injury and dyslipidemia,and combined exercise with Apocynin has better effect;3)Drinking alcohol during exercise exacerbates dyslipidemia,oxidative stress,with hepatocyte IL-6/p47^phoxdown regulated.