Up-regulation Of Hepatocyte Nuclear Factor 4α Expression Attenuates Experimental Hepatic Fibrosis

【Background and Objective】Hepatic fibrosis,characterized by deposition of extracellular matrix(ECM) in the Disse's space,is a pathological response to a variety of chronic liver diseases,which is recognized as a dynamic and reversal process.Therefore,blocking,inhibition or even reversal of hepatic fibrosis is a major target for the treatment of chronic liver disease.Generally,it has been accepted that activated hepatic stellate cells(HSC), accompanying phenotypic transformation into myofibroblast-like cells,play the pivotal role in hepatic fibrogenesis.In the past decades,it is an important target on hepatic fibrosis therapy by repressing activation,proliferation and migration of HSC as well as inducing its apoptosis.Recently,several studies reported that HSC could represent a epithelial phenotype and play a significant role in inducing liver stem cell differentiation, maintaining hepatocyte proliferation and function.In addition,it has been reported that hepatic parenchymal cells,such as hepatocytes and biliary epithelial cells,participated in the process of hepatic fibrosis.Therefore,it is unreasonable to repress the activation and proliferation of HSC in hepatic fibrosis therapy,and new strategy on additional fibrotic mechanism and treatment should be reestablished.Epithelial-to-mesenchymal transition(EMT) is the process that epithelial cells gradually lose their epithelial signatures while acquiring the characteristics of mesenchymal cells in cell morphology,structure,biological function,adhesion and migration ability.More importantly,some researches revealed that EMT was a predominant mediator to participate in the process of organ fibrosis in kidney,lung and liver.Substantial advances have been made to highlight that during fibrosis,the heterogeneous pool of(myo-)fibroblasts can be supplemented by EMT from cholangiocytes and potentially also from hepatocytes.Therefore,we believed that extension of EMT concepts in the hepatic fibrogenesis will provide a new therapeutic target for hepatic fibrosis.Hepatocyte nuclear factor 4(HNF4) is a member of the nuclear hormone receptor family of transcription factors.HNF4αis a main phenotype of HNF4 which plays an important role not only in regulating hepatocyte differentiation and maintaining liver-specific functions including energy metabolism,xenobiotic detoxification,bile acid synthesis,serum protein production,but also in controlling epithelial phenotype of hepatocyte.Importantly,HNF4αregulates the expression of genes encoding proteins involved in all major types of cell junctions including tight junctions,adhesion junctions, gap junctions,desmosomes,as well as epithelial polarization and cytoskeletal organization.Recently,some studies demonstrated that expression of HNF4αwas closely associated with EMT.The expression of HNF4αdecreased obviously while transforming growth factor-β1(TGF-β1) induced an EMT state in mouse hepatocytes in vitro. Overexpression of HNF4αcould inhibit the process of EMT induced by Snail.In addition, its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition, suggesting that HNF4αwas a dominant regulator of the epithelial phenotype.Based on the above results,we presume that HNF4αmight interrupt EMT of hepatocytes to maintain its epithelial phenotype and biological function;more importantly,its ectopic expression may induce MET of the activated HSC as well as facilitate hepatocytes regeneration.In this study,we detected the expression of HNF4αin the process of hepatic fibrosis and examine the effect of HNF4αon hepatic fibrosis by tail vein injection of AdHNF4α. Furthermore,we detected expression of EMT related genes in rat hepatocytes stimulated by TGF-β1 as well as activated HSC after AdHNF4αinfection in vitro,in order to elucidate the anti-fibrotic mechanism of HNF4α,which would provide a noval effective strategy for the treatment of hepatic fibrosis.【Methods】1.HNF4αeffect on experimental hepatic fibrosisAdHNF4αand control adenovirus-AdGFP replication-deficient recombinant adenoviruses were packaged in 293 cells respectively.Then adenoviruses were stored after purification by cesium chloride(CsCl) gradient centrifugation and determination of the viral titers.Dimethylnitrosamine(DMN) induced model:Sixty male Sprague-Dawley(SD) rats were divided into 4 groups randomly.Group 1(n=10) was served as a normal control,and the rats in next three groups(n=50) were hepatic fibrosis models induced by 1%DMN(10μg/kg) three times per week for 5 weeks intraperitoneally.Ten rats were sacrificed at 2 and 4 weeks respectively after DMN injection.Four weeks after DMN injection,rats in group 2 (10 rats),3(n=10) and 4(n=10) were infused with saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein,respectively.At the end of study(2 weeks after gene delivery),the animals were sacrificed for analysis of liver function and histology.Immunohistochemical staining was used to detect collagen typeⅠandⅢ.For the semiquantitative analysis, stained sections were measured by an image analyzer.Bile duct ligation(BDL) induced model:The 48 male SD rats were divided into 4 groups randomly.Each group composed 12 rats.Group 1 was treated with sham surgery served as the control,and the rats in next three groups were hepatic fibrosis models induced by BDL for 3 weeks.Common bile ducts of models were dissected bluntly and ligated.After ligated for 2 days,rats in group 2,3,4 were infused with saline,4×10~9 pfu AdGFP or 4×10~9 pfu AdHNF4αvia tail vein injection,respectively.Three weeks after BDL,the animals were sacrificed for examinations of liver function and immunochemistry.Expression of HNF4αin the normal liver and the fibrotic liver induced by DMN was detected by real time RT-PCR and immunohistochemistry staining.In addition,mRNA expression of EMT related genes was detected by real time RT-PCR.2.Effects of HNF4αgene on hepatocytesPrimary hepatocytes derived from male SD rats were cultured for 2 days,then the culture medium was replaced by serum-free medium containing 2 ng/ml TGFβ1. Simulately,the adenoviruses AdGFP or AdHNF4αwere added with 10 MOI.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,albumin(ALB),glutamine synthetase (GS),cytochrome P4501A2(CYP1A2),E-cadherin,Vimentin,Snail and collagenⅠmRNA in the cells was detected by real time RT-PCR respectively after adenovirus infection for 48 and 72 hours.3.Effects of HNF4αon HSC cell phenotype and biological characteristicsThe rat HSC cell line HSC-T6 was infected by AdGFP and AdHNF4αwith 50 MOI respectively.Cell morphology was observed at different time points.Total RNA and protein in different groups were extracted and the expression of HNF4α,ALB,GS, CYP1A2,E-cadherin,Vimentin,HNF4α,Snail,collagen typeⅠ,α-SMA and tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA in the cells was detected by real time RT-PCR after adenovirus infection for 48 and 72 hours.On the other hand,cell proliferation in HNF4αtransfection group was compared with that in the control using MTS method.4.Statistical AnalysisStatistical analysis of values was performed with SPSS software(11.0 version),with a P value＜0.05 considered significant and P＜0.01 as very significant.【Results】1.HNF4αeffect on experimental hepatic fibrosis(1) Real time RT-PCR and immunohistochemistry staining indicated that the expression of HNF4αin the fibrotic livers decreased gradually after DMN injection, compared with that of the normal liver.In addition,the expression of Vimentin and Snail was enhanced in the fibrotic liver,while expression of E-cadherin decreased significantly (P＜0.05).(2) HNF4αinhibits the development of hepatic fibrosis induced by DMNSerum level of ALT in AdHNF4αinfusion group,saline infusion group and AdGFP infusion group was 113.2±21.6 U/L,163.8±19.4 U/L and 165.2±27.1 U/L,respectively (P＜0.05).In addition,PT level reduced in AdHNF4αgroup(23.8±1.41 s),compared with that in saline group(29.4±1.63 s) and AdGFP group(27.4±1.96 s).Furthermore,less amount of liver hydroxyproline was detected in AdHNF4αgroup(217.16±42.51μg/g), compared with that of saline infusion group(341.07±81.20μg/g) and AdGFP group (361.37±112.83μg/g)(P＜0.05).Based on the analysis of image scan technique,we found that the area of ECM in AdHNF4αgroup reduced to 49%of that in AdGFP group.The expression of collagen typeⅠand typeⅢdecreased significantly after AdHNF4αinfusion.(3) HNF4αsuppresses hepatic fibrosis induced by BDLIn AdHNF4αinfusion group,serum levels of ALT and AST(86.4±13.9 U/L, 523.6±90.0 U/L) decreased significantly compared with that in AdGFP infusion group (127.8±13.9 U/L,726.8±129.7 U/L,P＜0.05).Semiquantitative analysis revealed that AdHNF4αtreatment reduced fibrotic areas by 48%(P＜0.05).Moreover,the expression of collagen typeⅠandⅢalso decreased significantly after AdHNF4αinfusion.(4) HNF4αinhibits the process of EMT in liver tissueReal time-RT PCR demonstrated that the expression of Vimentin and Snail in liver infected with AdHNF4α,was down-regulated compared with that in AdGFP treated liver, while E-cadherin expression was up-regulated significantly(P＜0.05).2.Effects of HNF4αon hepatocytes EMT(1) Hepatocytes morphology had no obvious changes after TGF-β1 addition for 24 hours.TGF-β1-treated hepatocytes represented a spindle-shaped morphology 48 hours later.After TGF-β1 stimulation for 72 hours,the alteration of hepatocytes morphology became more obviously.Real time-PCR indicated that the expression of Vimentin,Snail and collgen typeⅠmRNA was up-regulated in TGF-β1-treated hepatocyte(P＜0.05),while the expression of E-cadherin,HNF4α,ALB,GS and CYP1A2 was suppressed(P＜0.05). These results demonstrated that TGF-β1 could induce an EMT state in rat primary hepatocyte in vitro.(2) Cellular morphology of hepatocytes treated by TGF-β1+AdHNF4αpreserved a more cuboidal/hexagonal shape compared with that of TGF-β1+AdGFP treated ones.In addition,the number of viable hepatocytes in TGF-β1+AdGFP group reduced significantly. Real time-PCR showed that the expression of mesenchymal phenotype genes in AdHNF4αinfected hepatocytes,including Vimentin,Snail and collgen typeⅠmRNA,was down-regulated(P＜0.05).On the other hand,the expression of liver-specific genes and epithelial phenotype genes such as HNF4α,ALB,GS,CYP1A2 and E-cadherin were up-regulated significantly(P＜0.05).These results imply that HNF4αcould interrupt the EMT process of primary hepatocyte induced by TGF-β1.3.Effects of HNF4αon HSC cell phenotype and biological characteristicsReal time-PCR revealed that the expression of Vimentin,Snail,collagenⅠ,α-SMA and TIMP-1 decreased significantly in HSC-T6 infected with AdHNF4α(P＜0.05). Interestingly,the expression of liver-specific genes including GS,CYP1A2 and AFP enhanced(P＜0.05).More importantly,E-cadherin expression(epithelial phenotype gene) was also elevated(P＜0.05) after AdHNF4αinfection.Cellular morphology of activated HSC treated by AdHNF4αcould turn from a spindle shape into a more cuboidal/hexagonal shape.Additionally,proliferation of HSC-T6 by HNF4αgene delivery was inhibited according to MTS method(P＜0.01). 【Conclusion】All of our results revealed that:1.The expression of HNF4αwas gradually down-regulated in the development of hepatic fibrosis.2.Gene delivery of HNF4αin vivo was an effective strategy to attenuate experimental hepatic fibrosis by reducing ECM deposition and promoting hepatocyte function.3.HNF4αcould interrupt EMT process in TGF-β1 treated hepatocytes,preserve their epithelial morphology,as well as improve hepatocyte function.4.HNF4αcould inhibit the biological characteristic of HSC and up-regulate liver-specific genes by inducing MET process of the activated HSC,.5.HNF4αameliorates hepatic fibrosis by interrupting EMT of hepatocytes and HSCs, which could lead to improve hepatocyte function and inhibit the activity of activated HSCs.