The Mechanism Of CD39-adenosine-A2B Signaling Pathway In The Improvement Of DCD Liver Ischemia-reperfusion Injury By Hypothermic Oxygenated Perfusion

Background:Liver transplantation is the only effective treatment for end-stage liver disease.However,an extreme shortage of donor organs causes many patients to die while waiting for liver transplant treatment.To alleviate the shortage of organs for transplant,the use of liver donation after cardiac death(DCD)has increased.However,compared with liver donation after brain death(DBD),the additional in situ heat ischemia time during recovery in DCD liver transplantation increases the risk of graft-related complications in the recipient,resulting in a poor prognosis and poor tolerance of DCD donor livers to conventional static cold storage(SCS).Hypothermic oxygenated perfusion(HOPE)is a new organ preservation method emerging in recent years,and its role in alleviating ischemia reperfusion injury(IRI)of DCD donor liver has been increasingly recognized.However,the specific molecular mechanism by which HOPE regulates liver IRI in DCD liver transplantation remains unclear.Purinergic signaling pathway is closely related to liver diseases and is involved in pathophysiological processes such as liver inflammation,IRI and rejection after transplantation.CD39 is an extracellular nucleoside enzyme that plays a key role in regulating purinergic signal transduction by decomposing extracellular ATP and ADP into AMP,which is converted to adenosine by CD73 and binds to extracellular adenosine receptors for antiinflammatory and tissue protection.A2 B is the least sensitive adenosine receptor and functions only under pathological conditions such as hypoxia or ischemia.In this study,we explored the specific protective effect of HOPE on DCD liver and whether the mechanism was the regulation of CD39-adenosine-A2 B signaling pathway.Methods:A rat DCD liver model,HOPE perfusion system and isolated perfusion rat liver model system were established to simulate the clinical DCD liver transplantation process.Liver injury was assessed by detecting perfusate AST,ALT and TM levels,liver tissue H&E staining and CD31 immunofluorescence(IF)staining;Oxidative stress injury was assessed by detecting MDA and SOD content and ROS staining in liver tissue;Inflammatory responses were assessed by detecting IL-6 and TNF-αlevels in liver tissue and immunohistochemical(IHC)staining for MPO and CD68;Apoptosis was assessed by detecting apoptosis-related proteins(Bax,Bcl-2 and Caspase 3)and TUNEL staining;Adenosine in liver tissues was determined by High performance liquid chromatography(HPLC);The expressions of CD39,CD73,A1,A2 A,A2B and A3 were detected by Western blot and q T-PCR;The expression and distribution of CD39 and A2 B in liver cells were investigated by immunofluorescence double-label co-staining;Adeno-associated virus 8(AAV8)was constructed and transfected into rats to regulate the expression of CD39;PSB1115 was used to inhibit the expression of A2 B.Finally,four pairs of clinical liver transplantation specimens were collected for tissue H&E staining and CD31 IHC,and the expression of CD39 and A2B in the tissues were detected.Results:Compared with CS,HOPE reduced AST,ALT,and TM levels in rat DCD liver perfusate,and alleviated liver cell necrosis and vacuolization,interhepatic sinus congestion,and hepatic sinus endothelial injury;HOPE reduced MDA and ROS content,increased SOD level and alleviated oxidative stress injury in DCD liver tissue of rats;HOPE reduced the levels of tissue pro-inflammatory cytokines TNF-αand IL-6,reduced the infiltration of tissue neutrophils and macrophages,and alleviated the inflammatory response of rat DCD liver;HOPE inhibited the expression of pro-apoptotic protein Bax and Cleaved caspase 3,but promoted the expression of anti-apoptotic protein Bcl-2,reduced the number of TUNEL-positive cells,and alleviated the apoptosis of rat DCD liver cells;HOPE promoted the expression and adenosine production of CD39,CD73 and A2 B,and CD39 was mainly expressed in hepatic sinusoidal endothelial cells and A2 B was mainly expressed in hepatic cells;AAV8 effectively inhibited or promoted the expression of CD39.Promoting the expression of CD39 enhanced the protective effect of HOPE on rat DCD liver,while inhibiting the expression of CD39 weakened the protective effect of HOPE on rat DCD liver;Drug blocking of A2 B expression eliminated the effect of CD39 overexpression in HOPE on alleviating liver injury of DCD in rats;Both liver cells and hepatic sinusoidal endothelial cells were damaged after IRI in clinical liver transplantation specimens,and IRI can promote the expression of CD39 and A2B in tissues.Conclusions:HOPE can inhibit IR-induced oxidative stress,inflammatory response and apoptosis in rat DCD liver,thereby alleviating DCD liver injury.Mechanistically,HOPE can promote the expression of CD39 on sinusoidal endothelial cells of DCD liver,promote adenosine production,and then activate adenosine A2 B receptor on liver cells,inhibit oxidative stress,inflammatory response,cell necrosis and apoptosis induced by IR,and play a protective role on rat DCD liver.In clinical liver transplantation,IRI can induce hepatic sinusoidal endothelial cells and hepatocytes injury,and promote the expression of CD39 and A2 B.