| Background and ObjectivePrimary biliary cholangitis(PBC)is an autoimmune cholestatic liver disease characterized by the existence of anti-mitochondrial antibody against the pyruvate dehydrogenase complex and chronic non-suppurative destructive cholangitis,with an apparent injury to intrahepatic biliary epithelial cells(IBEC),finally causing biliary cirrhosis.Some studies have found that the epithelial-mesenchymal transition(EMT)is closely related to the development of liver fibrosis.Inhibiting the EMT can reduce or reverse hepatic fibrosis,even delay the process of cirrhosis of the liver.Therefore EMT has drawn wide attention in the research on liver fibrosis.IL-17A is a newly discovered pro-inflammatory cytokines,which has been recently identified as a key cytokine involved in numerous autoimmune processes.However,its role in PBC remains unclear.Does IL-17A induced IBEC-EMT contribute to the pathogenesis of biliary fibrosis in PBC?Taken together,it’s worthy to focus on the contribution of IL-17A to IBEC-EMT and biliary fibrosis and the possible mechanism in PBC.In order to understanding this question,this study observed the role of IL-17A in IBEC-EMT and biliary fibrosis in vivo and vitro.Firstly,we detected the pathological changes of liver tissue of PBC patients.Immunohistochemical staining will be used to detect the level of biliary fibrosis,IL-17A,Th17 cells and IBEC-EMT.Secondly,we will investigate the signaling mechanism mediating the regulatory role of IL-17A in IBEC-EMT,and eventually help looking for blocking PBC liver fibrosis development strategies and methods to provide the experimental basis.Content1、Clinical and pathological analysis of PBC cases and immunohistochemistry of liver tissue confirmed the existence of IBEC-EMT,bile duct fibrosis and high expression of local IL-17A in peripheral blood of patients with PBC,and changes in peripheral blood IL-17A and other inflammatory cytokines.The incidence of PBC is related;2、Observe IL-17A-induced IBEC-EMT by IBEC culture in vitro and explore its possible molecular mechanisms.2.1 Establishment of EMT classic cell model:IBECs were cultured with different concentrations of TGFβ1,and the changes in mRNA and protein expression levels of EMT-related markers were detected at different times,mainly to verify that IBEC could induce EMT,and cells were given for the next experiment.Preparation,reagents,conditions,methods,etc.2.2 The best time and concentration of IBEC-EMT induced by IL-17A:IBECs were cultured with different concentrations of IL-17A at different times.The morphology of the cells was observed,and the changes of mRNA and protein expression levels of EMT-related markers were detected to confirm IL-17A.The IBEC-EMT can be induced to lay the foundation for the follow-up molecular mechanism exploration.2.3 IL-17A interferes with the culture of IBECs,and then silences the expression of IBEC proteins with siRNA.The expression of relevant mRNA and protein in signal pathways may be detected to explore the molecular mechanism of IBEC-EMT induced by IL-17 A.3、Effect of IL-17A on IBEC-EMT induced by classical cytokine TGF-β1.The optimal IL-17A and TGF-β1 levels co-operate with IBECs,and the best time point for Western Blotting was to detect the changes of EMT-related proteins,and to clarify the synergistic or antagonistic effects of both on IBEC-EMT.Methods1、The following serum biomarkers and cytokines were detected by ELISA:IL-17A,AMA-M2,IL-6,IL-8,IL-23,MIP3a/CCL20,and TGFβ1 were detected.2、The following serum specific proteins were detected by rate nephelometry:IgG,IgA,IgM,C3,C4,ASO,RHF,CRP,light chain kappa,light chain lambda,free KAP,free LAM.3、Liver histopathological staining:The clinical pathology was observed by HE staining.4、Liver tissue immunohistochemistry:The expression of IBEC-EMT and fibrosis markers in the hepatobiliary tissue of the experimental group and the control group was detected,mainly including CK7,CK19,Vimentin,E-cadherin,IL17A,and IL17AR.5、The main application techniques were included for the molecular mechanism of IBEC-EMT induced by IL-17A:cell culture technology,Quantitative real-time RT-PCR,Western blotting,siRNA interference.Result1、There are obvious abnormalities in many physiological and biochemical indicators of PBC patients,and there are various levels of inflammatory cytokines in serum.2、HE staining analysis of histopathological changes and staging of 9 cases of PBC(3 in stage I,4 in stage Ⅱ,2 in stage III)and 4 cases of normal liver tissue.Further immunohistochemical findings revealed intrahepatic bile duct epithelial cell epithelium in PBC patients.The marker E-cadherin was significantly decreased,and the interstitial marker Vimentin was significantly increased.Meanwhile,the expression of IL-17A and IL-17RA increased.3、The optimal concentration of IBEC-EMT induced by TGFβ1 was 5ng/ml,and the best time was 48h.4、The optimal concentration of IL-17A induced IBEC-EMT was 50ng/ml,and the best time was 48h.5、IL-17A interferes with the culture of IBEC,and transfection with siRNA-Actl can reverse the IBEC-EMT process.At the same time,a variety of proteins on the downstream signaling pathway are abnormally expressed.6、IL-17A and TGF-β1 co-interfering culture induced IBEC-EMT obviously than single application.Conclusion1、Abnormal physiological and biochemical indicators of PBC patients,peripheral blood serum levels of IL-17A and other inflammatory cytokines increased,and the pathogenesis of PBC.2、IBC-EMT and intrahepatic bile duct epithelial cells IL-17A and IL-17RA are highly expressed in patients with PBC.IL-17A may be involved in intrahepatic bile duct IBEC-EMT of PBC.3、IL-17A can induce IBEC-EMT in vitro.Actl is one of the key proteins in the EMT cell signaling pathway induced by IL-17A in IBECs.P65/p-P65 is an important molecule downstream of IL-17A-induced IBEC-EMT signaling pathway.Induction of IBEC-EMT by IL-17A is through the IL-17A-IL-17RA-Actl-NF-κB signaling pathway.4、In combination with IBEC at optimal concentrations of IL-17A and TGF-β1 and time,IBEC-EMTs act synergistically. |
PDF Full Text Request