| Objective: Liver transplantation is the only effective method treating end-stage liver disease.However,the severe shortage of the donors restricts the surgery on liver transplantation and results in a lot of death of patients who are waiting for the transplantation surgery.Donors after cardiac death(DCD)can expand the pool of donors,in China,DCD will become the most important source of organ donation as there is no legal standard for brain death.Comparing to donors after brain death(DBD),the DCD donors are prone to getting poor early graft function(PEGF),primary non-function(PNF)and ischemic biliary tract disease for experiencing warm ischemia injury,which leading to dysfunction of grafts for long-term survival.A lot of studies showed that ischemia-reperfusion injury(ischemia-reperfusion injury,IRI)is the main cause for intrahepatic bile duct disease and graft dysfunction.How to reduce IRI,raise the utilization of donor organs,expand the pool of donors,reduce the complications after surgery,improve the life quality of recipients has become one of the most promising research direction for DCD liver transplantation.The research of mechanism about IRI now mainly focuses on ROS outbreaking,intracellular calcium overload,inflammation and so on.The recent studies revealed that cell autophagy is also involved in the mechanism of IRI,but how autophagy can impact on IRI should be further researched.Cell autophagy can maintain cellular homeostasis by degrading the abnormal proteins and damaged organelles,decreasing the injury on cells and getting the decomposed amino acids as the necessary substance for metabolism.Under physiological condition,autophagy plays a very important role on maintaining cellular homeostasis,but autophagy can also be excessively inducted under the condition of severe hypoxia,infection or other pathological states,which leading to programmed cell death.At present,it is controversial for the role of autophagy in liver IRI.There is little report on the role of autophagy playing in organ transplantation,and the limited coverageof the role of autophagy in liver IRI is inconsistent.Glycogen synthase kinase 3?(GSK-3?)is a serine / threonine protein kinase,which is deemed to be a key kinase which determines the fate of cells.It plays an important role in a variety of signal transduction pathways,embryonic growth,cell differentiation,apoptosis and tumor formation.GSK-3? widely exist in mammalian eukaryotic cells,but whether GSK-3? plays an important role in autophagy of cells or not is unclearly.Once IRI in liver transplantation can be further reduced by regulating autophagy,GSK-3?probably can be served as a new drug target for improving the prognosis of transplanted patients.The objective of this study includes 1.investigating the expression of GSK-3? and the changes in autophagy of hepatocytes during IRI in liver transplantation.2.investigating the effect of autophagy on hepatocyte survival and the effect of GSK-3? on autophagy during IRI.3.investigating the pathways of GSK-3? regulating autophagy.4.providing a new drug target for DCD liver transplantation.Methods: DCD liver transplantation model was used to observe the expression of GSK-3? and the changes in autophagy of hepatocytes during IRI in vivo.The hypoxia-re oxygenation model was used to observe the effect of autophagy on the survival of hepatocytes and the effect of GSK-3? on autophagy in vitro.In vivo,small animals and large animal DCD liver transplantation model were established.The liver transplantation was performed on 50 SD rats by orthotopic liver transplantation in 25 cases as small animal transplantation model.Cardiac arrest was induced by clamping the basilar part of the heart and block the troracic aorta of the dornor rats.The hepatic superior vena cava were anastomosed by "magnetic ring method".The portal vein and subhepatic inferior vena cava were reconstructed by Kamada double sleeves method.We set the groups according to the time after reperfusion by 0 hours,3 hours,6 hours,12 hours,24 hours,each group included 5,another normal control group also included 5,all the groups were collected liver tissue and venous blood samples.The liver transplantation was performed on 20 Bama miniature pigs by orthotopic liver transplantation in 10 cases as large animal transplantation model.Asphyxia was used to induce cardiac death.The liver tissue was collected from the donor's laparotomy,and the venous blood was taken as the normal control.The liver tissue was collected at 0 hours,3 hours,6 hours,12 hours and 24 hours after transplantation.All the groups were collected liver tissue and venous blood samples.Immunohistochemical staining and western blotting were used to detect the expression of LC3,p62,GSK-3? and AMPK.The changes of serum enzymes after liver transplantation,including AST,ALT were analyzed and liver tissue HE staining was used to observe the morphological changes of liver cells.GSK-3?-WT(wild-type),GSK-3?-K85R(inhibitor)and GSK-3?-S9A(activator)were transfected into human normal hepatocytes(L02),and autophagic activator group was set as a positive control.The expression of LC3,p62 and AMPK waa detected by immunofluorescence and western blotting after hypoxia-reoxygenation culture.The proliferative of hepatocyte activity was detected by MTS.Results: In the SD rats DCD liver transplantation,the expression of LC3 reached the peak at 6 hour,then decreased gradually;the expression of p62 decreased gradually,it showed that the expression of p62 reduced significantly at 6 hour after the transplantation,which meant the autophagy was enhanced guradually after reperfusion.GSK-3?,AMPK and autophagy varied consistently.The ALT and AST increased gradually in the DCD liver transplantation after reperfusion,and reached the peak at 6 hour,then decreased gradually.No necrosis was observed in the transplanted hepatocytes.In the porcine DCD liver transplantation,the expression of LC3 reached the peak at 12 hour,then decreased;the expression of p62 decreased gradually,it showed that the expression of p62 reduced significantly at 12 hour after the transplantation,which meant the autophagy was enhanced guradually after reperfusion.GSK-3?,AMPK and autophagy varied consistently.The transplanted liver cells at 0 hour,3 hour,6 hour,12 hour showed no significantly differences comparing with the control group,but a part of liver cells dead after transplantation for 24 hours.The ALT and AST increased gradually in the DCD liver transplantation after reperfusion,and reached the peak at 24 hour,then decreased gradually.No necrosis was observed in the transplanted hepatocytes.The proliferative activity in the GSK-3?-K85 R group and autophagic activator group was increased,and the expression of LC3,AMPK was up-regulated and the expression of p62 was down-regulated in the GSK-3?-K85 R group and autophagic activator group,which indicated that GSK-3?-K85 R could promote the autophagy and increase hepatocyte activity.The up-regulation of AMPK is consistent with the ability of autophagy.The proliferative activity in the GSK-3?-WT group and GSK-3?-S9 A group was decreased,and the expression of LC3 was down-regulated and the expression of AMPK and p62 did not change significantly,which indicated that GSK-3?-WT and GSK-3?-S9 A could inhibit the autophagy and decrease hepatocyte activity.Conclusions: In the DCD liver transplantation,autophagy was gradually increased with the prolongation of reperfusion time,then decreased.Autophagy protects hepatocytes from IRI.Up-regulation on the expression of GSK-3? can enhance autophagy.The expression of GSK-3? and AMPK is consistent with autophagy,which indicating that GSK-3? may regulate autophagy through AMPK-mTOR pathway.GSK-3? may provide a new drug target for clinical research. |
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