| Background and objectives:Non-anastomotic bile duct stricture (NABS) is the main biliary complication of livertransplantation, its pathological basis is the abnormal wound healing after epithelial injuryand the subsequently biliary fibrosis. Cold preservation reperfusion injury (CPRI) isconsidered as an independent risk factor of biliary fibrosis in graft, but the mechanismremains to be clarified. Recent studies found that biliary epithelial cells (BEC) function asthe heterologous matrix producing cells through epithelial-mesenchymal transition (EMT)which contributes to the biliary fibrosis. EMT refers to the epithelial cells gradually lost itsphenotype accompanied with the aquisition of mesenchymal characteristics under specificconditions, which plays an important role in embryonic development, tumor metastasis andepithelial wound healing. Previous studied demonstrated that BEC EMT is the pathologicalmechanism of biliary fibrosis in primary biliary cirrhosis and primary sclerosing cholangitis.Same results were also found in renal tubular and lung epithelial cells. Meanwhile, EMTwas observed in mature BECs after stimulated by cytokine/proinflammatory in vitro, aswell as in the liver graft of rats after transplantation. Results suggest that BEC EMT mayparticipate in the process of CPRI-induced biliary fibrosis after liver transplantation.CPRI is the inevitable pathological process of liver transplantation, and interleukin-6(IL-6) is the main responsive factor in BEC. IL-6stays at a low level in BEC under normalconditions. Diversified pathological factors lead to sharply increase in IL-6expression andthe consequent phosphorylation of STAT3via its binding to the receptor gp130. Activationof IL-6/gp130/STAT3signaling pathway was found to be involved in BEC proliferation,wound healing, and barrier formation, while IL-6-/-mice was vulnerable to impaired biliaryepithelial integrity due to its insufficient repair and barrier functions. Previous studiesfound that either treatment with CPRI or rhIL-6enhanced BEC proliferation in the rat livergraft via phosphorylation of STAT3, the accumulation of peri-biliary myofibroblasts and collagen deposition were observed simultaneously with the prolongation of the coldpreservation duration and increasing dosage of rhIL-6, acompanied with co-expression ofboth epithelial and mesenchymal markers in BEC. Results suggest that CPRI induces BECEMT via activation of IL-6/STAT3signaling pathway, which contributes to transition intothe matrix producing cells in biliary fibrosis after liver transplantation.As an important member of newly defined trefoil peptide, trefoil factor family3(TFF3)is a small polypeptide molecule which play an important role in the wound healing processof the gastrointestinal epithelium. Recent researches suggest that TFF3was regulated byIL-6/STAT3signaling system and enhances BEC migration, which participates the biliaryepithelial wound healing. In addition, TFF3was found to locate mainly in BEC of large bileducts in portal area and intrahepatic biliary trees, which also consistent with the lesiondistribution of NABS after liver transplantation. Therefore, TFF3may play a key role in theregulation of CPRI-induced BEC EMT and the subsequent biliary fibrosis.Based on the above theoretical basis and research results, we hypothesize that CPRIinduces BEC EMT via activation of IL-6/STAT3signaling pathway and its downstreammolecule TFF3, which plays a critical role in the regulation of BEC EMT. The presentstudy was to investigate the effect and mechanism of IL-6/STAT3/TFF3signaling system inthe process of CPRI-induced BEC EMT. The results were expected to further clarify thepathogenesis for biliary fibrosis in liver graft, and to provide a new target for clinicaltreatment of NABS after liver transplantation.Methods:1. Liver specimens of patients underwent retransplantation due to NABS from2002Nov to2011Jan were collected from department of pathology of our hospital. Afterpreliminary screening, the serial sections were analyzed by using immunohistochemicalstaining. The expressions of epithelial markers (CK19, E-cadherin) and mesenchymalmarkers (a-SMA, Vimentin) in BEC were determined carefully, with simultaneousdetermination of TFF3expression and its location.2. Further experiments were perfomed in vitro which began with the simulation ofCPRI process in cultured hBEC. QRT-PCR and WB analysis were applied to determine theexpressions of IL-6/STAT3/TFF3signaling pathway and the epithelial/mesenchymalmarkers. Then, the addition of rhIL-6or neutralizing anti-IL-6, STATTIC (a specific inhibitor of STAT3phosphorylation), rhTFF3, and transfection of TFF3siRNA wereperfomed respectively to inhibit/enhance the signaling activation followed by thequantitation of mRNA and protein expressions. Finally, TFF3adenovirus was constructedand transfeted to hBEC, wound healing assay was applied to determine its direct effect onBEC migration.3. Experiments in vivo were performed in real disease conditions. Homologous maleSprague Dawley rats were subjected to orthotopic liver transplantation with reconstructionof the hepatic artery. Rats with different durations of graft cold preservation (1h,6h,12h)were sacrificed at1,3,7, and14days after transplantation, sham operation was used ascontrol groups. Bile duct tissues of rats from a total of16groups (n=5) were detected byusing QRT-PCR and WB analysis. The expressions of IL-6/STAT3/TFF3signaling pathwaywith epithelial markers (CK19, E-cadherin) and mesenchymal markers (a-SMA, Vimentin)were determined quantitatively.Results:1. Immunohistochemical staining in human liver sections showed co-expressions ofthe epithelial markers (CK19, E-cadherin) and the mesenchymal markers (a-SMA,Vimentin) in BEC, with the coinstantaneous high expression of TFF3which located in thesame areas. Results suggest that BEC EMT do exist in the liver graft, and TFF3may beinvolved in the regulation of this process.2. QRT-PCR and WB analysis showed a signifcant up-regulation of IL-6/STAT3/TFF3signaling pathway in hBEC treated with either rhIL-6or CPRI, accompanied with thedown-regulation of CK19, E-cadherin and up-regulation of a-SMA, Vimentin. The effect ofCPRI was inhibited signifcantly after signaling system blockage by adding neutralizinganti-IL-6/STATTIC/TFF3siRNA. In contrast, the addition of exogenous rhTFF3couldrevert the defect of hBEC EMT caused by TFF3gene silencing. In addition, a significantlyenhanced hBEC migration was observed after transfection with the TFF3adenovirus in ourwound healing assay. Results suggest that CPRI induces BEC EMT via activation ofIL-6/STAT3signaling pathway and its downstream molecule TFF3, which plays a criticalrole in the regulation of BEC EMT.3. QRT-PCR showed significant up-regulations of IL-6and TFF3mRNA,accompanied with down-regulation of CK19, E-cadherin and up-regulation of a-SMA, Vimentin in the bile duct tissues of rat liver grafts. These dynamic changes were stronglyassociated with the cold preservation durations of graft. Coincident changes of proteinswere validated by WB analysis. In addition, increasing expression of p-STAT3wasobserved in protein level. Results suggest that the activation of IL-6/STAT3/TFF3signalingpathway is associated with BEC EMT, and CPRI is considered as the primary initiator.Conclusions:CPRI induces BEC EMT via activation of IL-6/STAT3signaling pathway and theconsequent up-regulation of TFF3, which directly contributes to BEC EMT. This process ofwound healing responds to injury should be considered as a potential mechanism of biliaryfibrosis in the liver graft. |
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