| 1. PurposeBiliary complication has a serious impact on the survival and life quality of liver transplanted recipient, which is recognized as a weakness of liver transplantation (Achilles'heel). With the deepening of people's understanding of the biliary complications and improvement of surgical anastomosis technology, the incidence of bile duct anastomotic complications is on the decline, and Biliary complication caused by non-surgical technical factors is still one of the main factors that restrict the long-term effects of liver transplantation. The Ischemic-type biliary lesions (ITBL)with the features of focal or diffusal graft biliary tree's stricture , expansion, and damage is the main type of non-surgical transplantation bile duct complications, which occurrence rate is 2%-19%. Although it is recognized that cold preservation and reperfusion injury (CPRI) are the main reason for its illness, the specific mechanism is still unclear. In recent years, people pay attention to the hydrophobic bile salt's effect on the injury of the bile duct. Hydrophobic bile salt can not only interfere with lipid metabolism of biofilm by "detergent", but also induce the cells to apoptosis, which is highly toxical.95% of bile salt pool is composed of hydrophobic bile salt. Under normal conditions, human body has a set of self-protection mechanism to neutralize hydrophobic bile salt cytotoxicity. For instance, lecithin and bile salt can be combined to form micelle to reduce its cytotoxicity, and hydrophilic bile salt contained in the bile can resist hydrophobic bile salt cytotoxicity by enhancing the stability of the membrane, and so on. In addition, the secretion of the major components of bile completely depends on the Hepatobiliary membrane transporter (HMTs) capillary in the canalicular membrance of hepatocytes. If HMTs expresses abnormally, it could lead to cholestasis and bile duct injury. Research has suggested there is imbalance of bile in the early stage of liver transplantation. On the basis of the comprehensive study results at home and abroad in recent years, we pay more attention to "toxic bile" hypothesis's effect on transplanted liver's cold preservation and reperfusion injury. Therefore, we propose the following idea: If we can regulate the composition of bile positively and increase the proportion of bile lecithin and hydrophilic bile salt, will it help resist hydrophobic bile salt's cytotoxicity and diminish the injury of biliary tree after liver transplantation?3-hydroxyl-3-methyl Coenzyme A (HMG-CoA) reductase inhibitors (simvastatin) are in wide application because of its definite effect on the major components of blood lipids, and have a good risk-benefit ratio and unique cholesterol -lowering benefits. Some recent studies show that statin drugs have a significant impact on the phospholipid secretion of rat's liver, the long-term treatment lasting more than five days can induce the synthesis of liver phosphatidylcholine (PC) and promote the discharging of bile phospholipid, but have no effect on the secretion on the bile salt.Hydrophobic bile salt has highly cellular toxicity and plays an important role in bile duct injury. It is of important practical significance and clinical application value to change the composition of bile positively and prevent bile salt's toxic effect on hepatocytes and epithelial cell of bile duct. This study intends to aim at exploring the prevention and treatment strategies, builds a rat's liver transplantation model for transplantation problems of bile duct, studies the change rules of biliary major components after CPRI and its relativity with bile duct injury, and also studies expression and functional changes of phospholipid transferring protein to offer new ideas for prevention and cure of clinical transplantation of biliary complications.2. MethodsIn order to verify our assumptions, the design is as follows: First, establish rat's liver transplantation model of hepatic artery reconstruction, set simvastatin handling group, control group and sham group, and implant handling and control group's donor liver into the receptor after 12 hours' UW liquid preservation; obtain samples of the rat's liver, blood and bile after 14 days; Subsequently, detect the composition and changes of bile salt in the bile sample with the application of high-performance liquid chromatography (HPLC), and further analyze the changes of phospholipid (PL) and total bile acid (TBA) in bile and serum, evaluate transplantation liver rat's changes of bile composition after simvastatin treatment, then observe the pathological changes in liver tissue and explore the relationship between these changes and phospholipid secretion of bile; Finally, detect expression and functional changes of phospholipids transfer protein with the application of immunohistochemistry and real-time PCR method and explore simvastatin's functional mechanism on rat liver transplantation phospholipid changes.3. Results1. The rat's original liver transplantation model of hepatic artery's reconstruction applies to the study of biliary complications, which has a better operational quality and repeatability;2. Phospholipid components of rat's bile increase significantly after Simvastatin treatment, which has statistical significance(P <0.05) compared with the sham and control groups; have no effect on bile salt composition in the bile, lower indirectly bile salt and phospholipid ratio in the bile, and reduce the effects of transplanted hepatolithiasis damage factors;3. The rat's serum r-GT, ALP of Simvastatin treatment group decreases significantly after operation, which has statistical significance (P <0.05); and the changing trend has negative correlation with phospholipid secretion in bile;4. In liver HE and Masson stain, we find the control group has obvious liver inflammation, serious damage to bile duct, and a lot of duct hyperplasia; while simvastatin treatment group has minor pathological changes;5. Immunohistochemistry shows that phospholipid transfer protein (Mdr2 P-glycoprotein) increases significantly in expression in the rats of simvastatin treatment group, which are mainly located in the region around the portal vein, while Mdr2 P-glycoprotein's expression of the rats in the control group weakens significantly; sham rats Mdr2 P-glycoprotein's expression is weak;6. The expression of Taqman probe real-time PCR detection on Mdr2 mRNA in the rats of simvastatin handling group has a significant increase, which is of statistical significance (P <0.05).4. Conclusion1. Simvastatin can effectively increase the secretion of phospholipid composition in rat's bile after transplantation, but has no effect on the bile salt, and can significantly reduce the ratio of bile salt and phospholipid; 2. The increase of Phospholipid composition in bile significantly improves the function of the transplanted liver, decreases the pathological changes in liver tissue, and effectively protects the transplanted liver and bile duct; and the mechanism which increases phospholipid secretion may be realized through the regulation of Mdr2 gene.
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