Effect Of BMMSCs Combined With Normothermic Machine Perfusion On The Quality Of Rat DCD Liver And Its Mechanism

Aim: To investigate the effects of bone marrow mesenchymal stem cells(BMMSCs)combined with normothermic machine perfusion(NMP)on the quality of rat DCD donor liver and its mechanism.Methods: Extraction of BMMSCs by adherent method.Rat thoracic aortas were clipped to obtain DCD livers,and a rat NMP system was established.The DCD livers were grouped by preservation method: Normal,static cold storage(SCS),NMP,and BMMSCs plus NMP;storage time was up to 8 h.An IAR20 cell oxidative stress injury model was established in vitro by simulating DCD oxidative stress injury,and co-culture with BMMSCs for 6 h.Liver function in outflow perfusate was detected by biochemical methods;liver tissue histopathology was observed by hematoxylin–eosin staining;mitochondria ultrastructure was observed by transmission electron microscopy;hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase d UTP nick end labeling or Annexin V-FITC/PI staining;CD14,CD68 and myeloperoxidase(MPO)expression was detected by immunofluorescence;endothelin-1(ET-1),endothelial nitric oxide synthase(e NOS),inducible nitric oxide synthase(i NOS),von Willebrand factor(v WF),intercellular adhesion molecule(ICAM-1),intervascular adhesion molecule-1(VCAM-1),JUN N-terminal kinase(JNK),p-JNK(Thr183 / Tyr185),NF-κB(p65),p-NF-κB(Ser36),AMP-dependent protein kinase(AMPK)α,p-AMPK(Thr172),acetyl-Co A carboxylase(ACC)and p-ACC(Ser79)were detected by immunohistochemistry and western blot;ET-1,NO,VCAM-1,ICAM-1,TM and PAF levels in the outflow perfusate were tested by ELISA;oxidative stress level was detected by MDA and GSH kits;ROS level was detected in cells DCFH-DA fluorescent probe method;mitochondrial membrane potential was detected by JC-1 fluorescent probe method.Results: Compared with SCS,BMMSCs combined with NMP and NMP alone significantly improved DCD liver function and liver histological damage,reduced hepatocyte apoptosis.It was further found that: 1.BMMSCs combined with NMP improved DCD liver microcirculation: Improved liver ET-1 / NO balance and microcirculation perfusion,compared with SCS,BMMSCs combined NMP significantly inhibited the expression of ET-1,i NOS,and NO in the liver,and promoted the expression of e NOS(P < 0.05);inhibited intrahepatic macrophage activation intercellular adhesion,improved endothelial damage,compared with SCS,NMP combined with BMMSCs significantly down-regulated the expression of CD14 and CD68 in liver macrophages,inhibited ICAM-1 and VCAM-1 expression and v WF expression in the liver,and has a better effect than NMP alone(P < 0.05).2.BMMSCs combined with NMP relieved DCD liver oxidative stress injury,relieved liver cell mitochondrial damage and improved mitochondrial membrane potential level(P < 0.05),and BMMSCs combined with NMP was significantly better than NMP alone;BMMSCs combined with NMP could significantly inhibit JNK-NF-κB pathway to reduce oxidative stress and promote AMPK activation to to protect damaged mitochondria,thereby reducing mitochondrial damage and increase mitochondrial function in DCD liver(P < 0.05).In cell models,BMMSCs significantly reduced cell apoptosis,inhibited ROS release,relieved cell mitochondrial damage and improved mitochondrial membrane potential levels in the IAR20 cell oxidative stress model;BMMSCs also significantly downregulated the JNK-NF-κB signaling pathway and promoted AMPK activation in the IAR20 cell oxidative stress model.This confirmed again the mechanism by which BMMSCs play a protective role.Conclusions: In the situation of liver donor shortage,DCD donor liver is an effective method to expand the available donor pool.This research proved that: BMMSCs combined with NMP could more effectively improve the quality of DCD donor liver.The following mechanisms of BMMSCs play a protective role:(1)Inhibited macrophage activation and intercellular adhesion,improved endothelial damage and microcirculation perfusion;(2)Inhibited JNK-NF-κB signaling pathway and reduced DCD liver oxidative stress injury,and promoted AMPK activation to reduce mitochondrial damage and increase mitochondrial function.The preservation of DCD liver by BMMSCs combined with NMP provided protective effective new method for improving the quality of DCD liver,and provided experimental evidence for the use of clinical DCD liver.