Activation Of β-catenin And Its Cellular Localization And Differentiation Induced By Pathological Microenvironment Of Mouse Hepatic Fibrosis

Background and purposeIt has been proved that cell death caused by a variety of reasons willinduce the regeneration and repairing of the tissues, which depend on thecommunication between the cells and its microenvironment. If thepathological tissue damaged and cell loss occurred repeatedly, the abnormaltissue structures, such as liver cirrhosis, glandular epithelial hyperplasia,abnormal metaplastic etc., will appear after the continual regeneration andrepairing process. Because the destruction of the inherent differentialmicroenvironment of normal cells, further differentiation and maturation ofthe proliferating cells will thus be interfered or lead to abnormal proliferation.China is a liver disease-prone area. chronic liver injury caused by a variety ofpathogenic factors （viruses, alcohol, parasites, chemical damage, etc.） thatexist for long trem or repeated inflammatory activity will finally develop to liver cirrhosis and liver cancer, which must pass through the pathologicalprocess of liver fibrosis, therefore, the cell proliferation, differentiation andits molecular regulation under the microenvironment of fibrosis is the key tounderstand the mechanism of liver cancer genesis.The existing studies suggest that liver fibrosis is a result of the interactionof the apoptosis of damaged hepatic cells and activation of hepatic stellatecells （HSC）. The activated HSCs will express α-smooth muscle actin（α-SMA） and a variety of extracellular signal transduction pathway proteins,and produce a large number of collagen-based extracellular matrixcomponents and cytokines, result in the changes of the microenvironmentand structure of the liver tissue. Multiple signaling pathways correlated withthe process of liver fibrosis, including the Wnt/β-catenin pathway whichregulates the proliferation and differentiation of cells, but the localization androles of β-catenin, the key effector molecule of Wnt/β-catenin pathway, inthe process of liver fibrosis is obscure. In this study, the traditional CCl₄hepatic injury model, histopathologic methods and molecular biologymethods will be used to dynamically observe the expressive characteristicsand localization of β-catenin during the progress of hepatic fibrosis, toconfirm the cell types in which the β-catenin signal was activated. The rolesof β-catenin in the pathological changes of liver structure and cellularmicroenvironment will be explored. Methods1.Animal modelHealthy male Kunming mice （n=45） were randomly divided into controlgroup （n=15） and experimental group （n=30）. The experimental group wasrandomly divided into injury1wk group, injury4wk group, injury8wk group.The mice of the experimental groups were injected subcutaneously with CCl₄liquid （6mL/kg body weight） for the first time, after then, injected with50%CCl₄-corn oil suspension fluid （6mL/kg body weight） every3days again,twice a week. The mice of control group were injected subcutaneously withan equivalent dose of corn oil, twice a week.2. The preparing of the experimental specimensMice in each group were sacrifced at the end of1,4and8weeks.Themiddle lobe of liver tissues were dissected and part of them were quicklyfrozen in liquid nitrogen and used to extract RNA and protein, the other partwere fixed at the40g/L neutral formalin and embedded with paraffin. Thetissue array paraffin block and slide were prepared.3. Experimental methodsThe liver pathological changes were estimated and compared by H&Estaining and Masson staining. The expression of Desmin, a-SMA andβ-catenin in the liver tissue were detected by immunohistochemistry, RT-PCRand Western Bloting methods and analyzed statistically. The cell types ofβ-catenin positive cell were determined to evaluate its significance by β-catenin and CK19immunofluorescence double labeling methods.Results1.H&E stainingLiver lobular architecture of the control group was normal and no celldegeneration and necrosis can be found. The damage degree of the livers inexperimental groups were aggravated along with the administration. A littleinflammatory cells and a small number of necrotic cells can be found in the1wk liver tissue; many vacuolar degeneration of liver cells and fibroplasiabetween the hepatic lobule were appeared in the liver tissues of the4wkgroup, many newly proliferating cell mass can also be found in the portalarea around lobules. in the liver tissues of the8wk group, lobular architecturewas destructed and the fibrous septa surrounding the liver tissue and form thefalse lobules with more freshmen bile duct.2. Massson-stainingIn the portal area of liver tissues in control group, the green collagenfibres can be found in the vessel wall, but increased gradually in the livertissues of experimental groups. The hyperplasia of collagen fibres appered inthe8wk liver tissues and form the septa and false lobules.3. Expression of DesminImmunohistochemical results showed that a few Desmin positiveexpressing cells scattered in the liver tissues of control group and located around the hepatic sinusoid. The positive cells number was increased with theprolonged injury time and arrived its supreme at4wk. RT-PCR resultsshowed that the expression of Desmin mRNA appeared in both the controlgroup and experimental groups, The positive cells in the liver tissues of theexperimental groups at all time point were significantly higher than that ofcontrol group （P <0.001）, and had significant difference when compared eachother within1wk,4wk and8wk groups（P <0.001）.4. Expression of α-SMAImmunohistochemical results showed that only a few α-SMA positivecells can be found in the vascular wall of liver tissues in the control group; inthe experimental group, α-SMA positive cells were distributed beside theliver cells and liver sinusoids. The α-SMA positive cells gradually increasedwith the prolonged injury time and arrived its peak at4wk. The RT-PCRresults showed the α-SMA expressed in all the control and experimentalgroups with a significant difference （1wk vs. control group, P <0.05,4wk,8wk vs. control group, P <0.001）. The comparison of α-SMA expressionwithin1wk,4wk and8wk gropus were also different significantly （P <0.001）,the expressive level of α-SMA expression in4wk group was significantlyhigher than that of the other two groups（P <0.001）.5. Expression of β-cateninRT-PCR results showed the β-catenin expression appeared in all thecontrol and experimental groups. A decreased β-catenin mRNA level were found in the liver of experimental group at4wk and8wk after CCl₄treatment（P<0.01）. There are no significant difference can be found between injury1wk and the control group （P>0.05）, but have the significant differencebetween4wk or8wk and control groups （P<0.01）, the expression ofβ-catenin in4wk group was the lowest one. The Western blot results showedthat the β-catenin expressed in the liver tissues of all groups, a significantlyincreased level of β-catenin protein were appeared in the experimental groupat injury4and8wk （P <0.001）, but no significant difference between injury1wk and the control group（P>0.05）. The immunohistochemical resultsshowed that the membrane of liver cells and bile duct epithelial cells werepale marked with β-catenin antibody. Besides the basal expression ofβ-catenin in liver cell membrane, a dark staining of β-catenin was seen in thecytoplasm of liver sinusoidal endothelial, the intrahepatic newborn cell massand the bile duct epithelial cells in injury4wk group, some individualhepatocytes also have the positive expression of β-catenin in its cytoplasm.6. Immunofluorescent double labeling of β-catenin and CK19Immunofluorescence double labeling results showed that the expressionof β-catenin and CK19can be found in the control group, both of which werelocated in the bile duct epithelial cells; the double labeling result of1wkgroup was the same with the that of control group; the higher expression ofβ-catenin in the newborn cell mass can be seen in4wk group. The β-cateninand CK19were co-localizated or apperaed alone in the cytoplasm of new-cell mass and biliary epithelial cells in the4wk group; themajorityepithelial cellsof bile duct in the8wk liver tissue appeared the co-expression of β-cateninand CK19.Conclusions1. The mouse liver fibrosis model induced by CCl₄was constructedsuccessfully.2. β-catenin positive cells in the fibrosis liver induced by CCl₄mainlylocalized in the neonatal biliary epithelial-like cell mass and the cytoplasm ofbile duct epithelial cells, its mRNA and protein expression were notsynchronized and which suggesting there may be an accumulation ofβ-catenin caused by the decreased degradation of the protein.3. The proliferation of bile duct epithelial-like cells can be induced during theliver fibrosis, both the β-catenin and CK19were co-expressed in these cellswhich belong to hepatic stem cells （oval cells）.