Mechanism Of IRF1 In Alcoholic Fatty Liver Disease And The Intervention Of Xie Zhuo Tiao Zhi Tea

ObjectiveAlcoholic fatty liver disease(AFLD)is a liver disease caused by long-term heavy alcohol consumption.As the disease progresses,AFLD can be further developed into steatohepatitis and liver fibrosis/cirrhosis.Interferon regulatory factor 1(IRF1)is a transcription factor which plays an important role in immune response,cell necrosis,DNA damage and tumor.However,its relationship with liver metabolic diseases,especially AFLD,remains unclear.The purpose of this project is to reveal the role of liver parenchymal cells IRF1 in the occurrence and development of AFLD,reveal its potential biological mechanism,and explore the role of Xie zhuo Tiao zhi tea in the intervention of AFLD.Methods1.The 8-week-old male C57BL/6J SPF mice were used as the research object to construct Lieber-DeCarli and NIAAA AFLD mouse models.The mRNA and protein levels of IRF1 were detected by qPCR and Western blot.2.Hepato-specific adeno-associated virus type 8(AAV-8)was used as a vector to construct a short hairpin RNA(shRNA)vector that could effectively silence IRF1 gene and package the virus.The animal model of liver-specific knockdown of IRF1 was constructed by injecting the virus through the tail vein.The mice were divided into four groups:control group(n=8),AFLD model group(n=8),IRF1 knockdown group(n=8)and AFLD model group with IRF1 knockdown(n=8).Body weight,liver weight,food intake and lipid levels in feces were measured.Plasma ALT,AST,blood glucose,blood lipid and other routine biochemical indicators were detected by enzyme method to explore the effect of IRF1 knockdown on AFLD.3.The triglyceride detection kit was used to detect whether IRF1 knockdown or overexpression could affect the lipid accumulation of alcohol-induced AML-12 hepatocytes;4.AML-12 mouse liver cells cultured in vitro were used as the research object to establish the model of fatty liver induced by alcohol(300 mmol/L).Proteasome inhibitors MG132(10μmnol/L),lysosome inhibitors CQ(20 μmol/L)and BafAl(100 nmol/L)were used to inhibit the degradation of IRF1 protein.5.The E3 ligase of IRF1(F-box and WD repeat domain containing 7,FBXW7)was clarified through co-immunoprecipitation technology and protein stability experiments.The expression of FBXW7 in AFLD was detected by qPCR and Western blot.6.Endoplasmic reticulum stress(ER stress)was induced by ER stress activators such as toxic carotene Tg(2 μmol/L)and lamycin Tm(5 μmol/L).ER stress signaling pathway IRE1a specific inhibitors STF083010(40 μmol/L)and SP600125(30 μmol/L),PERK specific inhibitor GSK2606414(300 nmol/L)and ATF6 specific inhibitor Ceapin-A7(10 μmol/L)were used inhibited the signal transduction,and Western blot assay was used to detect the protein levels of FBXW7 and IRF1,to explore the mechanism of ER stress signaling pathway regulating FBXW7 and IRF1.7.Lipid metabolizing enzymes in mouse liver tissues were detected by qPCR to detect whether IRF1 affected the expression of these key metabolizing enzymes,and to clarify the signaling pathway of IRF1 regulating alcohol-induced lipid accumulation;8.The 8-week-old male C57BL/6J SPF mice were used as the research object to establish Lieber-DeCarli AFLD mouse model,and then the mice were given the decoction of Xie zhuo tiao zhi tea by gavage.The mice were divided into three groups:control group(n=6),AFLD model group(n=6)and AFLD+Xie zhuo tiao zhi tea group(n=6).The following indexes were used to determine the influence of turbid tea on AFLD.The main indicators included:body weight,liver weight,food intake and lipid level in stool;Plasma ALT,AST,blood glucose,blood lipid and other biochemical indexes were detected by enzymatic method.QPCR and Western blot were used to detect the expressions of IRF1,FBXW7 and ER stress pathways related molecules in the liver tissues of the three groups of mice,so as to explore whether Xie zhuo tiao zhi tea can affect the AFLD process through ER stress pathways.Results1.Animal experimental studies showed that the expression of IRF1 at the protein level was significantly down-regulated in Lieber-DeCarli and NIAAA AFLD mouse models(P<0.01).2.In AFLD mouse model,the specific knockdown of IRF1 protein in liver significantly increased liver lipid accumulation(P<0.01),and the expression of ALT(P<0.05)and AST(P<0.01)in serum was up-regulated,which promoted alcohol-induced liver injury and accelerated the course of AFLD disease.3.After AML-12 cells were exposed to alcohol(300 mmol/L)for 12 h,IRF1 protein expression was significantly down-regulated(P<0.01);Using this model,the overexpression of IRF1 protein in the liver cells of AML-12 mice significantly reduced the accumulation of lipids in the liver cells(P<0.01).4.Experimental studies showed that IRF1 protein was degraded through ubiquitin-proteasome pathway,resulting in down-regulated expression of its protein level.Alcohol promotes ubiquitination of IRF1 protein.5.In vitro and in vivo immunoprecipitation experiments confirmed that IRF1 directly interacts with FBXW7,and FBXW7 promotes ubiquitination modification of IRF1 protein and regulates the stability of IIRF1 protein.6.Mechanism studies have shown that activation of ER stress can promote the up-regulation of FBXW7 expression and the degradation of IRF1 protein.Intervention studies using the ER stress signaling pathway IRE1a specific inhibitors STF083010(40 μmol/L)and SP600125(30μmol/L),PERK specific inhibitor GSK2606414(300 nmol/L)and ATF6 specific inhibitor Ceapin-A7(10 μmol/L)showed that PERK inhibitors inhibited alcohol and Tg up-regulation of FBXW7 and reversed the degradation of IRF1 protein by alcohol and Tg.7.The down-regulation of IRF1 expression in AFLD prevented IRF1 from entering the nucleus,and then blocked the expression of IRF1 target gene PPARa at the mRNA and protein levels,inhibited the β-oxidation of lipids,and promoted the accumulation of lipids in hepatocytes.8.The intervention of Xie zhuo tiao zhi tea in AFLD mouse model proved that Xie zhuo tiao zhi tea could effectively improve the liver injury of mice and reduce the accumulation of lipids in the liver.Mechanism studies have shown that Xie zhuo tiao zhi tea can inhibit the degradation of IRF1 protein by blocking the conduction of ER stress signaling pathway,and then initiate the expression of PPARa through nucleation,promote fatty acid β-oxidation,and thus improve AFLD.ConclusionSpecific knockdown of IRF1 in mouse liver promotes alcohol-induced lipid accumulation and promotes the development of AFLD.Alcohol activates the PERK-ATF4 signaling pathway,inducing the up-regulation of FBXW7 expression and the ubiquitination degradation of IRF1 protein.After alcohol induced degradation of IRF1 protein,it lost its function as a nuclear transcription factor and could not enter the nucleus to initiate the expression of key genes related to lipid metabolism such as PPARα,inhibit fatty acid β-oxidation,promote the accumulation of lipid in cells,and then induce the occurrence and development of AFLD.By blocking the conduction of ER stress signaling pathway,Xie zhuo tiao zhi tea inhibits the degradation of IRF1 protein,improves alcohol-induced liver injury,and reduces the accumulation of lipids in the liver.These studies suggest that IRF1 is a potential target for AFLD.