Effectiveness Of β-catenin In Acute Liver Injury

Background &Objective:β-catenin is the main component of the canonical Wnt signaling pathway , which is targeted for regulating cell growth, regeneration and differentiation. Induced by Wnt ligand, cytoplasmicβ-catenin molecules formulate transcription complex with Tcf4/Lef and translocate into nucleus. Nuclearβ-catenin regulates cell growth and apoptosis by switch on transcription and expression of downstream target genes such as c-Myc, Cyclin D1 and Survivin. Meanwhile,β-catenin interacts with transmembrane proteins of the cadherin family to promote cell-cell adhesion. Numerous physiologies and pathologies in liver closely correlate with abnormal change of Wnt/β-catenin pathway, especially withβ-catenin mutation. Studies imply thatβ-catenin plays a significant role in regulation of hepatocyte development, proliferation and apoptosis.Mutations ofβ-catenin also show relations with hepatocellular carcinoma and hepatoblastoma. However, there have been no functional research addressing the requirement of Wnt/β-catenin pathway, especiallyβ-catenin, in acute liver injury.Acute liver injury is a severe clinical syndrome associated with high mortality. Autoimmune hepatitis,viral hepatitis,alcohol consumption and hepatotoxins have been identified as trigger factors of acute liver injury. Massive hepatocytes necrosis and apoptosis is an important pathobiologic feature in acute liver injury. The Fas antigen is a cell surface protein which mediates apoptosis. After binding with specific ligand or antibody, Fas antigen induces apoptosis by switching on caspase cascade. Administration of anti-Fas antibody(Jo2) quickly killed mice through massive hepatocyte apoptosis. Bacteria lipopolysaccharide (LPS) is a strong inducer which could promotes macrophages to secrete TNF-αand nitric oxide to promote oxidative stress which woule induce acute liver failure. D-galactosamine (D-GalN) could sensitize rodents to LPS induced liver injury. LPS plus D-GalN could induce acute liver injury in mice. This research uses hepatocyte-specificβ-catenin knockout mice as a tool to establish the liver injury modol induced by Jo2 and LPS/D-GalN respectively to explore the role ofβ-catenin in acute liver injury.Materials&Methods: 1.B6.129-Ctnnb1tm2Kem/KnwJ (carrying the floxed allele at intron 1 and 6 ofβ-catenin) mice and B6.Cg-Tg(Alb-cre)21Mgn/J (albumin promoter-driven Cre recombinase) mice were obtaind from Jackson Laboratory in America. Alb-Cre andβ-cateninloxP/loxP mice were crossed twice to obtain hepatocyte specificβ-catenin knock out mice. Genotype was identified via PCR, Western blot and immunohistochemistry . 2.Male Alb-cre;β-cateninloxP/loxP (β-catenin KO)mice, weighing 18-23g,at 6-8 weeks of age, were randomly divided into three groups which receive either Jo2 (Jo2 group,n=6) or LPS/D-GalN(LPS group,n=6), and untreatment group(n=4). Mice with heterozygousβ-catenin hepatocytes (Alb-Cre;β-cateninloxP/+) and mice lacking the Cre recombinase (β-cateninloxP/loxP andβ-catenin loxP/+) were grouped together as control littermates (Control). Jo2 group mice were injected intraperitoneally with Jo2(0.5ug/g body weight) and LPS group mice were given a single intravenous injection of LPS(80ug/g body weight) plus D-GalN(800ug/g body weight) .Serum and liver samples were collected at 6h after injection. 3. (1) Serum samples were used for measurement of serum ALT and AST; liver sections were subjected to hematoxylin-eosin staining .These measurements were used for studying how liver specific loss ofβ-catenin impact on Jo2 and LPS/D-GalN induced liver injury. (2) Measure the level of inflammation factors(TNF-α,IL-6,IFN-γ)via Real-time PCR to investigate the influence of liver specific loss ofβ-catenin gene in Jo2 and LPS/D-GalN induced inflammation reaction in liver. (3) Liver sections were subjected to TUNEL staining and expression of apoptosis associated signaling molecules, including Caspase-3,PARP , Bcl-xL and NF-κB,were detected via Western blot or immunohistochemistry .(4).GSH lever of liver tissue was detected via GSH detection kit and iNOS mRNA expression was analysed by real-time PCR, which enable us to investigate the role ofβ-catenin in oxidative stress. 4. The data were analyzed by SPSS statistics software.Results:1. Generation of hepatocyte-specificβ-catenin knockout mice. Genotyping identified thatβ-catenin KO mice show ctnnb homozygosis(-/-) and Alb-Cre positive expression. Immunohistochemistry confirmed that Wt mice showed uniformβ-catenin expression in endothelial cells as well as liver parenchymal cells. However,β-catenin was completely absent in liver parenchymal cells ofβ-catenin KO mice. Western blot analysis showed an obviously lower expression ofβ-catenin inβ-catenin KO mice. 2.In Jo2 induced acute liver injury model,β-catenin KO mice showed significant higher AST and ALT lever compared with control group mice,P<0.05. Real-time PCR revealed a little higher level of IL-6 and IFN-γ. HE staining(200X) showed significant sinusoid inflationand congestion, extensive acidophilic necrosis, focal hemorrhages, hepatic lobula structure disorder and slightly lymphocyte inflammatory cells infiltration inβ-catenin KO mice; in control group, HE staining show slightly sinusoid inflation and congestion, limited acidophilic necrosis and focal hemorrhages, hepatic lobula structure nearly integrity , slightly lymphocyte inflammatory cells infiltration. Immunohistochemistry show higher rate of nucleus positive staining ofβ-catenin in KO group than in control group. TUNEL analysis showβ-catenin KO group have more numbers of apoptotic cells per high-powered field than control group(P<0.05). Western blot displayed higher expression of cleavaged caspase-3, PARP and a lower expression of Bcl-2 inβ-catenin KO group.3. In LPS/D-GalN induced acute liver injury model,β-catenin KO mice show significant lower AST and ALT lever compared with control group mice(P<0.05). Real-time PCR revealed lower level of IL-6 and iNOS ofβ-catenin KO group than control group whereas show a higer level of Bcl-xL. HE staining(200X) showed dispersed sinusoid inflationand, congestion and inflammatory cells infiltration. Hepatocyte lamellar acidophilic necrosis, focal hemorrhages inβ-catenin KO mice; in control group, HE staining showed serious sinusoid inflation and congestion, massive acidophilic necrosis, hemorrhages and lymphocyte inflammatory infiltration, hepatic lobula structure disorder. Immunohistochemistry showed little difference of nucleus positive staining of P65 betweenβ-catenin KO group and control group. TUNEL analysis showed thatβ-cateninKO group have fewer numbers of apoptotic cells per high-powered field than control group (P<0.05). Western blot revealed lower expression of cleavaged caspase-3 and PARP . Bcl-2 shows a little higher expression compared to control group.Liver tissue GSH lever was significant higher inβ-catenin KO group than Control group.Conclusions:1.Degree of liver injury induced by Jo2 is significantly higher in hepatocyte-specifecβ-catenin knockout mice than in control group, which indicate a protective role ofβ-catenin in Fas mediated apoptosis. 2. Liver injury induced by LPS/D-GalN is less severe in hepatocyte-specificβ-catenin knockout mice than control group, which indicate that loss ofβ-catenin in hepatocyte lower the susceptibility to LPS/D-GalN induced liver injury.The mechanism may depends on the severe oxidateve stress potentiated byβ-catenin.