Study On Correlation And The Role Of FoxA1/foxA2 In Ischemic-Type Biliary Lesions After Liver Transplantation

Introduction:Biliary tract complication is the leading cause of dysfunction of allograft following liver transplantation,among which the ischemic-type biliary lesion(ITBL)is the most difficult,considering to be the "Achilles heel".Previous clinical practice and research have shown that ITBL is the result of multiple injury factors,and there are three main factors related to ITBL,including,injury induced by cytotoxic bile salts,and immunological-mediated injury,but the exact etiology and pathogenesis of ITBL are still not clear.Therefore,the prevention and treatment meanings of ITBL are very limited.Forkhead-boxa1A1(FOXA1)and Forkhead-boxa1A2(FOXA2)are also known as the hepatocyte nuclear factor-3?/?(HNF3?/?),which is an important member of the transcription factor family-Fox family,being expressed in most tissue of human body,such as esophagus,breast,pancreas,lung,bladder,and prostate,etc.FOXA1 and FOXA2 are also expressed in liver cells,and the study showed that FOXA1 and FOXA2 are important transcriptional factors for liver development and maintenance of normal physiological functions(especially for bile secretion).Based on the clinical pathological observations of a large number of liver biopsy,the expression of FOXA1 and FOXA2 in the biliary epithelial cells(BEC)is lower than normal.therefore,we suspect that low expression of FOXA1/A2 in BEC is closely associated with the occurrence of the ITBL after liver transplantation,and FOXA1 and FOXA2 may have been involved in the process of ITBL,which has not been reported before.In order to analyze the relevance between expression of FOXA1 and FOXA2 in BEC and ITBL,and the possible roles in ITBL,firstly,Formalin-Fixed and Paraffin-Embedded liver tissues were used and immunohistochemical methods were applied to observe the expression of FOXA1 and FOXA2 in liver,and Secondly,the HIBEpi C cells were used to explore the influence factors for expression of FOXA1 and FOXA2 in BEC and possible roles of FOXA1 and FOXA2 in the development of ITBL.Objectives:To analyze the correlation between FOXA1/FOXA2 expression and ITBL,and analyze the correlation between FOXA1 and FOXA2 positive expression and severitygrade of ITBL.To investigate the role of FOXA1 and FOXA2 in the development of ITBL after liver transplantation and a possible mechanism.Methods:In this study,the localization and expression of FOXA1/FOXA2 protein in liver tissues were detected by immunohistochemistry using Formalin-Fixed and Paraffin-Embedded liver tissue,analyzing the correlation between FOXA1/FOXA2 expression and ITBL.The relationship between the expression of FOXA1/FOXA2 and the severity of ITBL was also analyzed.The factors influencing the expression of FOXA1/FOXA2 were investigated,and the role and possible mechanism of FOXA1/FOXA2 in ITBL were discussed through experiments in vitro,by Western blot,real-time PCR and other experimental methods.Results:1.Immunohistochemical staining results showed that FOXA1 and FOXA2 were positively expressed in hepatocytes and biliary epithelial cells of ITBL group and the non-ITBL group,locating at the nucleus of liver cells and biliary epithelial cell.Expression rates of FOXA1 and FOXA2 in the biliary epithelial cells of ITBL group were significantly lower than which of non-ITBL group(p<0.05).2.A quantitative analysis of immunohistochemical staining results showed that expression of FOXA1 in BEC of ITBL had no obvious correlation with severity of ITBL(p=0.093),but expression of FOXA2 was negatively correlated with the severity grade of ITBL(p=0.025).3.The results of experiments in vitro showed that high concentration of bile salts,inflammatory factors such as IL-1 and PGD2,could cause injury and cell apoptosis of BEC.4.Stimulation of high concentration of bile salt could cause a down-regulation of FOXA1 and FOXA2 expression in BEC.And IL-1 and some other inflammatory factors could cause a down-regulation of FOXA2 expression in BEC.Conclusions:1.FOXA1 and FOXA2 were positively expressed in hepatocyte and intrahepatic biliary epithelial cells in ITBL and non-ITBL liver tissues after LT.Expression of FOXA1 and FOXA2 in ITBL were significantly lower than that in non-ITBL,and low expression of FOXA1 and FOXA2 were closely related to the occurrence of ITBL after LT.2.Expression of FOXA2 in BEC was negatively correlated with the severity grade of ITBL.3.The results of experiments in vitro showed that high concentration of bile salts,inflammatory factors such as IL-1 and PGD2,could cause injury and cell apoptosis of BEC.4.Stimulation of high concentration of bile salt could cause a down-regulation of FOXA1 and FOXA2 expression in BEC.And IL-1 and some other inflammatory factors could cause a down-regulation of FOXA2 expression in BEC.5.High concentration of bile salts,IL-1 and other inflammatory factors may regulate the over-expression of IL-6 by down-regulating the expression of FOXA1 and FOXA2 in BEC,causing dysplasia of BEC,leading to the adverse proliferation of the intrahepatic bile ducts,and promoting the occurrence of ITBL.