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Mechanisms of Adenosine A2A Receptor Agonist Induced Delayed Liver Transplantation Protection in Rats

Author: WuLinZuo
Tutor: ZengZhong；YuZhiYong
School: Kunming Medical College
Course: Surgery
Keywords: Adenosine A2A receptor CGS21680 preconditioning late phase ischemia/reperfusion injury liver transplantation rat
CLC: R657.3
Type: Master's thesis
Year: 2010
Downloads: 41
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Abstract


BACKGROUNDStarzl performed the first clinical liver transplantation in 1963,but the patient died due to excessive blood loss.In 1983,National Institute for Health agency announced that liver transplantation is an effective treatment for end-stage liver diseases,should be promoted. Since then,the liver transplantation had a rapid development.Today,liver ischemia/ reperfusion（I/R） injury is one of the confronted important problems and considered a major cause of graft injury,causing liver dysfunction and even failure post transplantation.The conventional liver resection and the treatment of severe liver trauma will also cause liver ischemia-reperfusion injury experienced if we need block the hepatic blood flow.How to effectively protect the liver from ischemia-reperfusion injury has been a research focus.In recent years the rise of the ischemic and pharmacological preconditioning of the liver demonstrated a strong protective effect,become promising new treatments.Compared with ischemic preconditioning,pharmacological preconditioning has many advantages,such as: safety threshold large,without special equipment,simple operation and easier clinical application.Studies have shown that ischemic preconditioning has dual-phase:the early phase and late phase.Because of the delayed protection of late phase in long duration,wide scope, so more clinical value.The research of finding the best preconditioning drug to prevention ischemia-reperfusion injury will be the focus of future.Adenosine is an endogenous human cells throughout the nucleoside.Adenosine receptor （AR） is a G protein coupled glycoprotein,which was divided into 4 subtypes:AI、A2A、A2B and A3.Adenosine,primarily through its receptor,coupling second messenger such as cAMP and Ca2+,play a key role in cell signal transduction network.Recently,a growing number of studies show that Adenosine2A receptor(A2AR) is the most critical receptor which mediate the warm ischemia-reperfusion injury protective effect of liver ischemic preconditioning and some pharmacological preconditioning.The study of A2AR-mediated hepatic ischemia reperfusion injury murch confined to the liver warm ischemia reperfusion injury and the early stages of preconditioning（within 12 hours after preconditioning）.However,whether it has effective for the cold ischemia-reperfusion injury of liver transplantation and whether it has delayed protection effective for the cold ischemia-reperfusion injury of liver transplantation are unclear（within 24-72 hours after preconditioning）.OBJECTIVEThrough the establishment of rat orthotopic liver transplantation model,to investigate whether the adenosine A2A receptor agonist CGS21680 could induce the late phase of preconditioning against cold ischemia-reperfusion injury in rat liver transplantation and its possible mechanism,to provide a possible method for clinicians to prevent and treat of cold ischemia-reperfusion injury after liver transplantation.MATERIALS AND METHODS1.Animal Group:Fifty-six Male Sprague Dawley（S-D） Rats weighing 200-250g were used and divided randomly into four groups:①sham group:only free liver ligament②CGS group:donors received CGS21680,an Selective A2AR agonist（i.n.） 24h prior to harvest.③CGS+ZM group:donors received CGS21680,an Selective A2AR agonist and ZM241385,an A2AR specific inhibitor（i.n.） 24h prior to harvest.④ischemia and reperfusion group:donors received same volume of saline 24h prior to harvest.Donor rats were operated 24 hours after preconditioning.All liver grafts were harvested and stored with UW for 60min at 0-4℃.Separate groups of rats were killed at 6h after their vessels were unclamped,and liver samples were collected for further analysis.It has gone through 33h from preconditioning to obtaining samples.2.Animal Model:To establish an improved model of rat liver transplantation based on Kamada’s two-cuff technique.3.Detection:Separate groups of rats were killed at 6h after their vessels were unclamped, and liver samples were collected for further analysis.The hepatocellular function（ALT, AST） was analyzed.Detect the changes of plasma cytokines TNF-α,IL-6,IL-10.Detect the changes of liver tissue MDA level and SOD,MPO activity.The Bcl-2 mRNA and Caspase-3 mRNA levels were detected by RT-PCR.The apoptosis and necrosis of liver tissue were detected by flow cytomertry（FCM）.The hepatocellular damage was evaluated by liver histology.RESULTS1.The serum AST.AST levels were much lower in CGS group than that in I/R group and CGS+ZM group（P＜0.01）.There was no significant difference between I/R group and CGS+ZM group.2.The plasma cytokines TNF-α,IL-6 levels were much lower in CGS group than that in I/R group and CGS+ZM group（P＜0.01）.The plasma cytokines IL-10 levels in CGS group was significantly higher than that in I/R group and CGS+ZM group（P＜0.01）.There was no significant difference between I/R group and CGS+ZM group.3.The hepatic SOD activity in CGS group was significantly higher than that in I/R group and CGS+ZM group（P＜0.01）.The hepatic MPO activity was much lower in CGS group than that in I/R group and CGS+ZM group（P＜0.01）.There was no significant difference between I/R group and CGS+ZM group.4.The hepatic MPO level was much lower in CGS group than that in I/R group and CGS+ZM group（P＜0.01）.There was no significant difference between I/R group and CGS+ZM group.5.Flow cytometry show that the apoptosis and necrosis of liver tissue in CGS group were much lower than that in I/R group and CGS+ZM group（P＜0.01）.There was no significant difference between I/R group and CGS+ZM group.6.RT-PCR show that the Caspase-3 mRNA levels in CGS group was much lower than that in I/R group and CGS+ZM group（P＜0.01）,the Bcl-2 mRNA levels in CGS group was significantly higher than that in I/R group and CGS+ZM group（P＜0.01）.There was no significant difference between I/R group and CGS+ZM group.7.In I/R group and CGS+ZM group,H-E staining showed severe disruption of lobular architecture,significant periportal edema,congestion and necrosis,while in CGS group there was minimal degeneration.8.In I/R group and CGS+ZM group,electron microscopy showed severe mitochondria and endoplasmic reticulum swelling of hepatocytes accompanied by loss of microvillus;while in CGS group,both the cell nucleus and cellular organelles had no significant breakdown. CONCLUTION1.The adenosine A2A receptor agonist CGS21680 could induce the late phase of preconditioning against cold ischemia-reperfusion injury in rat liver transplantation, ameliorating hepatic damage,suppressing cytokines IL-6 and TNF-a release and improving liver function.2.It is possible that A2AR activate intracellular signal transduction network is the mechanism.It has a powerful protective effect to the ischemia-reperfusion injury after liver transplantation by anti-oxidation,anti-inflammatory,anti-apoptosis and the inhibition of platelet aggregation.

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