| BackgroundBiliary complications remain the major source of morbidity for liver transplant recipients. Owing to the improvement of surgical techniques, the incidence of anastomotic strictures has continued to decrease, with the result that the non-anastomotic form is now the main type of biliary complication. Non-anastomotic strictures in the presence of a patent hepatic artery have been described as ischemic-type biliary lesions (ITBL), recently called ischemic cholangiopathy.The molecular mechanisms involved in the pathogenesis of ITBL remain unclear. Recent year, increasing number of studies have indicated that an altered bile composition is associated with bile duct injury and with the subsequent development of non-anastomotic biliary strictures following human liver transplantation. However, the molecular mechanism by which the altered bile induces injury to the bile duct epithelial cells remains to be elucidated. It is well-known that bile salts solubilize membrane-bound lipids, leading to damage to cell membranes. However, at physiologically relevant concentrations, a simple detergent-like action involving disruption of the membrane is probably not relevant. The intracellular toxicity of bile salts, including death receptor-mediated and/or mitochondria-mediated apoptosis, may play a more important role in inducing the injury of bile duct epithelial cells following liver transplantation. Physiologically, the balance of bile acid influx and efflux in cholangiocytes is maintained by bile acid transporters to avoid the accumulation of toxic bile acids, which is a crucial step of the"cholehepatic shunt,"a process of bile acid absorption by cholangiocytes and subsequent excretion into the peribiliary capillary plexus, followed by resorption by hepatocytes and re-excretion in the form of bile. Several bile acid transporters have been identified in the cholangiocytes in humans and rodents. These transporters are widely expressed on enterocytes, and especially on the terminal ileocytes, where enterohepatic circulation is initiated. Studies show that these transporters will often undergo expressional and functional alterations and may be involved in intestinal metabolic and inflammatory diseases. Based on the above conception and evidence, we hypothesize that due to the expressional and functional alteration of inflammatory factors and nuclear receptor, the cholangiocyte bile acid transport may be disturbed and lost the self-protection from toxic bile, and subsequently inducing the accumulation of toxic bile and bile duct injury.ObjectiveThe current study focuses on the role of cholangiocyte bile acid transporters in bile duct injury after liver transplantation, and the possible mechanism.Methods and results:1. The immunohistochemistry assay on liver tissue section suggested that ASBT and IBABP were expressed exclusively on the large bile duct epithelial cells, while Ostαand Ostβwere expressed on both large and small bile duct. Quantitative PCR and Western blotting analysis showed that the expression levels of Asbt and Ilbp in large bile ducts were significantly higher than those in small bile ducts, while the expression levels of Ostαand Ostβin large bile ducts were similar with those in small bile ducts.2. Using rat liver transplantation model, we investigated the expression levels of cholangiocyte bile acid transporters ASBT, IBABP and Ostα/Ostβ, and evaluated the evaluation of bile duct injury histopathologically. The immunohistochemical assay showed that the expressions of these transporters on large bile ducts were significantly reduced after transplantation compared with the Sham group. Three days after transplantation, ASBT recovered but IBABP, Ostαand Ostβwere still suppressed. Western blot and quantitative PCR analysis showed that the expression levels of these transporters dramatically decreased after transplantation. It took seven to fourteen days for IBABP, Ostαand Ostβto recover, while ASBT recovered within 3 days and even reached a peak above the normal level seven days post operation. In the CP-12h group, the ratios of the ASBT/ILBP, ASBT/Ostαand ASBT/Ostβexpression levels were correlated with the injury severity scores of large but not small bile ducts.3. In the same animal model, we observed the changes of total bile acid (TBA) and phospholipids (PL) concentration in bile, and investigated the expression of FXR by quantitative PCR and Western blottingand, and analyzed its binding ability to promoter of Ostαand Osβgenes by EMSA. Result showed that cold preservation/reperfusion injury is associated with the elevated TBA/PL ratio which could induce the elevation of relative bile acid concentration in bile. It was also showed that the expression of FXR and its binding ability to promoter of target genes were inhibited after liver transplantation, and especially in the transplant group with long donor liver cold preservation time.4. In the in vitro study with cultured rat bile duct units, we observed that the bile with higher TBA/PL ratio, so called toxic bile, could induce the expression of ASBT,IBABP,Ostαand Ostβin normal bile duct. However, after the transfection with FXR shRNA, the expression of ASBT was still induced, while the expression of IBABP,Ostαand Ostβwere inhibited to various extent.Conclusions:1. The bile acid re-absorption may exclusively take places in large bile duct, as the initiator of'cholehepatic shunt'ASBT is just expressed in large bile ducts.2. The unparallel alteration of cholangiocyte bile acid transporters is significantly correlated with large bile duct injury after liver transplantation with prolonged donor liver preservation.3. The'toxic bile'could promote the rapid recover and subsequent elevation of ASBT expression, while the IBABP,Ostαand Ostβexpression were not promoted accordingly because of the inhibition of FXR, by which bile acid could induce the expression of IBABP,Ostαand Ostβ. |
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