The Protective Effects Of Minocyline On Cold Ischemia Reperfusion Injury In Rat Liver Transplantation

Part One The Establishment of Liver Transplantation Model in RatObjective: To establish a stable liver transplantation model in rat,which provide experimental basis for the study of protective effect of cold ischemia reperfusion injury by Minocycline in rat liver transplantation.Methods: Liver transplantation of SD rats were performed with modified Kamada’s two cuffs technique. And the survival and complications after liver transplantation were observed.Results: One hundred and sixty-five cases of liver transplantation in rat were performed in three phases. The surgical success rate in phase I, phase II and phase III were respectively 55%(33/60), 91.7%(55/60) and 100%(45/45). Moreover, the survival rate of a week was 85%(51/60) in phase II, We successfully established a stable liver transplantation model in rat for our project.Conclusion: Orthotopic liver transplantation in rats is a complex and delicate procedure. In order to establish a stable liver transplantation model in rat, efficient-sustainable training, patient and meticulous operation and skillful microsurgical techniques are indispensable. The sophisticated microsurgical technique and the delicate surgical manipulation is the key to successfully perform a rat orthotopic liver transplantation.Part Two The Protective Effects of Minocyline on Cold ischemia Reperfusion Injury in Rat Liver TransplantationObjective:(1)To explore the effects of different cold ischemia preservation time on the expression and activity of MMP-9 in rat liver graft and the injury of graft.(2)To investigate the protective effects of Minocyline on cold ischemia reperfusion injury in rat liver transplantation, and reveal the possible protective mechanism of Minocyline.Methods:(1) According to donor liver cold ischemia preservation time, SD rats were randomly divided into three groups: 30 mines group, 3 hours group, 6 hours group. At 24 hours after operation respectively harvested liver tissue and blood samples of each group to examine liver function(ALT, AST), tissue MPO activity, tissue MDA content and histopathologic changes. Real time-PCR was used to analyze the expression of MMP-9, TNF-a, IL-1β, IFN-γ and Bcl-2 in grafts, Location, expression and activation of MMP-9 in grafts were assessed by gelatin zymography and immunohistochemistry.(2) SD rats were divided into two groups randomly: Minocycline(M)group, control(C)group. At 24 h, 48 h, 72 h after liver transplantation, We respectively harvested liver tissue and blood samples of each group. Protein expression levels of Caspase 3 and Bcl-2 were assessed by Western Blot, besides, the other indicators and detection methods were same to Methods(1).Results:(1)The expression level and activity of MMP-9 were significantly higher in 6 hours group than those in 30 minutes and 3 hours groups after liver transplantation(p<0.05), and with the extension of cold ischemia preservation time of donor liver,the expression levels and activity of MMP-9 significantly increased in grafts. MMP-9 was located in endothelial cells mainly and a small mount of hepatocyte of portal area in 30 minutes group, the expressed regions and expression level of MMP-9 were amplified after reperfusion in 3 hours group and 6 hours group. The increased expression and activity of MMP-9 was postive relation to cold ischemia preservation time of donor liver were significantly associated with liver ischemia-reperfusion injury, as evidenced by elevated liver function(ALT, AST), expression of pro-inflammatory factors(IL-1β, IFN-γ), MPO activity, MDA content, histopathologic changes and reduced expression of Bcl-2 in grafts(p<0.05).(2)Compared with group C, the expression and activity of MMP-9,liver function(ALT, AST), expression of pro-inflammatory factors(IL-1β, IFN-γ,TNF-α), MPO activity, MDA content, and histopathologic changes significantly reduced and expression of Bcl-2 increased in grafts in group M at 24 h, 48 h and 72 h after liver transplantation(p<0.05). Compared with group C, the protein expression of Caspase 3 was significantly reduced(p<0.05), and Bcl-2 was significantly increased(p<0.05) at 24 h after reperfusion.Conclusion:(1)MMP-9 playes an important role in the process of cold ischemia-reperfusion injury. With the extension of cold ischemia preservation time of donor liver, the expression level and activity of MMP-9 significantly increase in grafts after reperfusion.(2)Through degrading endothelial basement membrane and extracellular matrix of hepatocyte, MMP- 9 promotes the migration and infiltration of neutrophils, the release of inflammatory mediator( IL-1β, TNF-α) of neutrophils, while inhibits the expression of Bcl-2 and promotes hepatocyte apoptosis, aggravates ischemia-reperfusion injury through multiple mechanisms.(3)By inhibiting the expression and activity of MMP-9, reducing migration, infiltrating and release of inflammatory mediators of neutrophils, as well as mitigating apoptosis of hepatocytes mediated by MMP-9, Minocycline significantly alleviates the cold ischemia and reperfusion injury in rat liver transplantation.