The Mechanism Of TXNIP/NLRP3 Pathway In Hypothermic Machine Perfusion Improving Donation After Cardiac Death Liver

Liver transplantation is an effective means of treating end-stage liver disease.The quality of donor liver is a key factor in determining the prognosis of liver transplantation.However,as the number of patients waiting for liver transplantation increases year by year,the lack of donor liver contributes to the extensive use of extended criteria donor livers such as donation after cardiac death(DCD)liver [1].Compared with the Donation after Brain Death(DBD)liver,the ischemia reperfusion injury(IRI)of this type of liver is more serious because of the presence of warm ischemic injury before the liver is retrieved.It is easy to cause primary non-function of the organ after transplantation,biliary ischemia and acute and chronic rejection after liver transplantation [1,2].Therefore,how to reduce DCD liver IRI,improve DCD liver quality and prognosis of liver transplantation patients has become a hot issue in the field of liver transplantation.Cold storage(CS)is still the most important means of preservation of donor organs.However,during CS,since the aerobic respiration has not completely stopped,resulting in the depletion of nutrients and the production of metabolic waste [3],CS does not effectively maintain and repair the DCD liver.In contrast,hypothermic machine perfusion(HMP)is a new type of dynamic organ preservation method.It can perform oxygenation perfusion of the liver through the portal vein under low temperature conditions,and can continuously provide a metabolic condition for the liver.Material and energy are supplied and accumulated toxic metabolites are removed [4].Animal experimental studies and clinical trials have shown that HMP can significantly improve the liver quality of DCD compared with CS,and can shorten the postoperative recovery time and reduce the incidence of severe postoperative complications such as liver PNF and biliary ischemia after transplantation [5,6].However,the specific molecular biological mechanisms by which HMP improves the liver quality of DCD are not clear.Previous studies have confirmed that the TXNIP/NLRP3 inflammasome pathway plays an important role in ischemia reperfusion injury.When cells are in oxidative stress,TXNIP is activated and binds to NLRP3 to activate the downstream inflammasome pathway,resulting in release of inflammatory factors and cell pyroptosis.HMP can significantly improve oxidative stress in DCD liver,so we hypothesized that HMP may have a mechanism to inhibit TXNIP/NLRP3 inflammasome pathway and thereby reduce DCD donor liver ischemia reperfusion injury.Therefore,based on the establishment of rat DCD model,liver HMP system and normothermic isolated perfusion rat liver model,we investigated whether the TXNIP/NLRP3 inflammasome pathway is involved in the mechanism of HMP improving quality of DCD liver.The results reveal the molecular mechanism of HMP to improve the liver quality of DCD liver,and provide an effective method and a strong theoretical basis for expanding the donor pool.Part? Protective Effect of Hypothermic Machine Perfusion on Rat DCD LiverObjective To investigate the protective mechanism of HMP in the liver of DCD,we need to first establish a stable HMP system and confirm its protective effect on DCD liver.Methods Eighteen SD rats were randomly divided into control group,CS group and HMP group.A rat donation after cardiac death(DCD)model was established in which the rat liver experienced 30 minutes of warm ischemia after cardiac arrest.The liver was then preserved by CS or HMP for 3 hours.The liver of the control group was retrieved directly without treatment.Three groups of livers were subjected to 1 hour of normal temperature in vitro reperfusion to simulate liver transplantation and then to assess liver damage.Results Compared with the CS group,HMP significantly reduced liver damage and improved liver function.The results of liver enzyme release,intrahepatic resistance,histology,apoptosis rate,oxidative stress and inflammatory response were all confirmed.The detection of hepatic oxygen consumption during HMP confirmed that hypothermic machine perfusion of inactive oxygenation also provided sufficient oxygen to the liver.Our results also showed that the damage of DCD liver after hypo-preservation(whether CS or HMP)was not significant.Normothermic reperfusion significantly activated liver injury in CS group.Liver in HMP group was repaired before reperfusion,and reperfusion injury was not serious.Conclusion Compared to traditional static cold storage,hypothermic machine perfusion has unique advantages in protecting DCD liver quality.Hypothermic machine perfusion provides sufficient oxygen to the liver during storage and replenishes energy,reducing hepatic sinus congestion and DAMPs release,thereby reducing damage to the liver during reperfusion.However,the specific molecular biological mechanism of hypothermic machine perfusion to alleviate DCD liver ischemia reperfusion injury remains unclear,which requires further exploration.Part ? Mechanisms of TXNIP/NLRP3 Inflammasome Pathway Improving the Quality of Rat DCD liver by hypothermic machine PerfusionObjective After confirming that HMP has a clear improvement in liver quality in rats with cardiac death,we explored whether the TXNIP/NLRP3 inflammasome pathway plays an important role in the mechanism by which HMP improves DCD quality.Methods Eighteen SD rats were randomly divided into control group,CS group and HMP group.A rat donation after cardiac death(DCD)model was established in which the rat liver experienced 30 minutes of warm ischemia after cardiac arrest.The liver was then preserved by CS or HMP for 3 hours.The liver of the control group was retrieved directly without treatment.Three groups of livers were subjected to 1 hour of normal temperature in vitro reperfusion.Liver specimens of three groups were obtained before and after in vitro reperfusion,followed by western blotting and co-immunoprecipitation to determine the activation of the TXNIP/NLRP3 inflammasome pathway.Results Compared with the control group,the western blotting results showed that the protein expression of the TXNIP/NLRP3 inflammasome pathway was significantly increased in the CS group after reperfusion.The co-immunoprecipitation showed that the TXNIP/NLRP3 complex formation in the CS group was significantly increased after reperfusion.Protein expression of the TXNIP/NLRP3 inflammasome pathway and TXNIP/NLRP3 complex formation were significantly inhibited in the HMP group after reperfusion compared with the CS group.Conclusion Hypothermic machine perfusion can repair the liver during reperfusion injury during liver preservation,and its mechanism is related to TXNIP/NLRP3 inflammasome pathway.Oxidative stress caused by reperfusion promotes the binding of TXNIP to NLRP3 and activates the downstream inflammasome pathway.By inhibiting oxidative stress,HMP inhibits the TXNIP/NLRP3 inflammasome pathway,thereby reducing the donor reperfusion injury and optimizing the quality of donor liver.Part ? Study on the Expression Pattern of TXNIP/NLRP3 Inflammasome Pathway in DCD LiverObjective To investigate the expression pattern of TXNIP/NLRP3 inflammasome pathway in the liver and to clarify the specific mechanism of HMP regulating TXNIP/NLRP3 inflammasome pathway.Methods Eighteen SD rats were randomly divided into control group,CS group and HMP group.A rat donation after cardiac death(DCD)model was established in which the rat liver experienced 30 minutes of warm ischemia after cardiac arrest.The liver was then preserved by CS or HMP for 3 hours.The liver of the control group was retrieved directly without treatment.Three groups of livers were subjected to 1 hour of normal temperature in vitro reperfusion.Liver specimens of three groups were obtained and subjected to immunofluorescence to determine the distribution of TXNIP and NLRP3 in liver cells.Results Immunofluorescence double labeling showed that both TXNIP and NLRP3 were mainly expressed in hepatocytes and less expressed in endothelial cells.Immunofluorescence single labeling showed that compared with the control group,TXNIP nuclear translocation was significant in CS group,and cytoplasmic NLRP3 expression was also significantly increased.Compared with CS group,TXNIP nuclear translocation and cytoplasmic NLRP3 expression increase in HMP group was significantly inhibited.Conclusion The TXNIP/NLRP3 inflammasome pathway plays a major role in hepatocytes.The specific mechanism by which HMP regulates the TXNIP/NLRP3 inflammasome pathway is as follows: HMP inhibits oxidative stress induced by reperfusion,thereby inhibiting nuclear translocation of TXNIP and increased cytoplasmic expression of NLRP3.Subsequently,HMP inhibits the binding of TXNIP and NLRP3 in the cytoplasm leading to inhibition of downstream inflammasome pathways.Since TXNIP and NLRP3 are less expressed in endothelial cells,we believe that HMP inhibits the expression of TXNIP in the liver by providing fluid shear stimulation and is not the main mechanism by which HMP inhibits the TXNIP/NLRP3 inflammasome pathway.