The Roles Of Kupffer Cells On Heme Oxygenase-1 Protecting Liver Against Ischemia/Reperfusion Injury In Rat Liver Transplantation

From the first report of Starzl,who performed clinical liver transplantation in 1963,orthotopic liver transplantation(OLT) is an effective treatment for end-stage liver diseases.Today,liver ischemia/reperfusion(I/R) injury is one of the confronted important problems and considered a major cause of graft injury,causing liver dysfunction and even failure post transplantation.The damage to the liver caused by I/R represents a continuum of processes that culminate in hepatocellular injury.These processes are triggered when liver is transiently deprived of oxygen and subsequent to reoxygenation,which inflicts direct tissue damage and initiates a cascade of deleterious cellular responses with concomitant release of oxygen-free radicals(OFR) and cytokines leading to cellular injury that culminates in the ultimate cell death and graft failure.OFR is one of the earliest and most important components of tissue injury after reperfusion of ischemic organs.The prime sources of OFR production in ischemic livers include cytosolic xanthine oxidase(XO),Kupffer cells(KCs) and adherent polymorphonuclear leukocytes(PMN).Endothelial cell(EC) damage results from free radicals produced from KCs and adherent PMN.Ultimately,this results in loss of microvascular integrity and reduction of blood flow('no-reflow phenomenon'). However,at present there is no treatment available to prevent liver I/R injury.Studies have shown that heme oxygenase-1(HO-1) exerts a cytoprotective function in a number of liver I/R injury.However,exact mechanisms by which HO-1 induction may lead to cytoprotection during I/R injury in organ transplantation have not been fully clarified.Three HO isoforms have been identified:inducible HO-1, also known as heat shock protein 32;constitutively expressed HO-2;and a related but less well-characterized HO-3.HO-1 is the rate-limiting step in the oxidative degradation of heme into biliverdin,carbon monoxide(CO) and free iron.Biliverdin is reduced to bilirubin(BR) by bilirubin reductase,and the free iron used in intracellular metabolism or sequestered in ferritin.Studies have also reported that CO protects graft livers against cold I/R injury associate with prolonged cold preservation. HO-1 is induced in a variety of organs during diverse stress-related conditions and is thought to provide cytoprotection.Thus,HO-1 is attractive target for anti-inflammatory therapies and potential candidate responsible for organs I/R injury.Kupffer cells,the resident macrophages of the liver,are the largest population of tissue macrophages and predominantly distributed in the lumen of hepatic sinusoids. KCs account for approximately 10～15%of all liver cells in number and for 80%of resident macrophages in the whole body.During the initial stages of reperfusion KCs are activated,producing morphologic changes that cause them to protrude into the sinusoids,contributing to the reduction of blood flow within the sinusoidal lumen. Activated KCs release a large amount of biologically active mediators such as proinflammatory and reactive oxygen species(ROS).Multiple lines of evidence have suggested that Kupffer cells are critical to the onset of liver I/R injury.Studies have shown that liver I/R injury can be attenuated by the suppression of KCs.Because activation of KCs results in the release of an array of inflammatory mediators,growth factors and ROS.Immunochemical studies with specific monoclonal antibodies have revealed that HO-1 express principally in Kupffer cells.However,little is known about the roles of KCs in HO-1 protecting liver cold I/R injury.In the present study,we investigated the roles of KCs and protective effect of HO-1 in liver cold I/R injury model.We'll provide rationally experimental evidence accordance with this study for further research.Methods: 1.Ex vivo rat liver tissue was perfused with collagenaseⅣand diced into pieces. The diced tissue was digested for 30 min at 37℃and following centrifuged to remove hepatocytes.Percoll density gradient centrifugation and selective adherence were combined.The cells were seeded onto tissue culture plates at a density of 2×10~6/ml and cultured in RPMI 1640 medium.Phagocytosis of ink and ED2 immunocytochemistry were used for cell identification.2.Male Sprague-Dawley(S-D) Rats weighing 220-250g were used and divided randomly into three groups:①Control group:no drugs were applied;②ZnPP treatment group:donors received ZnPP,an HO-1 inhibitor(i.p.) 24h prior to harvest;③CoPP treatment group:donors received CoPP,an HO-1 inducer(i.p.) 24h prior to harvest.All liver grafts were harvested and stored with saline solution for 90min at 4℃,and orthotopically transplanted into syngeneic S-D recipients.Separate groups of rats were killed at 6h after their vessels were unclamped,and liver samples were collected for further analysis.Kupffer cells were isolated from integral left liver tissue obtained from fresh specimens and were incubated for 48h.The supematant from Kupffer cells culture was collected,and total RNA or proteins were extracted from Kupffer cells.HO-1 expression in livers pretransplant was assessed,and the hepatocellular function(ALT,AST) was analyzed.The hepatocellular damage was evaluated by liver histology.The IL-6/TNF-αlevels of plasma and supematant were detected.CD14 mRNA expression levels of KCs were examined by RT-PCR and Western blot.Results:1.The average cell yield per gram of liver before plastic adherence was 2.1±0.3×10~6,and 1.5±0.1×10~6 following plastic adherence.The viability of the KCs after isolation was＞92%as determined by trypan blue exclusion.The purity of KCs identified by ED2 was＞90%.Cultures of isolated KCs were functionally intact and expanded irregularly.2.Livers harvested from donors that were pretreated with CoPP significantly up-regulated HO-1 mRNA expression levels as compared with Control or the ZnPP group.ZnPP treatment decreased HO-1 mRNA as compared with Control.Group pretreatment with CoPP significantly decreased Serum ALT/AST levels.Serum AST/AST levels were also significantly increased in the ZnPP treatment group as compared with both the Control and the CoPP group.The hepatocellular damage was evaluated by liver histology.Livers in ZnPP group showed a more severe hepatocyte necrosis,sinusoidal congestion and ballooning.In contrast,livers in CoPP group revealed almost complete preservation of lobular architecture.Plasma and supernatant cytokines(IL-6/TNF-α) concentrations were detected.ZnPP treatment significantly increased after reperfusion.However,the IL-6/TNF-αlevels in CoPP pretreated group were significantly lower,when compared to the Control group and ZnPP group.HO-1 mRNA expression levels of Kupffer cells with ZnPP pretreated were significantly down-regulated,and those with CoPP pretreated were up-regulated.A prominent increase of CD 14 mRNA and protein levels in Kupffer cells was detected in the ZnPP group as compared with the Control group or the CoPP group.Conclusions:1.The method of isolating and culturing Kupffer cells in this study is convenient, efficient and stable.The biological characters of the cultured Kupffer cells were retained,and the cells can be used to further study.2.HO-1 protects liver against I/R injury in rat liver transplantation,ameliorating hepatic damage,suppressing cytokines IL-6 and TNF-αrelease and improving liver function.It is possible that HO-1 inhibiting Kupffer cells activation is one of the mechanisms.