The Study On The Effects Of Succinate In The Ischemia Reperfusion Injury Of Liver Allograft During Liver Transplantation

Objective: Liver transplantation is the only effective treatment for end-stage liver disease.However,donor shortages severely restrict the development of liver transplantation.Lots of patients waiting for liver transplantation lose their lives while waiting.In order to solve the shortage of donors,the transplant centers in Europe and North America re-pay attention and begin to use DCD more and more to ease the contradiction between supply and demand.However,compared with DBD,DCD donors experience warm ischemic injury so that recipients are more prone to complications such as primary non-function(PNF),poor early graft function(PEGF)and ischemic biliary disease.In our country,DCD has become the most important source of organ donation in our country due to the absence of legal provisions on the standard of brain death.Therefore,how to reduce the ischemia reperfusion injury of the liver and assess the quality of the donor liver to decide whether to use it are the research hotspots in liver transplantation.Ischemia reperfusion injury(IRI)is the leading cause of organ loss after liver transplantation.The mechanisms of IRI include: reactive oxygen species burst,intracellular calcium overload,acidosis,inflammatory cell response,etc.Reactive oxygen species burst is considered as a promoter and plays an extremely important role in ischemia reperfusion injury.Reactive oxygen species overproduction mechanisms include: xanthine oxidase,inflammatory cells and mitochondria and so on.More and more literatures report the key role of mitochondrial respiratory chain in the overproduction of reactive oxygen species in ischemia reperfusion injury.However,the production mechanism of reactive oxygen species in mitochondria remains to be further studied.The latest article found that different organs in mice have the accumulation of succinate in the tricarboxylic acid cycle during ischemia,while the accumulated succinate during reperfusion is transmitted through the mitochondrial respiratory chain to produce large amounts of mitochondrial reactive oxygen species,leading to ischemia reperfusion injury.Therefore,during the process of ischemia reperfusion injury in theliver,if the accumulation of succinate is reduced in ischemic period,it is possible to reduce the reperfusion injury and thus to protect the liver.In the meantime,is it possible to predict the severity of reperfusion injury by measuring the amount of succinate accumulation at the end of the ischemic period,which is useful in evaluating organ function,reducing organ abandonment rate,or increasing effective organ usage?In ischemia reperfusion injury,there are many reports that MAPK pathway plays an important role in the heart,brain,kidney and other organs.Although there are many reports in the liver,it is controversial.At the same time,the role of the three classic MAPK pathways JNK,ERK,P38 is not the same in ischemia reperfusion injury of liver.Among the upstream stimulators of MAPK pathway,reactive oxygen species play a very important role.Therefore,the role of reactive oxygen species in the three MAPK pathway and how reactive oxygen species affect the MAPK pathway during liver ischemia reperfusion injury,are worth to study and verify.For liver transplantation,especially in China DCD era of liver transplantation,how to repair the liver and assess the function of the liver is crucial.At present,mechanical perfusion is the general trend of improving donor liver preservation and evaluating the quality of donor liver.However,the mechanism of mechanical perfusion and how to improve and evaluate the quality of donor liver require a large amount of experiments to explore and verify.Therefore,whether succinate,reactive oxygen species,MAPK pathway,mechanical perfusion can better protect DCD donor liver and evaluate the quality of donor liver is the purpose of this study.Methods: To establish a stable small animal and large animal liver transplantation model:SD rat magnetic ring double cannula liver transplantation model;Bama miniature pig DCD liver transplantation model.Validate ischemic succinate accumulation in rats and pigs by examining the succinate content of rats and pig livers during the ischemic period.The rat liver transplantation model was used.The content of succinate in the ischemia phase was intervened by the drugs DM and DS.The liver transplantation control group,DM group,DS group,DM + DS group,four groups(6 pairs)were enrolled in this study.The differences of succinate content in different intervention groups were explored.The severity of ischemia reperfusion injury was compared by ROS,MDA,hepaticenzymology and HE staining.Verify that succinate produces some degree of ischemi reperfusion damage through reactive oxygen species production.In addition,the expression of MAPK protein in liver transplantation group at different time point including before ischemia,warm ischemia 30 min,cold preservation 2h,reperfusion 1h,reperfusion 24 hours were determined.It is found that JNK pathway is closely related to ischemia reperfusion injury.The rat liver transplantation model was used in this study.The rats were randomly divided into 4 groups(control group,DS group,SP600125 group,SP600125 + DS group).The ROS,MDA,hepatic enzymology,HE staining,JNK pathway protein expression,MPTP opening degree and TUNEL were detected in each group to verify succinate,reactive oxygen species,JNK pathway,mitochondrial permeability transition pore,cell apoptosis.In addition,evidence of JNK pathway acting directly on mitochondria was found by immunoprecipitation of mitochondrial Sab and p-JNK in the liver transplantation group.Pig liver transplantation model,combined with hypothermic mechanical perfusion(HMP)and SP600125,are used to test the effect of pathway found in the experiment on large animal models of liver transplantation.The transplant groups were divided into 4 groups(6 pairs in each group):CS group(warm ischemia 30 min + cold preservation 4h),CS + SP600125 group(warm ischemia 30 min + cold preservation 4h +SP600125),HMP group(warm ischemia 30 min + cold preservation 2h + HMP 2h),HMP+ SP600125 group(warm ischemic 30 min + cold preservation 2h + HMP 2h +SP600125).Detect the ROS,MDA,serological examination,p-JNK and apoptotic protein expression,TUNEL,survival rate and other indicators to verify protection affect of the HMP and SP600125 in animal models,and determine whether it can be converted into clinical applications.Results:The livers of rats and pigs,like those of mice during warm ischemia,accumulate succinate and remain essentially stable after a slight decrease in cold storage.Through the intervention of medicine,It was found that ROS,MDA,hepatic enzymology and HE staining were correlated with the content of succinate in the end-ischemic period in each group.The more the amount of succinate was in the end of ischemia,The more ROS and MDA are produced,the worse liver function and HEstaining reflecting the severity of ischemia reperfusion injury.MAPK pathway expression at various time points during liver transplantation: p-JNK,p-P38 and p-ERK protein are almost no expression in the pre-ischemic,warm ischemia 30 min,cold preservation 2h period.Onlyp-JNK protein in the reperfusion 1h and 24 h were significantly increased,p-P38 increased only 24 h after reperfusion.p-ERK were basically no expression after reperfusion.It is found that SP600125 could inhibit the expression of p-JNK protein and alleviate ischemia reperfusion injury which was not affected by DS intervention through differences in liver transplantation groups treated with DS and SP600125.The expression of upstream protein of JNK pathway was increased,while the expression of p-JNK was still inhibited in the DS and SP600125 co-intervention group while the injury after transplantation was similar to that of SP600125 group.By co-immunoprecipitation,it is found that the direct interaction of p-JNK protein and mitochondrial scaffold protein Sab affect the function of mitochondria.In different groups of pig liver transplantation,HMP can reduce the content of hepatic succinate,and the results are better than cold preservation group in ROS,MDA,serological examination,HE staining,TUNEL,survival rate and so on.Compared with HMP group,the HMP+SP600125 group have better result in p-JNK protein expression,MPTP openness and apoptosis.Although the latter were superior in serology and survival rate,the difference is not statistically significant.Conclusions: Succinate was accumulated in rats and pigs during warm ischemia and remained stable after a slight decrease in cold storage.The accumulation of succinate at the end of ischemia results in the production of a large number of reactive oxygen species during reperfusion,resulting in ischemia reperfusion injury.Most of the reactive oxygen species affect the mitochondrial permeability transition pore through the JNK pathway,leading to apoptosis of cells.One of the protective mechanisms of hypothermic mechanical perfusion is to reduce the ischemia reperfusion injury by reducing the content of succinate in the end of ischemia.HMP combined with SP600126 can better improve the quality of liver and improve the prognosis of liver transplantation.