The Role Of Notch Signaling Pathway In The Regeneration Of Peribiliary Vascular Plexus In Ischemia-reperfusion Injury

[Background]Ischemia reperfusion injury refers to the restoration of blood flow and oxygen supply to tissue ischemia and hypoxia, not only failed to organ and tissue function recovery, but increased tissue and organ dysfunction and structural damage.Its mechanism is complex,will directly damages tissue and initiates a cascade of destructive cellular responses to inflammation,cell death and organ failure.IRI has damaged both in liver and biliary systemwith different degrees, and biliary system is more sensitively damaged than the liver parenchyma under IRI, thus bile is a important target organs of warm and cold IRI.IRI mainly damage biliary peripheral vascular plexus,injury endothelial cell and produce micro thrombosis in PBVP,caused biliary microcirculation.Ambient PBVP is constricted by secondary biliary inflammation, edema and fibrosis,caused a vicious cycle.Notch signaling pathway regulates many aspects of expression of transcription factors,a variety of cell proliferation,differentiation,apoptosis and angiogenesis,which it directly accept signals from adjacent cells and spread to the nucleus.According to many studies through the establishing organ ischemia-reperfusion model of rat heart, kidney and others,it indicates,notch pathway may regulate the ability of vascular regeneration of ischemic organ and is important for the prevention of IRI.But its role in PBVP regeneration in ischemia-reperfusion injury needs more study.[Objective]Among of γ-secretase inhibitor,DAPT is a specific inhibitor of notch signal transduction pathway,it can be effectively suppressed notch signaling pathway.In this study, to establish the biliary ischemia-reperfusion injury model of autologous orthotopic liver transplantation in rats pretreated with DAPT,we analyse changes of serum aminotransferase,morphological structure(vascular regeneration) and expression of periportal proteins about notch signaling pathway in biliary ischemia-repefusion,investigate the mechanism how notch signaling pathway affect PBVP regeneration after biliary ischemia-reperfusion injury,and provide a new theoretical basis and experimental evidence for the prevention of biliary complications after liver transplantation.[Method]1.According to the operation method narrated by Zhou J,to establish biliary ischemia-reperfusion injury model of autologous orthotopic liver transplantation in rats.2.48 male specific pathogen free Sprague-Dawley (SD) Rats weighing 250-280g were used and categorized randomly into three group:sham operation group(SO),ischemia reperfusion group(IRI), DAPT pretreatment group (DAPT).Anhepatic stage is 18 minutes and the time from portal reperfusion to reflowing hepatic artery is 30 minutes.Each group is divided into two equal subgroups randomly by portal reperfusion time,they are SO3d,SO7d,IRI3d,IRI7d,DAPT3d and DAPT7d(each subgroup n=8).In SO group,preoperative two hours give each rat peritoneal injection with lml saline,there were no drugs and ischemia-reperfusion injury,only commonly dissociated liver ligament,disarticulated subphrenic thicker veins and right upper adrenal vein,freed hepatic inferior vena cava to the level of right kidney vein,explored hilar structures,closed the abdominal cavity and unblocked artery.As a positive control group,they were applied with biliary ischemia-reperfusion injury and normal saline in the group.DAPT,4.3mg/kg,dissolved in DMSO and diluted in a 5% glucose solution for intraperitoneal administration.Rats were administered DAPT at a two hour before the biliary IRI model was established in the DAPT group.All animals were sacrificed after IRI to obtain blood sample(5ml) through aortaventralis and fresh liver tissue for further analysis.Serum level of ALT,GGT,AKP and STB were analyzed to evaluate hepatocellular function.Hepatocellular damage and angiogenesi were analyzed by hematoxylin-eosin staining and immunohistochemistry.Ultrastructural changes in periportal area were analysed by electron microscopy.The protein expression of notch signaling were assayed by immunohistochemistry.[Result]1.32 cases of biliary ischemia-reperfusion injury model of autologous orthotopic liver transplantation in rats were successfully established.2.The changes of liver function:Compared with the same time point subgroup in SO group,Serum ALT,ALP,GGT and TBIL level in IRI3d,DAPT3d and DAPT7d subgroup were significantly higher(P<0.05),the serum ALP,GGT and TBIL level in IRI7d were significantly higher(P<0.05),and the serum ALT in IRI7d was a little higher than SO7d,but the difference is not statistically significant (P>0.05).Compared with the same time point subgroup in IRI group,serum ALT,ALP,GGT and TBIL level in DAPT3d and DAPT7d subgroup were significantly higher(P<0.05).Compared between subgroups in each group:The difference between SO3d and SO7d subgroups is not statistically significant(P>0.05),serum ALT,ALP,GGT and TBIL level in 3d subgroup is significantly higher than 7d subgroup both in IRI and DAPT group(P<0.05).3.MVD in periportal zone:Compared with the same time point subgroup in SO group,The periportal MVD in IRI3d,IRI7and DAPT7d were significantly higher(P<0.05),the periportal MVD in DAPT3d was a little higher than SO3d,but the difference is not statistically significant (P>0.05).Compared with the same time point subgroup in IRI group,periportal MVD in DAPT3d and DAPT 7d were significantly lower(P<0.05).Compared between subgroups in each group,periportal MVD in 3d subgroup were significantly lower than 7d subgroup both in IRI and DAPT group(P<0.05).4.D114, Notchl, Hesl expression4.1 D114:Compared with the SO group,the peripotal D114 protein level in IRI3d subgroup were were significantly higher than SO3d subgroup(P<0.05).The same time,compared with the same time point subgroup in SO group,the peripotal D114 protein level in IRI7d,DAPT3d and DAPT7d subgroup were little higher,but the differences were not statistically significant(P>0.05).Compared with the IRI group,the peripotal D114 protein level in DAPT3d subgroup were significantly lower than IRI3d subgroup(P<0.05),however the difference of D114 protein level between in IRI7d and DAPT7d subgroup was not statistically significant(P>0.05)..4.2 Notchl:Compared with the same time point subgroup in SO group,the peripotal notch1 protein level in IRI3d,IRI7d and DAPT3d subgroups were significantly higher(P<0.05),DAPT7d was a little higher than SO7d,but the difference was not statistically significant (P> 0.05).Compared with the same time point subgroup in IRI group,the peripotal notch1 protein level in DAPT3d and DAPT7d were significantly lower(P<0.05).Compared between subgroups in each group,the notch1 protein level between SO3d and SO7d subgroup was no significant difference(P> 0.05),the difference of notch1 protein level between 3d subgroup and 7d subgroup was statistically significant both in IRI and DAPT group(P<0.05).4.3Hesl:Compared with the same time point subgroup in SO group,the peripotal hesl protein level in IRI3d,IRI7d and DAPT3d subgroups were significantly higher(P<0.0.5),DAPT7d is a little higher than SO,but the difference was not statistically significant(P> 0.05).Compared with the same time point subgroup in IRI group,the peripotal hesl protein level in DAPT3d and DAPT7d were significantly lower(P<0.05).Compared between subgroups in each group,the hesl protein level between SO3d and SO7d subgroup was no significant difference(P> 0.05),the difference of Hesl protein level between 3d subgroup and 7d subgroup was statistically significant both in IRI and DAPT group(P<0.05).5.Observation of cell ultrastructure in Electron Microscope:In two subgroups of SO group,it showed obviously the structure of biliary epithelial cells and endothelial cells were normal and there were clear nuclear membrane and nucleolus and normal organelle structure.Compared with the same time point subgroup in SO group,both in IRI and DAPT groups,the anoxic damage extent of biliary epithelial cells and endothelial cells were more significantly heavy,and DAPT group was more significantly heavy than IRI group in three days after operation,cell nucleus and cytoplasm were more heavy damaged in anoxic environment and abnormal red cells were showed in PBVP.However it showed obviously these changes have been turned more better in of postoperative seven days both in IRI and DAPT group.6.Observation by HE:In two subgroups of SO group,it showed obviously the structure of lobuli hepatis,bile duct and PBVP in periportal were clear.Compared with the same time point subgroup in SO group,both in IRI and DAPT groups,there were more heavy about injury of lobuli hepatis,bile duct and PBVP. However the damage extent in DAPT group was more heavy than in IRI group,there were cholestasis in bile duct and abnormal red cells and and microthrombosis in PBVP.These changes have been turned more better in of postoperative seven days both in IRI and DAPT group,meanwhile it showed obviously there were bile duct hyperplasia in periportal zone in DAPT group.[Conclusion]1.It was a reliable,workable and reproducible animal model that was biliary IRI model of autologous orthotopic liver transplantation in rats,According to the operation method narrated by Zhou J.This model was effective to avoid allograft rejection and immunosuppressive factors from interference experiments, help us more controllably study biliary IRI in rats.2.By expression of D114,notch1,hes1 and CD34 in periportal zone,it showed obviously that after underwent biliary IRI,activated notch signaling pathway is effectively promote angiogenesis formed around bile duct and has a important role in regulation regeneration of peripheral vascular plexus after biliary IRI.