| OBJECTIVE: Liver transplantation is the only effective treatment for many types of end-stage liver diseases.However,the shortage of donor organs severely restricts the development of liver transplantation,and a large number of patients die each year on the wait list.In order to solve the problem of donor organ shortage and enlarge the donation pool,many liver transplant centers began to pay attention to and use the Donation after cardiac death(DCD)in last decades.However,compared with the Donation after Brain Death(DBD),DCD undergoes an additional stage of warm ischemia during the acquisition process,and suffers more severe ischemia-reperfusion injury(IRI)after transplantation.Therefore,the risk of serious complications such as graft primary non-function(PNF)and ischemic-related biliary tract disease is higher in DCD recipients,comparing with DBD recipients.In order to improve the transplantation outcomes of DCD,how to store and repair DCD has become an emerging hot issue in liver transplantation surgery.In the process of liver transplantation,the donor organ suffers ischemia-reperfusion injury(IRI),and severe IRI is considered to be the main cause of graft loss after transplantation.For liver transplantation,the mechanism of IRI is very complicated and has not been fully revealed.The main mechanisms known include: activation of oxygen and nitrogen free radicals,intracellular calcium overload,acidosis,energy metabolism disorders,and activation of inflammatory responses.Recently,some studies have shown that there are Inflammasome activation in the liver tissue during the liver transplantation,and it is supposed to be related to liver injury,but the mechanism of injury is not fully understood.Inflammasome is a complex composed of a variety of proteins in a cell,and it is generally classified according to the type of receptor protein it contains,and representative ones are: NLRP inflammasome containing NOD-like receptor protein.AIM2 inflammasome containing the HIN200 protein receptor.The NLRP3 inflammasome is one of the most intensive and extensive studied members of the NLRP Inflammasome.It has been demonstrated that NLRP3 inflammasome play an importantrole in a variety of autoimmune diseases and chronic inflammatory diseases.Its well-recognized fuction is to promote the maturation of interleukin-1?(IL-1?).Recently,it has been confirmed that there are obvious NLRP3 inflammasome activation e in liver cells during liver transplantation,especially after reperfusion,but there is no clear report on the specific mechanism of NLRP3 inflammasome during the injury,and it need to be further studied.It has been reported that the use of the selective NLRP3 NLRP3 inflammasome inhibitor MCC950 in an animal model of coronary heart disease can significantly improve the prognosis of coronary heart disease animals.Inspired by the results of this experiment,whether NLRP3 inflammasome can be used as a therapeutic target,reducing liver IRI,and improving the prognosis of DCD liver transplantation is interesting and need to be studied.Hypothermic machine perfusion(HMP)is a novel liver preservation technique and is believed to partially repair damaged DCD organs and improve the prognosis of DCD recipients.Therefore,HMP has replaced the traditional static cold storage(SCS)as the preferred preservation method for DCD donor liver.However,the specific protection mechanism of HMP is not completely clear.The implementation conditions and parameters of HMP,the composition of the preservation solution are still controversial.In order to promote the development of HMP technology,it is important to test whether the addation of MCC950 to the perfusion solution of HMP can improve the preservation effect of HMP.Shortly,to reveal the injury-causing mechanism of NLRP3 inflammasome during liver IRI and to study whether NLRP3 inflammasome can be taken as a therapeutic target to improve the preservation of HMP is the purpose of this study.Research methods: animal models used in this study were orthotopic liver transplantation model of SD rats and miniature pigs: Rats were implanted with orthotopic DCD liver using magnetic ring and double cuff method,and the mini pig donations were preserved by HMP.SD rats were randomly divided into four groups: sham operation group,liver transplantation group,MCC950 group,IL-1Ra group,before the donations underwent WI,MCC950 group was given intravenous MCC950,IL-1Ra group was treated with IL-1Ra intravenously,a nature interleukin-1?(IL-1?)antagonist.MCC950 group and IL-1Ra group were intravenously injected with corresponding drugs after reperfusion.The IRI of liver tissue was evaluated according to changes in liver enzyme index,HE staining,various inflammatory factors in the blood,and TUNEL staining.The expression of NLRP3 inflammasome and the expression of apoptosis-related proteins and apoptotic pathways in each group were detected by Western blot.Bama mini pigs were divided into 3 groups(6 pairs in each group),control group,postoperative group(donations preserved by conventional HMP and MCC950 intravenously injection after surgery),perfusion + postoperative group(donations preserved by HMP with MCC950 in the perfusate and additional MCC950 intravenously injection after surgery).The IRI of liver tissue was evaluated according to changes in liver enzyme index,MDA,HE staining,expression of various inflammatory factors and TUNEL staining.Verify the protective effects of MCC950 in the perfusate of HMP on large animal models to determine whether they can be converted to clinical applications.RESULTS: During the liver transplantation of rats and mini-pigs,there was activation of NLRP3 inflammasome,which play an important role in the ischemia reperfusion injury.MCC950 had protective effects on liver ischemia-reperfusion injury in rats DCD livers.The serum levels of AST,ALT,IL-1? and TNF-? in the MCC950 group were lower,compared with that in the liver transplantation group.Meanwhile,the HE staining injury score decreased,and apoptosis decreased in the MCC950 group,compared with the liver transplantation group.Moreover,the protective effect of MCC950 on hepatic ischemia-reperfusion injury was significantly better than that of IL-1Ra,especially in reducing hepatocyte apoptosis.In the detection of apoptosis-related proteins,the level of cytochrome C(Cyt C)and cleaved caspase 3(CASP 3)in the liver tissue were significantly decreased in MCC950 group compared with IL-1Ra group.Further detection of the upstream protein of the mitochondrial apoptosis pathway revealed a significant decrease in the amount of activated Bid protein.In the process of mini-pig liver transplantation,MCC950 was added to the perfusion solution of HMP and repeated administration after operation.The effect of reducing IRI was better than that of normal HMP and MCC950 administration after surgery only.The performance was as follows: the perfusion + postoperative group had lower liver enzyme index,the serum levels of IL-1? and TNF-? and other pro-inflammatory factorsdecreased,the HE staining injury score decreased,and the apoptosis decreased.Conclusion: During the process of hepatic ischemia-reperfusion of liver transplantation,there is activation of NLRP3 inflammasome and play an important role.In addition to promotion of the pro-inflammatory factor IL-1? maturity,NLRP3 inflammasome activates the bid protein pathway to activate the mitochondrial apoptotic pathway to induce hepatocyte apoptosis.Before donations WI,MCC950 injection can effectively inhibit NLRP3 inflammasome activation after reperfusion,thereby protecting donations,preventing IRI,and improving the prognosis of DCD receptor.For better clinical transformation,the addition of MCC950 to mechanical perfusion preservation solution and repeated administration after surgery can also inhibit the activation of NLRP3 inflammasome.The addition of MCC950 to the perfusate improved the repair effect of HMP on the DCD livers,and improved the prognosis of DCD receptors. |
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