| Objective: Ethanol,as a recreational beverage,has been widely used all over the world for a long time,and China has become one of the most serious drinking countries in the world.At present,binge drinking and alcoholics increase significantly in China.Therefore,the negative effects and the disease caused by drinking are an important public health problem.Alcoholism is a series of clinical syndromes caused by binge and chronic intake of alcohol or alcoholic beverages.The disease caused by drinking involves multiple organ injuries,especially digestive system diseases.Acute pancreatitis is dangerous because it can cause hypoglycemia,hypothermia,shock and even death,which is one of the main causes of acute alcohol poisoning.Chronic alcoholism can form alcoholic liver disease(ALD).It is a basic disease of chronic alcoholism.Fatty liver is an early pathological change of ALD.And,this stage can be reversed by drug intervention and abstinence,which is the optimal stage to intervention for ALD in clinical.The role of oxidative stress in alcohol induced diseases is important.Transcription factor E2 related factor 2(Nrf2)is a core transcription factor regulating antioxidant defense genes and phase II detoxification enzymes,and plays an important role in the regulation of intracellular adaptive antioxidative response and redox homeostasis.Previous studies have shown that Nrf2 can reduce the incidence of ALD by regulating lipid metabolism and inflammatory response.However,the study of Nrf2 in alcoholic pancreatitis is still in the blank.Dimethyl fumarate(DMF),the Nrf2 activator,has not yet been used for the treatment of alcohol related diseases.Therefore,the effect of DMF on alcoholism and its mechanism need to be confirmed and discussed.In conclusion,we studied the effect and mechanism of Nrf2 on acute and chronic alcoholism by using Nrf2 global knockout mice,Nrf2 hepatocytes and myeloid specific knockout mice.DMF,the Nrf2 activator,was given to observe whether it had a protective effect on acute alcoholism.A comprehensive analysis of the mechanism of Nrf2 in alcoholism provides a new theoretical basis for the clinical treatment of alcoholism.Methods:1.Female 8-12 weeks Nrf2 global knockout mice(Nrf2-KO)and littermate wild-type C57BL/6 mice were randomly divided into Nrf2-WT high dose alcohol group,Nrf2-KO high dose alcohol group,Nrf2-WT low dose alcohol group,Nrf2-WT low dose group,Nrf2-KO low dose alcohol group,Nrf2-KO low dose group and control group and DMF intervention group,5-8 mice in each group.After gavage,the mortality of the two groups of mice was observed and the blood glucose and core body temperature were monitored dynamically.Primary hepatocytes separated from 12-16 weeks age male Nrf2-(L)KO and littermate Nrf2-KI C57BL/6 mouse,and cells were cultured with 200 mmol/L alcohol and 1 mg/mL of tunicamycin(TUN)for 0 h,2 h,6 h,12 h,18 h and 24 h.Cells were collected in time to detect the related proteins and genes expression levels of endoplasmic reticulum stress.The expression and activity of liver function,pancreatic function,alcohol metabolism related enzymes and the expression of NRF2 downstream genes were detected in mice.2.Male 12-16 weeks hepatocytes specific knockout mice(Nrf2-(L)KO)and littermate Nrf2-KI C57BL/6 mice were divided into eight groups: Nrf2-KI alcohol intragastric group,Nrf2-(L)KO alcohol intragastric group,Nrf2-KI high dose alcohol diet group,Nrf2-(L)KO high dose alcohol diet group,Nrf2-KI low dose alcohol diet group,Nrf2-(L)KO low dose alcohol diet group,Nrf2-KI low dose control diet group,Nrf2-(L)KO low dose control diet group,6-8 mice in each group.The level of liver function,damage and endoplasmic reticulum stress related protein expression and transcriptional level were detected in mice.3.Male 12-16 weeks Nrf2-(L)KO and littermate Nrf2-KI C57BL/6 mice were divided into 4 groups: Nrf2-KI control diet group,Nrf2-(L)KO control diet group Nrf2-KI alcohol diet group and Nrf2-(L)KO alcohol diet group;Nrf2 myeloid cell specific knockout mice Nrf2-(M)KO and littermate Nrf2-KI C57BL/6 mice were divided into 4 groups: Nrf2-KI control diet group,Nrf2-(M)KO control diet group Nrf2-KI alcohol diet group and Nrf2-(M)KO alcohol diet group,6 mice in each group.The levels of liver function,the expression and activity of enzymes related to alcohol metabolism were detected,also including inflammatory reaction,fibrosis and lipid metabolism related proteins and genes expression levels.Results: 1.Nrf2-KO mice exhibit hypoglycemia,hypothermia and rapid mortality by ethanol ingestion.Seventy percent of Nrf2-KO mice died within 36 hours after 6 g/kg BW ethanol administration while none died in the Nrf2-WT group.The level of blood glucose was less in Nrf2-KO mice in comparison to that of wild type(p < 0.05).We further measured the core temperature in mice,core temperature showed significant decrease in Nrf2-KO mice at 12 h and 24 h after ethanol exposure(p < 0.05).After 4.8 g / kg BW alcohol infusion for two times,compared with Nrf2-WT mice,the blood glucose level of Nrf2-KO mice was significantly lower than that of Nrf2-WT mice(p < 0.05)at 24 h after alcohol exposure.Moreover,after 12 h and 24 h alcohol by ingestion,Nrf2-KO mice were significantly lower than those of Nrf2-WT mice after alcohol exposure(p < 0.05).2.The effects of Nrf2 deficiency on alcohol metabolism in mice.The relative expression of Cyp2e1,Cat,Adh and Aldh2 mRNA was not different between the two genotype mice at basal level,while the Aldh1a1 mRNA level in Nrf2-KO mice was significantly lower than that in Nrf2-WT mice(p < 0.05).After alcohol treatment,the ALDH activity of Nrf2-KO mice decreased(p < 0.05),and was significantly lower than that of Nrf2-WT mice after alcohol exposure(p < 0.05).3.The deletion of Nrf2 aggravates alcohol induced liver injury in mice.After exposure to alcohol,there was no significant difference in HE staining between Nrf2-KO mice and Nrf2-WT mice.Alcohol exposure increased the level of ALT and AST(p < 0.05),further,the ethanol-induced elevation of plasma ALT and AST increase in Nrf2-KO mice compared to Nrf2-WT mice.4.The disruption of Nrf2 aggravates alcoholic pancreatic injury in mice.HE staining showed that the pancreatic injury was aggravated after alcohol exposure.Compared with Nrf2-WT mice,Nrf2-KO mice showed obvious pancreatic necrosis.Immunohistochemistry showed increased expression of Cleaved-Caspase 3 in pancreas.Alcohol exposure significantly increased the activity of AMS in mice(p < 0.05),but there was no difference between the two genotypes.The LPS level of Nrf2-KO mice in alcohol group was significantly higher than that in the control group(p < 0.05),and significantly higher than that of Nrf2-WT mice after alcohol treatment(p < 0.05).Alcohol treatment significantly increased the level of insulin in mice(p < 0.05),and compared with Nrf2-WT treated mice,the level of plasma insulin in Nrf2-KO mice increased significantly(p < 0.05).5.DMF significantly alleviates the fatal effect of acute alcohol exposure.Gclc,Nqo1 and Ho-1 mRNAs which are the downstream genes of Nrf2,in the DMF group were higher than those in the Veh group,and the difference in the expression of Gclc and Nqo1 mRNA was statistically significant(p < 0.05).Compared with the Veh group,the mice in group DMF were significantly higher than those in group Veh(p < 0.05)after alcohol gavage.During the whole experimental period,the body temperature of the DMF group was higher than that of the Veh group,and the difference of 24 h after the alcohol gavage was statistically significant(p < 0.05).At the end of the experiment,86% of the mice in the DMF group survived,while the group VeH was close to 40% mice.6.The effect of Nrf2 deficiency in hepatocyte on the general condition of mice after alcohol exposure Hepatocyte Nrf2 deletion aggravates the lethal effect of alcohol exposure in mice.There was no significant difference in the amount of food intake and body weight of the mice in each group(p > 0.05).There was no significant difference in body fat content in each group after 1 W and 6 W by ethanol liquid diet exposure(p > 0.05).There was no significant difference in the organ coefficient between each group(p > 0.05)for 28 and 42 days after feed ethanol liquid diet exposure.7.Effect of Nrf2 deficiency in hepatocyte on liver pathology and liver function in mice after alcohol exposure4.2% liquid feed was fed for 28 days,all mice had light liver injury,but there was no significant difference between the two genotypes.There was no significant difference in the levels of ALT,AST and MDA in each group(p > 0.05).After 42 day of alcohol liquid diet,the mice had moderate or severe liver injury.The results of ALT,AST and TG showed no significant difference between the two genotypes(p > 0.05).8.The effect of Nrf2 deficiency in hepatocyte on the expression of inflammation and fibrosis related genes in liver tissue of mice after alcohol exposureThere was no significant difference in the expression of inflammation related genes IL-6,IL-1β and F4/80,fibrosis related genes α-SMA,Collagen and Fibronectin mRNA in groups of mice fed with alcoholic liquid feed for 42 days(p > 0.05).9.The effect of Nrf2 deficiency in hepatocyte on the expression of lipid metabolism in liver tissues of mice after alcohol exposureThe expression of p-AMPK-beta1 and AMPK-beta1 in liver tissues of each group had no significant changes after 4.2% liquid feed for 28 days.10.Expression of liver tissue damage related protein expression in mice after alcohol exposure by Nrf2 deletion in hepatocyte.After 4.2% feeding for 28 days,there was no significant change in the expression of PCNA and PARP in each group of mice.The expression of Cleaved PARP increased with alcohol treatment,and the expression of Nrf2-(L)KO mice in alcohol group increased compared with that in alcohol group Nrf2-KI mice.11.The effect of Nrf2 deletion in myeloid cells on the general condition of mice after alcohol exposure.There was no significant difference in the amount of food intake,body weight and organ coefficient of the mice in each group(p > 0.05)for 42 days.There was no significant difference in body fat content in each group after 1 W and 6 W(p > 0.05).12.The disruption of Nrf2 in myeloid cells aggravates alcoholic fatty liver in mice.Ethanol liquid diet was fed for 42 days,Nrf2-(M)KO mice had more serious fatty liver compared with Nrf2-KI mice.The content of TG in liver of Nrf2-(M)KO mice was higher than that in Nrf2-KI mice(p < 0.05).There was no significant difference in ALT and AST between the mice in each group(p > 0.05).13.Effect of Nrf2 deletion in myeloid cells on lipid metabolism related protein and gene expression in liver of mice after ethanol liquid diets exposure.The expression of PPAR-alpha,PPAR-gamma-1,PPAR-gamma-2,PGC-1-alpha,p-AMPK-beta-1 and AMPK-beta-1 protein in liver tissues of all mice was not significantly changed.There was no significant difference in the expression of Srebp and Ppar-alpha mRNA between mice in each group(p > 0.05).14.Effect of Nrf2 deletion in myeloid cells on inflammation and fibrosis related protein and gene expression in liver tissue of mice after alcohol exposure.There was no obvious change in the expression of TGF beta 1 protein in each group.The levels of Tgf beta 1,alpha-SMA,Fibronectin and Collagen mRNA were not statistically significant(p > 0.05).There was no significant difference in the relative expression of Tnf alpha,IL-1 beta,IL-6,F4/80 and IL-10 mRNA between mice(p > 0.05).Conclusion:1.Nrf2 is a critical regulator of mortality in a binge ethanol-exposure mouse model.Global Nrf2 deficiency aggravated the lethal effect induced disruption of alcohol metabolism and promoting alcoholic pancreatic and liver injury.NRF2 activator DMF pretreatment protected against death from acute binge alcohol exposure by upregulating the ability of antioxidation.2.Loss of Nrf2 in hepatocytes could accelerate acute and chronic alcoholic death in mice.Nrf2 deletion in the hepatocytes has no significant effect on the onset of alcoholic fatty liver.3.The absence of Nrf2 in myeloid cells accelerated the progress of alcoholic fatty liver disease,not by affecting the fatty acid oxidation pathway of AMPK,PPAR-alpha,PPAR-gamma and PGC-1-alpha.The deletion of Nrf2 in myeloid cells has no significant effect on ethanol metabolism,inflammatory reaction and alcoholic liver fibrosis. |
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