The Research Of TSGL-Ig Attenuates Liver Ischemia-reperfusion Injury In Mouse Orthotopic Liver Transplantation

Background:Local sterile inflammation represents an innate immune signature of hepaticischemia-reperfusion injury(IRI),an inevitable event in organ procurement,transplantation,partial hepatectomy,trauma or shock.The IRI often results in hepaticnon-function or dysfunction at early post-transplant phase,and may lead to increasedincidence of acute and chronic rejection.In addition,IRI contributes to donororgan shortage as marginal livers are particularly susceptible to the ischemic insult.Liver endothelial cells(LECs)form the vascular wall of the hepatic sinusoid.LECs play protective rolescontrolling vascular homeostasis,inflammation,vascular tone,and toxicants clearance.Insult to hepatic endothelium during cold preservation contributes to liver IRI,bringingout low graft microcirculation,upregulation of adhesion molecule expression,oxidativestress,and further augmenting neutrophil infiltration and hepatocyte necrosis.The selectins,a family of lectin-domain glycoproteins expressed on endothelialcells,platelets and leukocytes mediate the initial capture and support leukocyte rollingon sinusoidal endothelial cells in the initial phase of IRI.The principal selectin ligand,P-selectin glycoprotein ligand-1(PSGL-1,CD 162)is the only known high-affinity ligand for P-selectin in the cell adhesion cascade.To gain further insight into putative role of adhesion molecule antagonism inhepatic inflammation and the acquisition of intracellular redox homeostasis,we haveconstructed a new soluble recombinant Ig fusion molecule,designated "TSGL-Ig" wherethe "T" stands for"tandem".The TSGL-Ig construct enhances the selectin binding properties ofthe previously rPSGL-Ig,that has been widely studied and shown to act as acompetitive antagonist for leukocyte binding to selectins both in vitro and in vivo.The oxidative stress caused by excessive production of reactive oxygen species(ROS),is vital for hepatocellular insult in the pathophysiology of liver IRI.TheKeap1-Nrf2-ARE signaling axis represents an essential trigger for adaptive oxidativestress response,in processing excessive ROS production,as well as maintaining the cellular redox balance and metabolism.Hereby,it will be imperative to illustrate the molecular mechanisms of ischemia and reperfusion injury under oxidative stress,and to establish new agent"TSGL-Ig" for therapeutic approach which can improve the prognosis of liver transplant patients.Objectives:The present study seeks to examine therapeutic effects and mechanisms by whichTSGL-Ig molecule targets hepatic IRI in a clinically-relevant mouse liver transplantmodel subjected to prolonged period of cold storage.Anticipating that TSGL-Ig wouldbind to selectins on activated endothelial cells,which play a key role in IRI,we testedwhether TSGL-Ig homeostatic function also involves Nrf2 translocation/activation inLECs.We wonder whether TSGL-Ig binding to selectins on endothelial cellsmay mitigate cellular damage in IR-stressed mouse liver grafts by exerting directcytoprotection on LECs in Nrf2-dependent manner,and find out an effective therapeutic approach which can improve the prognosis of liver transplant patients.Methods:1.Animals.Male 8-12 weeks old wild-type(WT)mice and Nrf2-deficient mice on a C57BL/6 background(backcrossed for at least twelve generations)were used.2.OLT Model.Livers from WT or Nrf2 KO mice were stored for 20 h at 4℃ in UW solution prior to transplantation into syngeneic hosts.Three groups of WT recipients were treated with TSGL-Ig,rPSGL-Ig,or control Ig at the time of liver harvest and immediately prior to reperfusion.3.Warm Liver IRI Model.In a mouse model of segmental(70%)hepatic warm IRI,the arterial/portal venous blood supply to the cephalad lobes was interrupted by an atraumatic clip for 90 min.4.Hepatocellular Function Assay.Serum alanine transaminase(sALT)levels were measured in blood samples to evaluate the injury of ischemia and reperfusion in the liver.5.Histopathology.Liver specimens(4μm),stained with hematoxylin and eosin(H&E),were analyzed blindly to measure the structure injury of liver.6.Myeloperoxidase Activity Assay.The presence of myeloperoxidase(MPO)was utilized as an index of neutrophil accumulation in the liver.7.Quantitative RT-PCR Assay.PCR were performed to analyse liver cytokine/chemokine program and Nrf2 downstream gene expression.8.Western blotAnalysis.Thesearetoanalyze Nrf2 expression in cytosolic and nuclear proteins from liver OLT samples.9.Cell culture.Mice LECs were isolated by in situ two-stage collagenase perfusion and cultured with complete DMEM for 24h.10.LDH/ALT Release Assay.LDH/ALT release into culture medium was measured to evaluate liverIRI.11.Statistical Analysis.All values are expressed as mean ± standard deviation(SD).Data were analyzed with an unpaired,two-tailed Student’s-t test;p<0.05 was considered statistically significant.Results:1.TSGL-Ig Improves OLT Survival and Ameliorates Hepatic IRI First,we found that treatment with TSGL-Ig(via portal vein at liver harvest and reperfusion)promotes survival in mouse recipients of syngeneic OLT subjected to prolonged ex vivo cold storage,compared with 40%survival in control Ig-treated group,over 90%of OLT recipients conditioned with TSGL-Ig remained alive at day 14 post-transplant.Then,we analyzed the hepatocellular function in IR-stressed OLT recipients by screening sera and liver graft samples and got the results that unlike in controls,mice given TSGL-Ig were IR-resistant,evidenced by reduced sALT levels and H&E staining.2.TSGL-Ig Depresses Neutrophil and Macrophage Sequestration in IR-stressed OLT We used immunohistology to analyze and quantitate cell trafficking patterns at 6h post-OLT.The results suggested P-selectin blockade with TSGL-Ig markedly decreased neutrophil and macrophage recruitment in ischemic OLT.Consistent with immunostaining data,MPO assay,reflecting hepatic neutrophil activity,was decreased in TSGL-Ig.3.TSGL-Ig Depresses IR-Induced Liver Cytokine/Chemokine Programs We use RT-PCR for chemokine and cytokine expression patterns in IR-stressed OLT.By 6h post-transplant,neutrophil/monocyte-derived pro-inflammatory chemokine(CXCL-1,CXCL-10)and cytokine(TNF-a,IL-1β and IFN-β)programs were uniformly suppressed(p<0.001)in TSGL-Ig treatment group as compared to Ig-control.4.TSGL-Ig Decreases IR-Induced Liver Oxidative Stress and Necrosis/Apoptosis Unlike IR-stressed control OLT characterized by abundant ROS levels,those treated with TSGL-Ig showed weak DHE staining,barely oxidized by superoxide TSGL-Ig diminished otherwise abundant hepatocellular necrosis and apoptosis,evidenced by decreased frequency of TUNEL+ assayshowed cells.In addition,TSGL-Ig therapy protected.LECs against IR-triggered necrosis/apoptosis.5.TSGL-Ig Differentially Regulates IR-induced LEC Activation The qRT-PCR-assisted analysis of control grafts by 6h or reperfusion has revealed strong gene induction of P-/E-selectin,VCAM-1/ICAM-1,which declined thereafter.TSGL-Ig abolished the expression of LEC-associated cell adhesion genes in IR-resistant OLT.6.TSGL-Ig Mediates LEC Cytoprotection in Nrf2-Dependent Manner Western blots have revealed that treatment with TSGL-Ig elevated both cytosolic and nuclear Nrf2 in IR-stressed OLT.Unlike in control OLT,TSGL-Ig increased the expression of Nrf2 downstream target genes,including Trx,GCL,NQOl,and HIF-1α(p<0.001).Primary LECs separated from WT livers were cultured with H202,an in vitro setting that mimics ROS-mediated ischemia insult in liver grafts.By employing immunofluorescence,we detected TSGL-Ig triggered translocation of Nrf2 transcriptional activator into LEC nucleus,with or without concomitant H202.Consistent with the ability of Nrf2 to regulate the induction of ARE target gene promoter regions,TSGL-Ig increased the expression of Trx,GCL,NQO1,and HIF-la in H202-stressed LECs(p<0.001)as compared with controls.We then employed primary LECs from Nrf2 KO mice to analyze mechanism of TSGL-Ig mediated cytoprotection.Indeed,compared with Nrf2-proficient LEC cultures,those with disrupted Nrf2 signaling were more susceptible to H202-stress,as measured by ALT/LDH release.Pretreatment with TSGL-Ig failed to protect Nrf2-deficient LECs against H202-stress,consistent with comparable ALT and LDH release in WT and Nrf2 KO cell culture groups.7.Nrf2 Signaling is Required for TSGL-Ig Hepatoprotection Against IR-Stress We employed Nrf2 KO mice as liver donors in our IRI-OLT model(20 h of cold storage).Indeed,unlike in WT,TSGL-Ig failed to protect Nrf2 deficient OLT against IRI,as measured by sALT at 6h.In addition,TSGL-Ig failed to mitigate IR-triggered hepatocellular damage after disruption of Nrf2 signaling,as documented by liver H&E staining;LEC activation(P-/E-selectin);and macrophage cytokine programs(TNF-α,IL-1β).We used murine model of hepatic partial warm IRI to confirm that TSGL-Ig protection against IR stress is Nrf2-dependent.WT and Nrf2 KO mice subjected to 90 min of hepatic warm ischemia were pre-treated with TSGL-Ig or control Ig.WT mice given TSGL-Ig were IRI-resistant,as compared to controls,evidenced by decreased sALT levels;well-preserved hepatic architecture;as well as depressed LEC(P-/E-selectin);and macrophage(TNF-a,IL-1β)activation.Consistent with our findings in cold-stored OLT,TSGL-Ig failed to abolish IR-mediated hepatocellular damage in Nrf2-deficient warm IR-stressed livers,as documented by sALT levels;liver H&E staining;and hepatic IRI cytokine signature/selectin expression patterns.Conclusion;1.TSGL-Ig could improve OLT survival and ameliorates hepatic IRI,decreases IR-Induced liver oxidative stress and necrosis/apoptosis,TSGL-Ig would be a novel agent applied in the protection of liver transplant organs.2.TSGL-Ig mediates LEC cytoprotection in Nrf2-dependent manner and Nrf2 signaling is required for TSGL-Ig Hepato-protection Against IR-Stress.