The Effect Of Gadolinium Chloride On The Expressions Of Bile Duct Apoptosis Related Factors Fas/Fasl After Early Ichemia Reperfusion Injury

Biliary complications (BC) accounts for 15% to 34% of the death after liver transplantation, It is more likely to occur after living liver transplantation and the transplantation from donor whose heart/brain-dead.In recent years,scholars at home and abroad have committed to expand the source of donors,including the use of "marginal" donor livers.However,the marginal donor livers may suffer more serious condition with liver ischemia reperfusion injury, and the risk of postoperative primary graft dysfunction(PGD), delayed function and ischemic biliary lesions (ITBL) increased significantly.Previous studies show that ischemia-reperfusion injury,infection,immunological rejection and other factors are the main reasons that lead to the morphological structure of transplantated biliary tract change and function impaire. An important cause of BC after transplantation are the biliary epithelial apoptosis and biliary microcirculation dysfunction caused by ischemia-reperfusion injury(IRI). Under hypoxic conditions,biliary epithelial cells have strong resistance to kill cells, after reperfusion,biliary epithelial cells was attacked by killer cells apparently,and basal levels of glutathione was lower,oxygen free radicals toxicity effects on biliary epithelial cells are 2.5 times stronger than liver cells.The regeneration of bile duct tissue after injury is low,so biliary ischemia-reperfusion injury as the important complication which will have an impact on the long-term effect of liver transplantation,has attracted the attention from clinical and researchers. Therefore,to avoid or mitigate the ischemia-reperfusion injury which is the key to improve the efficacy of liver transplantation and the quality of patients quality of life. There are fewer report about the injury mechanisms and protective aspect induced by extrahepatic bile duct ischemia-reperfusion injury,and the pretreatment of Kupffer cell inhibitor gadolinium has a significant protective effect on multiple organ ischemia reperfusion injury according to the reported literature,but the effect on the extrahepatic biliary system has not yet clear.The study simulated the entire process of clinical liver transplantation through the establishment of rat autologous transplantation model,to observe the expression level of the Fas, FasL and Caspase-3 on the ischemia-reperfusion injury early biliary epithelial cells from extrahepatic biliary system in rats after gadolinium chloride,and discuss the protective effect induced by gadolinium chloride on ischemia reperfusion injury,and do some initial study on its protection mechanisms.OBJECTIVEThis paper adopt simulate the process of liver transplantation in liver ischemia reperfusion injury and observe three gadolinium chloride protective effect of preconditioning on transplanted liver ischemia reperfusion injury by an rat autologous transplantation model, by studying the changes in serum bile enzyme,indicating related inflammatory factor activity,observing biliary pathology and morphological change,combing with biliary epithelial cell apoptosis related genes Fas/FasL expression level, to observe the gadolinium chloride protective effect on liver transplantation biliary ischemia reperfusion injury in rats,and preliminary discuss the mechanism of gadolinium chloride,then provide the new ideas and new target for clinical alleviation from the ischemia reperfusion injury.METHODS1.Established an rat autologous transplantation model referring to the method described by Zhou Jie.2.48 Sprague-Dawley (SD) rats (SPF level inbred male),body weight was 200-250g.They were divided into three groups randomly.①sham group (Sham):The rats get dorsal vein injection of an equal volume of physiological saline 24h before surgery, and then only received anesthesia,laparotomy, the abdomen was closed after liver free regulations.②Ischemia-reperfusion group (IRI):The rats get dorsal penile vein injection of an equal volume of physiological saline 24h before surgery,then have orthotopic autologous liver transplantation, and let hepatic arterial occlusion 30min until the cool lavage and the reflow portal finish.Close abdomen after opening hepatic arterial blood flow regulation.③Gadolinium chloride Preconditioning group(GdCl3+IRI):The rats get dorsal penile vein injection of an gadolinium chloride dorsal (lOmg/Kg) 24h before surgery,then have orthotopic autologous liver transplantation,and let hepatic arterial occlusion 30min until the cool lavage and the reflow portal finish.Close abdomen after opening hepatic arterial blood flow regulation.3.Each group animals were killed in 6h,12h after the hepatic artery reflow,abdominal aorta blood 5ml for detection.Compared bile enzymes and bilirubin (ALT, ALP, GGT, TBIL) change with each other group,and ELISA was used to detect blood plasma proinflammatory cytokine TNF-a levels and sFas levels.Got fresh liver,bile duct and liver tissue.Immunohistochemical staining was used to measure Fas,FasL protein expression level of extrahepatic biliary epithelial cells.Adoption Western blot to assay FasL, caspase-3 related protein of liver tissue and liver bile duct tissue.Transmission electron microscopy (TEM) was used to observe the hilar bile duct epithelial cells and extrahepatic biliary epithelial cell ultrastructure,as well as biliary epithelial cell mitochondrial damage and microvilli coverage.RESULT1.ModelingThe success rate of models’surgical was 98%,and successfully established 48 cases of autologous liver transplantation model.the surgery time was 53.8 ± 6.5min,and no liver period was 15.0 ± 1.8min.The rats survival rate after surgery was 100%.2.The level of postoperative serum ALT, ALP, GGT, TBILDetection of abdominal aortic blood after hepatic artery reflow 6h,12h for detecting the biliary enzyme.The levels of serum ALT,ALP,GGT,TBIL were significantly increased (P<0.05) in IRI group and GdCl3+IRI group compared with Sham group;Compare between GdCl3+IRI group and IRI group show that the levels of ALT,ALP,GGT,TBIL in GdCl3+IRI group were lower than IRI group,the difference there was statistically significant (P<0.05).3.The level of postoperative plasma TNF-α and sFasDetection of abdominal aortic blood after hepatic artery reflow 6h,12h,The levels of plasma TNF-α were significantly increased in GdCl3+IRI group and IRI group compared with the Sham group,the difference was statistically significant (P<0.05). After six hours of the hepatic artery reperfusion, the serum TNF-α level of GdCl3+IRI group and IRI group are respective 162.3+12.6ng/L,210.4+15.1ng/L. After twelve hours of the hepatic artery reperfusion, the serum TNF-α-level of GdCl3+IRI group and IRI group are respective 191.2±14.5ng/L、243.3±18.4ng/L. After postoperative two hours, the serum TNF-α level of GdCl3+IRI group are significant lower than those in IRI group(P<0.05).Compared with group of Sham, after six hours or twelve hours of the hepatic artery reperfusion, the serum sFas-level of GdCl3+IRI group and IRI group increase significantly at each time point (P<0.05), while after postoperative two hours, the serum sFas-level of GdCl3+IRI group higher than those in IRI group are respective 381.4±48.1ng/L、332.8±46.9ng/L at same time point, the difference means statistical significance(P< 0.05).4.The expression levels of Fas, FasL in hilar bile duct epithelial cells and between bile ducts epithelial cells interlobular biliary epithelial cells with immunohistochemical markersAfter hepatic artery reflow 6h、12h, the Fas, FasL expression levels and expression intensity in hilar bile duct epithelial cells and between bile ducts epithelial cells interlobular biliary epithelial cells was significantly increased in GdCl3+IRI group and IRI group compared with Sham group, differences were statistically significant (P<0.05). After six hours of the hepatic artery reperfusion, the positive rates of Fas, FasL in GdCl3+IRI group of hilar bile duct epithelial cells are respective 28.5%, 22.8%, and compared with the IRI group (32.5%,28.5%) are significantly decreased (P<0.05). After twelve hours of the hepatic artery reperfusion.The Fas, FasL expression levels in hilar bile duct epithelial cells and between bile ducts epithelial cells/interlobular biliary epithelial cells was significantly lower in GdCl3+IRI group when compared with IRI group, the difference was statistically significant (P<0.05).5.Western blot assay of FasL,Caspase-3 related protein content in hilar bile duct tissueAfter hepatic artery reflow 6h、12h,the FasL and Caspase-3 protein expression levels in hilar bile duct epithelium cells was significantly increased in GdCl3+IRI group and IRI group compared with Sham group,and the difference was statistically significant (P<0.05). After six hours of the hepatic artery reperfusion, the relative expression amount of Fasl in Hilar bile duct epithelial cells of GdCl3+IRI group and IRI group are respective 0.76±0.06,0.96±0.08, the relative expression amount of Caspase-3 are respective 0.80±0.07,1.02±0.09. The relative expression amount of Fasl,Caspase-3 are significant lower than those in IRI group, the difference means statistical significance(P<0.05).After twelve hours of the hepatic artery reperfusion, the relative expression amount of Fasl in Hilar bile duct epithelial cells of GdCl3+IRI group and IRI group are respective 0.69±0.06、0.87±0.07, the relative expression amount of Caspase-3 are respective 0.71±0.06、1.16±0.10, the relative expression amount of Fasl,Caspase-3 in Hilar bile duct epithelial cells of GdCl3+IRI group are significant lower than those in IRI group, the difference means statistical significance(P< 0.05).6.The biliary epithelial cell ultrastructure changes:On the study of the histopathological changes in the bile duct,using the transmission electron microscopy to observe the cell after hepatic artery reflow 6h. We found that biliary epithelial cells of Sham group had the normal structure,biliary epithelial cell of IRI group were swelling obviously with sparse microvilli,obviously swelling mitochondrial or the formation of vacuoles,matrix disappear,crest fracture disappeared,and visible apoptosis.The extent of damage of biliary epithelial cells in GdCl3+IRI group is better than IRI group.CONCLUSION1.The model simulates the entire process of liver transplantation,with the advantages of copying simple,reproducible,comparability,and fully discharge the influences made by the immune factors.In addition it can be more direct, more objective on observing the damage on the bile duct made by the cold ischemia,the relative heat ischemia or blood reperfusion.It is the ideal model for the study of biliary IRI.2.IRI can increase the Fas,FasL and Caspase-3 expression levels in the early stages.3.In the early stages of biliary IRI,applying GdCl3 pretreatment can reduced the Fas FasL protein expression significantly in bile duct epithelial cells.4.GdCl3’s protection mechanisms may includes:Inhibition of Kupffer cells’ activation; inhibition of oxidative stress, inhibition of endogenous apoptosis pathway by maintain the structure and function of Mitochondria;and inhibition of exogenous apoptotic pathway by inhibit Fas/FasL overexpression.