Study On The Impact Of Biliary Epithelial Cell Proliferation On The Activation Of Peribiliary Fibroblasts After Liver Transplantation

IntroductionLiver transplantation has developed dramatically and become a routine procedure for treatment of terminal liver failure. However, bile duct strictures remain the most common late complication of orthotopic liver transplanta. The incidence of anastomosis stenosis has been reduced significantly as a result of surgery technology development, making nonanastomosis the main bile duct complication after liver transplantation. Cold preservation and reperfusion injury to biliary epithelial cell (BEC, cholangiocyte) has been known to be the most dangerous contributing factor of the compliment.BEC is the epithelium lining the biliary tree. In human liver, small ducts are encircled by 4-5 BECs directly while large bile ducts are lined by more BECs and fibrous tissues beneath the epithelial layer that contain many fibroblasts. Fibroblasts are quiescent in normal liver and upon activation by injury, stimulus and cytokines, transform growth factor-β, which is secreted by other cells surrounding them, rapidly change into myofibroblasts. Myofibroblasts'persistent accumulation is the key factor contributing to the development of bile duct complication such as peribiliary fibrosis and bile duct stricture after liver transplantation. Most of BECs are quiescent in normal state and undergo pathological changes characterized with cell proliferation or loss induced by acute and chronic injury such as CPRI. Though BECs are able to be restored very close to the original state by proliferation of remaining BECs, they might have different function from normal ones because surviving cells might carry a legacy of DNA damage and senescence-related damage. For example, BECs secrete TGF-β1, Interleukin-6 (IL-6) and receptors of much cytokines, chemokines and angiogenesis factors. Therefore, BECs are not bystanders in intrahepatic affair but crosstalk with the periductual myofibroblasts through complex autocrine and paracine pathway and play an important role synergistically.in the development of some hepatobiliary disease such as peribliary fibrosis and biliary stricture.Interleukin (IL)-6 is a potent mitogen that regulates cell growth through signal transducers and activation of transcription-3 (STAT-3) in liver. It was reported that BECs possessed the IL-6 receptor complex and intact signaling mechanism leading to activation of STAT3. Meanwhile, CPRI led to increased IL-6mRNA expression in liver. Therefore, IL-6/STAT3 signal pathwaqay might be the mechanism of the regulation of BECs proliferation in the graft liver. It seemed that regulating BECs proliferation by intervention upon IL-6/STAT3 signal pathway might be a new sight of prevention and cure of peribiliary fibrosis as well as bile duct strict after liver transplantation. As a specific inhibitor of STAT3 activation, rapamycin (RPM) will be used widely in liver transplantation filed attribute to its potential antifibrosis by partly blocking the IL-6/STAT3 signal pathway.ObjectiveBeing sensitive to CPRI, BECs proliferated actively induced by CPRI (cold preservation and reperfusion injury) and played an important role in the development of biliary complication after liver transplantation. Based an animal model suitable for studying bile duct CPRI, BECs'morphous change and function injury conducted by CPRI were observed. The aims of the present study are to provide novel observations pertaining to the mechanism of proliferation of BEC after liver transplantation and elucidate the important role of IL-6/STAT3 signal pathway in the development of posttransplantation biliary complication such as peribiliary fibrosis and biliary sticture, and provided a new strategy for the treatment and cure of bile duct complication.Methods and ResultsConstructed and estimated an arterialized rat orthotopic liver transplantation model.Based the classic"two cuff"technique for orthotopic liver transplantation in rat, end-to-end anastomosis between the common hepatic artery of donor and recipient by the modified"Gao stent"was performed to reconstruct hepatic artery blood flow. Donor liver was preserved in UW solution at 4℃with 1 and 12hours respectively. Survival rat and pathological changes of the intrahepatic bile duct were carefully observed. It was shown that longer liver cold ischemia time resulted in recipients prominent bile duct injury but with moderate survival rate. Because of these attributes, the animal model was ideal for studying cold preservation and reperfusion injury of bile duct and was the base of our work.Evaluated the impact of CPRI on BEC proliferation and peribilary fibroblast activation. Based on the animal model with bile duct injury, impact of CPRI on BEC morphous and function was observed and the activation of peribiliary fibroblast was also detected. It was suggested that more severe BEC injury was showed in grafts with prolonged ischemia time, characterized by induction of BEC apoptosis and proliferation, liver function injury and cholestasis sign reflecting the increase of serum ALT, ALP and TBIL. Proliferating BEC initiated the phenotype change of fibroblasts into myofibroblasts by secreting TGF-β1, resulting in persistent and massive accumulation of myofibroblasts surrouding the bile duct, the key step leading to peribiliary fibrosis and bile duct stenosis after liver transplantationAnalyzed the regulation of BEC proliferation in liver graft through IL-6/STAT3 signal pathway. Exogenous rhIL-6 and RPM were administrated to the recipient rats and regulation of BEC proliferation through IL-6/STAT3 signal pathway was detected It suggested that CPRI increased IL-6mRNA expression in liver graft significantly. IL-6mRNA promoted BEC DNA synthesis potently by activating IL-6/ STAT3 signal pathway and regulated cell proliferation. RPM, a specific STAT3 inhibitor, restrained the BEC proliferation in the liver graft remarkably by partly blocking the signal pathway, which might relieve the accumulation of the myofibroblasts surrounding the bile duct and, therefore, might be used for the prevention and cure of peribiliary fibrosis and bile duct stenosis after liver transplantation.Conclusions1. Based the classic"two cuff"technique for orthotopic liver transplantation in rat, reconstruction of hepatic artery blood flow using modified"Gao stent"method and 12h donor liver cold ischemia time was suitable for studying the bile duct injury in graft liver induced by cold preservation and reperfusion.2. More severe BEC injury was showed in grafts with prolonged ischemia time, characterized by induction of BEC apoptosis and proliferation followed by obvious liver function injury and cholestasis symptoms. Proliferating BEC activated and initiated myofibroblasts accumulation arround the bile duct through secreting the cytokines TGF-β1, which might be the risk contributor to the peribiliary fibrosis and biliray stricture after liver transplantation.3. The activation of IL-6/STAT3 signal pathway might be the mechanism of the active BEC proliferation induced by CPRI after liver transplantaion.4. RPM was not only an immunosuppressant used synergistically with others in the organ transplantation field, but also the regulator of the BEC excessive proliferation in the graft liver with prolonged cold ischemia time by partly blocking IL-6/STAT3 signal pathway, which might be a new target for the prevention and the treatment of posttransplantation bile duct complication such as peribiliary fibrosis and bile duct structure.