The Role Of Forkhead Transcription Factor O1 In Alcohol Fatty Liver Disease

Objective: Alcoholic liver disease(ALD)is a chronic disease caused by long-term heavy drinking,with a histological spectrtun ranging from hepatic steatosis,steatohepatitis,fibrosis and ultimately to cirrhosis.Alcohol fatty liver disease(AFLD)is the early phase of ALD,which is characterized by increased accumulation of lipid in hepatic tissue.Large amounts of reseaches showed that Forkhead transcription factor O1(Fox O1)regulate various signaling pathways involved in lipid metabolism and oxidative stress response.FoxO1 was regulated by changes in subcellular localization coupled to post-translational modi?cations,including phosphorylation,acetylation and ubiquitination.When locating in cell nucleus,FoxO1 could bind to its target DNA sequences to regulate the expression of corresponding proteins.Microsomal triglyceride transfer protein(MTP)and adiponectin recpetor2(AdipoR2)were regulated by FoxO1.Our research group discovered that the mRNA and protein expression of SIRT1,AdipoR2 was suppressed by alcohol in the rats' hepatic tissue.FoxO1 might be the junction of SIRT1 and AdipoR2.Long-term alcohol intake suppressed the activity of SIRT1,resulting in the decreasing level of deacetylated FoxO1 and increasing level of acetylated FoxO1,which promotes the nuclear exclusion of FoxO1,attenuating its transcriptional activity.Then the expression of its downstream proteins AdipoR2 was down-regulated,followed by disorder of a series of its downstream lipid metabolism signaling pathway.MTP,one of the downstream protein of FoxO1,was involved in the transfer of TAG from their site of synthesis on the membrane of the endoplasmic reticulum to the site of very low density lipoproteins(VLDL)assembly.The attenuating transcriptional activity of FoxO1 caused by alcohol might influence the expression of MTP,which could lead to lipid accumulation in liver.Thisresearch aims to discuss the function of FoxO1 in the pathogenesis of alcoholic fatty liver disease.Methods:Fifty healthy male Wistar rats weighting 200±20g were fed for seven days normally.Then ten of them were randomized into the normal control group.Other forty rats were to develop the model of AFLD.The model were treated by intragastric alcohol of increasing concentration 30%-60%,5-9g·Kg-1·d-1.At the end of 4th,8th,12 th and 16 th week,ten,nine,eight and eight rats were sacrificed respectively.Portions of rat livers were fixed in10% neutral-buffered formalin overnight,and then were embedded in paraffin.The rest hepatic tissue were frozen in liquid nitrogen immediately,then stored in-80? fridge.RT-qPCR was applied to determine the mRNA expression of FoxO1.Immunoprecipitation was applied to extract the acetylated protein.The protein levels of total FoxO1,acetylated FoxO1 and MTP were determined by Western-blot.Immunohistochemical was applied to compare the different content of FoxO1 in nucleus and cytoplasm.Results:1 Histopathological changes of hepatic tissue: With the procession of AFLD,adipose degeneratio was aggravating gradually in hepatic tissue,indicating the AFLD model was founded successfully.The expression of FoxO1 protein in hepatic tissue by immune-histochemistry staining : In the control group and the early age of AFLD,there is no significant increase for the expression of FoxO1.With the procession of AFLD,the expression of FoxO1 protein increased gradually,especially in cytoplasm,compared with the normal control group.2 The mRNA level of FoxO1 were gradually up-regulated both in 4W,8W,12 W and 16 W,along with the stimulation of alcohol,compared with the normal control group(F=127.56,P<0.01).3 The expression of target protein by western blot :The protein expression of total FoxO1 didn't increase at early phase,but it showed a trend of increase in general.No significant statistical differencewas found between 0th week and 4th week,4th week and 8th week.There were significant statistical difference between the 0th week group and 8th week group,0th week group and 12 th week group,0th week group and 16 th week group(F=268.56,P<0.01).There were a lot expression of MTP in hepatic tissue of rats in normal control group.The protein expression of MTP were gradually down-regulated both in 4W,8W,12 W and 16 W,along with the stimulation of alcohol(F=285.242,P<0.01).There were a little expression of Ac-FoxO1 in hepatic tissue of rats in normal control group.The protein expression of Ac-FoxO1 were gradually up-regulated both in 4W,8W,12 W and 16 W,along with the stimulation of alcohol.And the level of acetylation of FoxO1(Ac-FoxO1/total FoxO1)were gradually up-regulated,too(F=172.73,P<0.01).Conclusions: With the procession of AFLD,the mRNA level of FoxO1 were gradually up-regulated,and the total FoxO1 protein were gradually up-regulated,but MTP,which were the downstream protein of FoxO1,were gradually down-regulated,adipose degeneratio was aggravating gradually in hepatic tissue.In the further research,the level of acetylation of FoxO1 were found gradually up-regulated along with the stimulation of alcohol,making FoxO1 located in cytoplasm,indicating that the decrease of MTP was related to the increase of the level of acetylation of FoxO1,and the acetylated FoxO1 reduced its transcriptional activity.So the expression of its downstream protein MTP were gradually down-regulated,resulting lipid accumulate in liver,which indicating the important role FoxO1 played in AFLD.Therefore,in the procession of AFLD,the up-regulated level of acetylation of FoxO1 caused by alcohol attenuate its transcriptional activity,leading to the decreasing expression of MTP,causing lipid accumulation in liver,which probably is one of the pathogenesis of AFLD.