Hepatocyte Nuclear Factor 4α Reverses Liver Cirrhosis In Rats

【Background and Objective】Hepatic cirrhosis is the advanced stage of fibrosis, compromising a significant morbidity and mortality worldwide. It is defined as a diffuse process characterized by fibrosis and the conversion of normal liver architecture into structurally abnormal nodules. There is now a substantial body of evidence to show that liver fibrosis is an active, dynamic process and no doubt at all on the reversibility of hepatic fibrosis. However, it is still controversial on the possibility of cirrhosis reversion. Although there are now some evidences in human liver disease and animal models indicating that liver cirrhosis could regress over time, most studies demonstrated different extent of reversal of fibrosis in cirrhosis, but not complete reversal of cirrhosis itself. And complete reversion of cirrhosis has never been clearly demonstrated.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.This study was to investigate the therapeutic effect and the mechanism of HNF4αon hepatic cirrhosis.【Methods】1. Amplification of replication-deficient recombinant adenovirusesAdHNF4αand control adenovirus-AdGFP replication-deficient recombinant adenoviruses were infected in 293 cells respectively. Then adenoviruses were stored after purification by cesium chloride (CsCl) gradient centrifugation and determination of the viral titers.2. Treatment of experimental hepatic cirrhosis in ratThe distinct model of early stage hepatic cirrhosis was induced by TAA: Male Sprague-Dawley (SD) rats were divided into 4 groups randomly (8 rats in each group). Group 1 served as a normal control that received intraperitoneal saline injection, rats in groups 2, 3 and 4 were injected intraperitoneally with 10% TAA (0.2g/kg) for three times per week (once per 2 days) up to 15 weeks. The distinct model of advanced stage hepatic cirrhosis was induced by TAA for 18 weeks in the same way. After the last injection of TAA, rats in groups 2, 3 and 4 were infused with saline, 5×10~9 pfu AdGFP or 5×10~9 pfu AdHNF4αvia tail vein, twice a week, for three weeks. 2 weeks after the end of infusion, the animals were sacrificed for analysis of morphological and histological study.3. Histological examination and immunohistological stainingAll paraffin-embedded liver tissues were stained with H&E, Masson's trichrome, and Sirius red staining. Three fields were selected randomly from each section, and three rats from each group were examined. Immunohistochemistry were performed on paraffin-embedded liver sections according to manufacturer's recommendation.4. Measurement of hepatic hydroxyproline contentTotal hepatic hydroxyproline levels were determined in the hydrolysates of liver samples as previously described.5. Statistical analysisThe arithmetic mean and standard deviation (s.d.) were calculated for the data, and statistically evaluated using 2-tailed unpaired t-test. P < 0.05 was considered statistically significant.【Results】1. HNF4αreverses experimental hepatic cirrhosisIntroduction of AdHNF4αreversed the early stage of hepatic cirrhosis. Morphological study showed that, in the early stage of hepatic cirrhosis (15 weeks), the hardness of liver increased and the surface showed granular or nodular. Pathological examination showed that liver cells were degenerated and necrosis. The necrosis site was marked in the area of surrounding hepatic lobules. Inflammatory cells were infiltrated into liver periportal and fibrous tissue was hyperplasia. The formation of pseudolobule could be observed in liver widely and the pseudolobules were re-segretated by fibrous tissues. Regeneration nodules of hepatocytes could be observed obviously. The liver of AdHNF4αtreated rats is soft, the surface is smooth. HE, Masson and Sirius Red staining showed that HNF4αtreated rats significantly reduced the degree of liver fibrosis and the pseudolobule disappeared, suggest that HNF4αcould reverse early stage of hepatic cirrhosis. Real time PCR and immunohistochemical analysis showed that, along with the development of cirrhosis, HNF4αexpression was down-regulated. Less amount of liver hydroxyproline was detected in AdHNF4αgroup, compared with that of AdGFP group (P<0.05). The Real time PCR results showed that the hepatic function related factors ALB, GS and CYP1A2 increased in AdHNF4αinfused group (P<0.05), which means HNF4αcould protect hepatic function. Meanwhile, the expression of collagenⅠ, collagenⅢ,α-SMA, TGF-?1, FSP1 significantly decreased compared with AdGFP group (P<0.05).But the treatment of AdHNF4αcould partially ameliorate the advanced hepatic cirrhosis. In the advanced stage of hepatic cirrhosis (18 weeks), the hardness of liver increased and the surface showed granular or nodular. Furthermore, a large number of ascites formate, indicates that the rats entered the decompensated stage of hepatic cirrhosis. In AdHNF4αgroup, the formation of pseudolobule could be observed in liver. Indicate that HNF4αcould only reduce the degree of fibrosis, but could not reverse the advanced hepatic cirrhosis.2. HNF4αinhibits ERK signal pathwayAccording to the real time PCR and Western blot results, HNF4αdecreased ERK1, ELK1 mRNA in BRL-3A and HSC-T6(P<0.05), but MEK1 increased compared with AdGFP(P<0.05). In addition, HNF4αreduced both phosphorylated ERK1 protein and phosphorylated JunD protein, but ERK1/2 and JunD protein expression was not affected.Real time PCR and IHC revealed that the expression of HNF4αin liver decreased gradually with the formation of liver cirrhosis. Real time PCR revealed that ERK1, ELK1 significantly decreased(P<0.05)and MEK1 increased slightly(P<0.05). IHC revealed that the expression of p-ERK, ERK1/2, p-JunD, JunD decreased compared with control groups.3. HNF4αregulates expression of TIMP-1 and MMPsWe then analyzed the levels of TIMP1 and MMPs by Real time PCR and Western blot in BRL-3A and HSC-T6 cell lines. Real time PCR revealed that TIMP-1 decreased, MMP2, MMP9, MMP13 upregulated(P<0.05). Western blot in HSC-T6 revealed that TIMP-1 decreased. But when the effect of HNF4αstopped, TIMP-1 was upregulated.Real time PCR revealed that the expression of TIMP-1 significantly decreased and MMP2 increased slightly(P<0.05), MMP9 and MMP13 were not affected in liver. IHC revealed that the expression of TIMP-1 and MMPs decreased in compared with control groups.【Conclusion】1. The expression of HNF4αwas gradually down-regulated during the development of hepatic cirrhosis.2. Gene delivery of HNF4αin vivo could reverse early stage of cirrhosis, and improve hepatocyte function. But the treatment of AdHNF4αcould partially ameliorate advanced stage of hepatic cirrhosis.3. The effect of HNF4αon liver cirrhosis was achieved by down-regulating the expression of ERK and TIMPs and enhancing MMPs activity.