Protective Effect And Mechanism Of Hypothermic Machine Perfusion In The Rat DCD Liver Transplantation

Objective To develop a stable model of static cold storage(SCS)and(or)hypothermic machine perfusion(HMP)on liver grafts of rats from donor after cardiac death(DCD)in orthotopic liver transplantation and study the protective effect of HMP during it,and investigate its possible mechanism.Methods Ninety male Sprague-Dawley(SD)rats(250?320g)were randomly divided into three groups:hypothermic machine perfusion(HMP)group with 30 min of in situ warm ischemia+3-h static cold storage followed by 1-h hypothermic machine perfusion in HTK solution(4?)(HMP group,n=30),static cold storage(SCS)group with 30-min in situ warm ischemia and 4-h static cold storage in HTK solution(4?)(SCS group,n=30),and normal control experiments with rat livers cold flushed and stored in HTK solution(4?)immediately after procurement,i.e.,without in situ warm ischemia and minimal static cold storage(45 min)was granted to allow cuff placement before liver transplantation(Normal Control group,n=30).The effects of hypothermic machine perfusion and static cold storage were evaluated in the rat DCD liver transplantation.The morphological changes were observed by hematoxylin and eosin(H&E)staining and electron microscopy(EM),and the apoptotic morphological by Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling(TUNEL)assay.The MDA content was measured with thiobarbital acid.TRX activity was measured by the insulin disulfide reduction assay,and the cleaved caspase 3 protein expression by immunohistochemistry and the TXNIP,JNK,p38,VCAM1,caspase 3 protein expression by western blot.The combination state of TRX-ASK1 was tested by immunoprecipitation.ALT and AST contents in the plasma 16 hours after orthotopic liver transplantation were measured with commercial testing kits,and Interleukin-2(IL-2)and tumor necrosis factor alpha(TNFa)levels with Elisa testing kits.Results The MDA content in liver tissue 1 h after unclamped the portal vein during orthotopic liver transplantation in the HMP group was(0.65±0.17)nmol/mg protein,while in the SCS group[(0.98±0.21)nmol/mg protein,P<0.05]was increased as compared with the HMP group.The MDA content in liver tissue 1 h after unclamped the portal vein during orthotopic liver transplantation in the normal control group was(0.43±0.07)nmol/mg protein.The ALT and AST levels in the plasma tested 16 hours after orthotopic liver transplantation in the HMP group were(2.04±0.41)IU/g liver,(2.61±0.59)IU/g liver respectively,while in the SCS group[(4.51±1.07)IU/g liver,P<0.01],[(6.35±1.21)IU/g liver,P<0.01]were increased as compared with the HMP group.The ALT and AST levels in the plasma tested 16 hours after orthotopic liver transplantation in the normal control group were(0.27±0.08)IU/g liver and(0.67±0.17)IU/g liver.The IL-2 and TNFa levels in the plasma tested 16 hours after orthotopic liver transplantation in the HMP group were(410.43±57.62)pg/ml,(0.92±0.21)pg/ml,while in the SCS group[(659.61±233.49)pg/ml,P<0.05],[(1.87±0.59)pg/ml,P<0.05]were increased as compared with the HMP group.The IL-2 and TNFa levels in the plasma tested 16 hours after orthotopic liver transplantation in the normal control group were(209.51±23.56)pg/ml and(0.21±0.04)pg/ml.H&E staining showed that the structure of liver tissue 1 h after unclamped the portal vein during orthotopic liver transplantation in the HMP group was more complete than the structure of liver tissue 1 h after unclamped the portal vein during orthotopic liver transplantation in the SCS group,and hepatic necrocytosis in the HMP group was less than in the SCS group.There were a small number of cytoplasmic vacuoles and inflammatory cells infiltration in the HMP group,while a large number of cytoplasmic vacuoles and inflammatory cells infiltration in the SCS group.Electron microscopy showed that the mitochondria in hepatocytes were mild edema and the hepatic sinus endothelial cells were swelling in the HMP group,while the mitochondria in hepatocytes were swelling and the hepatic sinus endothelial cells were swelling and deformed and broke away from the surface of hepatocytes in the SCS group.The arrangement of hepatocytes was regular and the hepatocytes were mild edema in donor liver tissue 1 h after unclamped the portal vein during orthotopic liver transplantation in the normal control group.There were few hepatocyte necrosis and cytoplasmic vacuolation and inflammatory cells infiltration in donor liver 1 h after unclamped the portal vein during orthotopic liver transplantation in the normal control group.Electron microscopy showed that the morphology of the mitochondria in hepatocytes was almost complete and no swelling and shrinking of hepatic sinus endothelial cells in donor liver 1 h after unclamped the portal vein during orthotopic liver transplantation in the normal control group.Tunel assay showed a more significant reduction in apoptosis in the HMP group than in the SCS group.The insulin disulfide reduction assay showed a more significant increasement of TRX activity in the HMP group than in the SCS group.The immunohistochemistry showed the cleaved caspase 3 protein expression in the HMP group decreased more significantly than in the SCS group.HMP depressed TXNIP,p-JNK1,p-JNK2,p-p38,VCAM1 and cleaved caspase 3 protein expression as showed in western blot.The immunoprecipitation showed that after using the ASK1 antibody for precipitation the expression of TRX in the HMP group increased more significantly than in the SCS group.Conclusion Hypothermic machine perfusion can reduce hepatic necrocytosis and apoptosis of rat DCD liver,and can decrease the IL-2 and TNFa level,and reduce hepatic oxidative stress injury,as well as enhance the TRX activity and increase the combination of TRX and ASK1.HMP can also depress the expression of TXNIP,p-JNK1,p-JNK2,p-p38,VCAM1,as well as reduce the expression of cleaved Caspase 3,and protect rat hepatocytes,and improve the quality of DCD liver.Its mechanism may be related to the increase of TRX activity and the depression of TNF-ASK1-JNK/p38 signaling pathway and the reduction of the production of inflammatory cytokines.