The Role Of Frizzled-4 In The Interstitial Remodeling Of Liver Injury And Intervention Influence Of Loureirin

Introduction:Liver fibrosis is a reversible wound self-healing response which refers to a variety of chronic pathogenic factors that leading to hepatocytes inflammation and necrosis,the imbalance between degradation and deposition of collagen and other extracellular matrix(ECM),leading to abnormal proliferation of liver connective tissue and angiogenesis in the pathological state.The activation of hepatic stellate cells is the central critical step at the progress of liver fibrosis.The cell proliferation,migration and contractility are significantly increased in activated hepatic stellate,which secreted and gathered to the excessive deposition of collagen and other extracellular matrix in Diss gap,and also generated a variety of pro-angiogenic factor to promote the reconstruction of hepatic mesenchyme.WNT signaling is a conserved pathway involved in cell differentiation and fate determination during embryo-genesis and late stages of development.WNT signaling constitutes one of the most critical biological processes during cell fate assignment.The canonical signaling WNT/?-catenin signaling pathway transduction needs binding extracellular WNT ligand proteins to transmembrane receptors of frizzled family and interacting with co-receptors of the "low densitylipoprotein-related protein"(LRP)family.Cells stimulation by WNT protein induces dishevelled recruitment at the membrane and blocking the GSK-3?,Axin and APC inhibiting complex.These events result in the release of ?-catenin which translocates to the nucleus where it forms dimers with the“LEF/TCF”DNA binding factors(lymphoid enhancer factor 1/T-cell factor).Ultimately,regulate the transcription and expression of target genes that related to cell survival,proliferation,division,migration,and other extracellular matrix synthesis and degradation.The emerging research of Wnt/?-catenin signaling shows that there is a certain relationship among the Wnt/?-catenin pathway and activation of hepatic stellate cells and liver fibrosis.Blocking this signaling pathway is a effective way to inhibit the activation of hepatic stellate cell and to defer the process of chronic hepatitis and liver fibrosis.Consequently,Frizzled-4/LRP5 probably play a key role in WNT/?-catenin signaling pathway,and the studies of its intervention can provide novel molecular targets for the treatment of anti-liver fibrosis.Our previous research shows that loureirin and PAE are effective in anti-liver fibrosis,but the molecular target about the intervention for hepatic fibrosis is not clear.So,the major purpose of this study is to explore the role of Frizzled-4 receptor in the interstitial remodeling of liver injury and intervention influence of loureirin.Objective:To explore the effect of loureirin and PAE on the expression of Frizzled-4 acceptor,the cell proliferation,the reconstruction of liver mesenchyme,and the synthesis and secretion of vascular cytokines.Methods:In vitro studies:Isolated hepatic stellate cells from Sprague-Dawley rat,activating and inducing primary hepatic stellate cell from qHSC to aHSC.Then,the cultured aHSC were treated with different concentrations of loureirin A/B or APE,the inhibitory rate of aHSCs proliferations were measured by MTT assays,the expressions of Frizzled-4 were measured by western blot and RT-PCR analysis,and the content of ?-SMA,TGF-?1,and VEGF in the cultured aHSCs'supenatant were measured by enzyme-linked immunosorbent assay(ELISA).In vivo studies:Establishing the rat-model of acute liver injury that induced by thioacetamide(TAA),to explore the content of angiogenic related cytokines in serum,the degree of liver damage,and the expression of Norrin/Frizzled-4 signal in liver tissue.Animal experiment showed that the contents of VEGF and CD 105 is lower in the serum of the normal control group than the acute liver injury group that induced by thioacetamide than;in the injury group,the tissue is swelling with inflammatory necrosis and the expressions of Norrin/Frizzled-4 signaling proteins is increased(P<0.05).Conclusion:Loureirin and PAE can inhibit the proliferation of hepatic stellate cells effectively,they also possibly affect the activated hepatic stellate cell maintain their phenotype and slow down the process of liver fibrosis by inhibiting the secretion of TGF-?1 VEGF and a-SMA,and reducing the expression of Frizzled-4 receptor in WNT/?-catenin signaling pathway.Frizzled-4 receptor is highly expressed in tissues of acute injury liver,which may be related to the activation of WNT/?-catenin signaling pathway and involved in the tissue repair and the interstitial remodeling of liver injury.