| 【Background and aim】 Most of chronic liver diseases follow a common pathological process: chronic liver inflammation, liver fibrosis, and cirrhosis. At present, liver fibrosis is considered as a reversible proces. Hence, in-time controling of liver fibrosis could block the development of chronic liver diseases toward cirrhosis. Most of recent studies showed that the activation of hepatic stellate cells(HSCs) plays an important role in liver fibrosis. Therefore, it is traditionally acceped that inhibition of HSC activation and proliferation might reverse liver fibrosis. In recent years, emerging studies revealed that HSCs not only promote hepatic progenitor cells(HPCs) differentiation and hepatocytes regeneration, but also differentiate into hepatocytes and cholangiocytes, indicating that HSCs play an important role in liver regeneration. Therefore, the treatment of liver fibrosis targeting HSCs alone presents certain limitations. Generally, liver fibrosis is caused by a variety of cells in liver. There has been evidence showing that the activation of HSCs could be induced by the apoptosis of hepatocytes, which eventually led to the hepatic fibrosis. Furthermore, recent studies have also indicated that hepatocytes might convert into myofibroblasts through epithelial mesenchymal transition(EMT) involving the process of liver fibrosis. The hepatocyte nuclear factor family, including HNF1, HNF3, HNF4, HNF6 and CCAAT / enhancer binding protein, is mainly expressed in liver. These transcription factors form a complex transcriptional regulatory network, and participate in the liver differentiation, liver development and the maintenance of hepatocyte function. Our previous studies showed that HNF1 alpha and HNF4 alpha could significantly inhibit the activation of HSCs. HNF4 alpha could not only inhibit the EMT process in both hepatocytes and HSCs, but also enhance the liver function. Meanwhile, HNF1 alpha inhibited hepatic inflammation and liver fibrosis by regulating the expression of tyrosine phosphatase SHP-1. Transcription factor FOXA2, also known as HNF-3 beta, belongs to the forkhead transcription factor superfamily and is predominantly expressed in liver, lung, pancreas and gastrointestinal. FOXA2 can regulate the expression of many hepatocytes specific genes in liver, involving in liver development, metabolism and hepatic function maintainance.Briefly, FOXA2 has similar functions with HNF1 alpha and HNF4 alpha. Recently, many studies have showed that FOXA2 expression is reduced in different types of liver diseases. In addition, FOXA2 could regulate the expression of numerous inflammatory cytokines in liver. However, the role of FOXA2 in the process of liver fibrosis has not been reported. Considering the importance of liver fibrosis in the chronic liver disease, herein we first analyzed the expression level of FOXA2 in patient cirrhotic livers and liver tissues of CCl4-administrated mice with different degree of liver fibrosis, as well as the expression of FOXA2 in hepatocytes and HSCs, so as to explore the relationship between FOXA2 expression and liver fibrosis. To explore the relationship between FOXA2 and liver fibrosis, different types of vectors were constructed to up-regulated the expression of FOXA2 in liver, hepatocytes and HSCs respectively. Moreover, mice with hepatocyte specific FOXA2 deletion of in were used to assess the effects of FOXA2 on liver fibrosis. Finally, microarray was applied to screen the differentially expressed genes regulated by FOXA2 in the hepatocytes to investigate the molecular mechanism underlying the role of hepatocyte FOXA2 in liver fibrosis, and thus clarifying whether FOXA2 could serve as a new target in clinical treatment of liver fibrosis.【Methods】 1. The expression of FOXA2 in liver fibrosis The expression of FOXA2 in normal liver and cirrhotic liver of patient was detected by immunohistochemistry. We also compared the expression of FOXA2 in liver tissue of CCl4 model mice with different degrees of fibrosis by Real-time RT-PCR, western blot and immunohistochemistry. Meanwhile, the expression of FOXA2 in hepatocytes and HSCs was detected by Real-time RT-PCR and western blot, respectively. 2. The effect of FOXA2 overexpression on liver fibrosis Firstly, we constructed the FOXA2 overexpression plasmid p CDH-FOXA2 with lentiviral expression vector p CDH-CMV-MCS-EF1-cop GFP. The FOXA2 expressing lentivirus system consists of three plasmids, including p CDH-FOXA2, p MD2.G and ps PAX2. The validated LV-FOXA2 was injected into the tail vein of CCl4 model mice. Then, we compared the expression of FOXA2, collagen I and α-SMA in FOXA2 group and control group. Furthermore, H&E staining and Sirius red staining were performed to assess the pathological change in mice liver. Moreover, the hydroxyproline kit was used to detect the content of hydroxyproline in mice liver. In addition, the expression change of IL-6, TNF-α and TGF-beta 1 in liver tissues of mice were detected by Real-time RT-PCR. 3. Effect of hepatocyte FOXA2 overexpression on liver fibrosis By using adeno-associated virus vector p ENN-AAV-TBG-PI-RBG, we successfully constructed hepatocyte-specific plasmid p AAV8-TBG-FOXA2 for FOXA2 overexpression. Then, the adeno-associated virus was injected in the tail vein of CCl4 model mice. To confirm the specificity of AAV8-TBG-FOXA2, we imported the virus into normal mice through tail vein and isolated the primary hepatocytes and other cells from the mice liver followed by FOXA2 expression analysis using Real-time RT-PCR and western blot. Moreover, the expression changes of FOXA2, Collagen I and α-SMA in FOXA2 group and control group were compared by Real-time RT-PCR, Western blot and immunohistochemistry. Furthermore, H&E and Sirius red staining were conducted to evaluate the pathological change in the mice liver from each group. The content of collagen was then quantified using the software. In addition, hydroxyproline kit was used to detect the content of hydroxyproline in mice liver of each group. Finally, the expression changes of IL-6, TNF-α and TGF- beta 1 in liver tissues of mice were detected by Real-time RT-PCR and immunohistochemistry. 4. Effect of FOXA2 deletion on hepatic fibrosis in hepatocyte-knockout mice FOXA2 lox P/lox P mice were gained from FOXA2 lox P mice crossing. Then the AAV-TBG-Cre were injected into CCl4 mice through the tail vein to specifically knockout the expression of FOXA2 in hepatocyte. After mice were sacrificed, the expression changes of FOXA2, Collagen I and α-SMA in FOXA2 group and control group were determined by Real-time RT-PCR, Western blot and immunohistochemistry. Besides, H&E and Sirius red staining were performed to evaluate the pathological change in the liver of each group, and the content of collagen was quantified using the software. Moreover, hydroxyproline kit was used to test the content of hydroxyproline in mice liver of each group. 5. Up-regulation of FOXA2 expression in HSCs and its effect on liver fibrosis HSCs-specific FOXA2 overexpression plasmid p LVX-GFAP-FOXA2 and lentiviral expression vector p LVX-GFAP-IRES-Zs Green were constructed. After lentivirus packing, we concentrated and purified the virus and injected it into the tail vein of normal mice. The CCl4 was injected into the mice intraperitoneally one week post virus injection. Furthermore, the primary hepatocytes and HSCs were isolated from normal mice administrated with the lentivirus LVX-GFAP-FOXA2, and the specific expression of the FOXA2 was identified by Real-time RT-PCR assay. Besides, the expression of FOXA2, collagen I and α-SMA of FOXA2 group was compared between experimental group and control group by Real-time RT-PCR, Western blot and immunofluorescence. Then H&E and Sirius red staining were conducted to evaluate the pathological change of liver in each group. 6. Effects of FOXA2 up-regulation on hepatocyte gene expression profile in CCl4 model mice. We isolated the primary hepatocytes from CCl4 model mice injected with AAV8-TBG-FOXA2 via the tail vein, and extracted the total RNAs from the cells. After quality check of RNA, gene chip hybridization experiment was carried on to screen the differential genes after FOXA2 overexpression. Finally, the Gene Ontology DAVID(GO), KEGG, BIOCARTA, BBID and other databases were used to obtain the information on the functional gene classification and the related-signal transduction pathway. 7. Statistical analysis All data are presented as mean ± SD. Statistical calculation was performed using the Statistical Program for Social Sciences software 18.0 version. A value of P < 0.05 was considered statistically significant, and P < 0.01 was considered very significant.【Results】 1. FOXA2 expression negatively correlated with the progression of hepatic fibrosis The results of immunohistochemistry showed that the expression of FOXA2 in human cirrhotic liver was significantly lower than that that in normal livers(P<0.01). Meanwhile, FOXA2 expression decreased progressively during the development of hepatic fibrosis in CCl4-administrated mice by immunohistochemistry. Furthermore, the expression of FOXA2 in liver tissues of CCl4 model mice was significantly lower than that in normal mice(P<0.01). However, Real-time RT-PCR and western blot assay revealed that FOXA2 expression was down-regulated in hepatocytes and up-regulated in HSCs(P<0.01). 2. Non-specific overexpression of FOXA2 ameliorated liver fibrosis The primary mouse hepatocytes were infected with LV-FOXA2 and control virus in vitro after separation from mouse liver. Real-time RT-PCR and western blot assay showed the significant overexpression of FOXA2, indicating that the lentivirus was successfully constructed. Moreover, the lentivirus LV-FOXA2 and control virus were injected into CCl4 model mice through tail vein. We found that overexpression of FOXA2 in CCl4 model mice liver had no significant effect on the body weight of mice in FOXA2 group compared with control group(22.782±0.6585 vs 22.290±1.2478), while liver weight(1.350±0.2090 vs 1.894±0.3868) and the ratio of liver weight to body weight(0.059±0.0095 vs 0.084±0.0134)were obviously higher in FOXA2 group. Furthermore, Real-time PCR and western blot assay indicated that non-specific overexpression of FOXA2 could reduce the expression of Collagen I and α-SMA in the liver of CCl4 model mice. Besides, results of Sirius red staining suggested that overexpression of FOXA2 could significantly reduce hepatic collagen deposition(P<0.01). In addition, hydroxyproline assay showed that non-specific FOXA2 overexpression significantly reduced the level of hydroxyproline in liver of FOXA2 group compared to control group(376.810±47.5108 vs 249.926±42.9257). We also determined the expression of inflammation-related genes, and the result showed that TGF-beta 1 significantly reduced in FOXA2 group compared to control group, while IL-6 and TNF-α exhibited no significant difference between the two groups. 3. FOXA2 overexpression in hepatocytes could ameliorate liver fibrosis First of all, we used Realtime RT-PCR and western blot to identify the specific expression of AAV8-TBG-FOXA2, and the results showed that the virus could up-regulate FOXA2 expression in hepatocytes rather than other cell types. Then the adenovirus associated virus AAV8-TBG-FOXA2 and control virus were injected into CCl4 model mice through tail vein. We found that overexpression of FOXA2 in hepatocytes of CCl4 model mice liver could significantly increase the body weight of mice in FOXA2 group compared with the control group(20.828±0.9264 vs 22.647±0.5900), as well as in liver weight(1.072±0.0987 vs 1.382±0.1724) and the ratio of liver weight to body weight(0.051±0.0035 vs 0.061±0.0080). Furthermore, Real-time RT-PCR and western blot showed that overexpression of FOXA2 in hepatocytes could reduce the expression of Collagen I and α-SMA in liver of CCl4 model mice. Sirius red staining suggested that overexpression of FOXA2 in mice liver could significantly reduce hepatic collagen deposition compared with control group(1.832±0.3559 vs 1.126±0.2511, P<0.001). In addition, hydroxyproline assay suggested that overexpression of FOXA2 in hepatocytes could significantly reduce the level of hydroxyproline in liver of mice compared to control group(547.800±85.8522 vs 354.125±155.384, P<0.05). Moreover, we detected the expression change of inflammation-related genes, and Real-time RT-PCR and immunohistochemistry assay showed that TGF-beta 1, IL-6 and TNF-α levels were significantly reduced in FOXA2 group compared to control group. 4. Hepatocyte-specific ablation of FOXA2 promoted liver fibrosis CCl4 was injected into hepatocyte-specific FOXA2 knock-out mice and the control mice. The body weight appears slightly reduced in FOXA2 knock-out mice compared to control mice(5.610±2.1738 vs22.391±3.2161), but no significant difference was observed(P = 0.07). Liver weight of mice in knock-out group decreased significantly(1.587±0.3028 vs 1.182±0.2307) and the ratio of liver weight to body weight was consistently decreased(0.061±0.0068 vs 0.053±0.0055). Moreover, FOXA2 expression in mice of knock-out group decreased by 88% compared with control group(P<0.01) at the m RNA level, while expression of collagen I increased up to 1.97 times(P< 0.01) and α-SMA exhibited 3.65 times(P< 0.01) higher than that of control group. Western blot and immunohistochemistry assay showed that FOXA2 significantly reduced in the liver of mice from knock-out group, while expression of collagen I and α-SMA was increased in compared with control group. Furthermore, H&E and Sirius red staining suggested that connective tissue increased significantly in the liver of mice from knock-out group. In addition, the hydroxyproline content was higher in the liver of mice from knock-out group than that from control group(126.634±19.6342 vs 185.516±28.7116, P<0.01). 5. FOXA2 overexpression in HSCs had no effect on liver fibrosis Real-time RT-PCR assay suggested that infection of lentivirus LVX-GFAP-FOXA2 could significantly increase the expression of FOXA2 in HSCs but not in hepatocytes. Besides, overexpression of FOXA2 in HSCs had no significant effect on α-SMA expression in normal mice. In CCl4 mode, overexpression of FOXA2 in HSCs did not affect the body weight and the ratio of liver weight and liver weight(P>0.05). Moreover, overexpression of FOXA2 in HSCs could marginally promote the expression of collagen I and α-SMA in CCl4 model mice compared with control group, but statistical difference in two groups was not significant(P>0.05). Sirius red staining and H&E staining showed that HSC-specific overexpression of FOXA2 had no significant effect on liver pathology. In addition, immunofluorescence assay indicated that overexpression of FOXA2 in HSCs had no significant influence on the expression of α-SMA in liver of CCl4 model mice. 6. FOXA2 overexpression in hepatocytes affected the gene expression profile in liver of CCl4 model mice Agarose gel electrophoresis assay showed that the quality of RNA sample met the required criterion. Gene chip screening demonstrated that there were 3219 up-regulated genes and 713 down regulated genes between control group and normal group, while 282 up-regulated genes and 279 down-regulated genes between FOXA2 group and control group. Through KEGG analysis, the pathways involved in liver fibrosis were obtained, which includ MAPK signal pathway(15 genes), PI3K-Akt signal pathway(7 genes), cell adhesion molecules(7 genes), apoptosis(5 different genes), p53 signaling pathway(4 genes), NF kappa-B signaling pathway(4 genes), VEGF signaling pathway(2 genes), TNF signaling pathway(2 genes), TGF-beta signaling pathway(2 genes), Erb B signaling pathway(2 genes), Hedgehog signaling pathway(1gene) and JAK-STAT signal pathway(1 gene).【Conclusion】 1. The expression of FOXA2 is decreased in the liver fibrotic tissues. Interestingly, it was decreased in hepatocytes and increased in HSCs. 2. Overexpression of FOXA2 could significantly ameliorate the liver fibrosis.3. Hepatocyte-specific ablation of FOXA2 aggravates liver fibrosis in mice and hepatocyte-specific overexpression of FOXA2 significantly alleviates liver fibrosis, while HSC-specific overexpression of FOXA2 exhibited no significant effect on liver fibrosis. 4. The expression of FOXA2 affects the process of liver fibrosis and the hepatocyte would be an important target for the treatment of liver fibrosis. |
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