Role Of High Mobility Group Box-1 Protein Inducing Endothelial Cell Activation And Gut Mucosal Barrier Dysfunction During Murine Severe Acute Pancreatitis

IntroductionSevere acute pancreatitis(SAP) has become focus of clinical research because of its high mortality and severe features.SAP's pathogenesis is extremly complicated,and now it is believed to be a systemic inflammation involved with many kinds of inflammatory factors and cells,such as neutrophils,mononuclear macrophage,and endothelial cell.As more the more information about SAP has been obtained by clinicans and researchers,people gradually realized that systemic inflammation response syndrome(SIRS) and multiple organ failure(MOF) following SIRS are two main causes of death after SAP.Decreasing the severity of SIRS can reduce the possibility of MOF,therefore decreasing mortality.More and more evidence indicate that pro-inflammatory factors excreted by inflammatory cells are the key factor to lead to SIRS.Researchers suspect that monoclonal antibodies and antagonists to these cytokines may be benefit to the inflammation during SAP.Althougt later research prove these conclusions,therapeutic results are not ideal.Plenty of cytokines which can act and induce each other constitute a complex network,so the strategy that only acts on a single or several cytokines has resulted in limited success.How to regulate the production of cytokines and inhibit the development of SIRS and MOF is very important to management of SAP.Although there are numerous cytokines,signal transduction pathways that can regulate the production of these cytokines arc rare.Hfective inhibition or regulation to some key pathways may control the production of pro-inflammatory cytokines, furthermore,interrupt the development of SIRS and MOF.So,it is very important to investigate the role of signal transduction during the progression of severe acute pancreatitis.In severe acute pancreatitis(SAP),multiple organ dysfunction syndrome(MODS) in the early phase and complications of infection(infected pancreatic necrosis and sepsis) in the late phase are contributors to high mortality in SAP.MODS is a consequence of the systemic inflammatory response syndrome,and it is conceivable that release of humoral mediators from the excessive activated macrophages/ monocytes and neutrophils may lead to the remote organ injury.Complications of infection are thought to be a result of bacterial translocation from the gastrointestinal tract,and breakdown of intestinal integrity is considered to be implicated in the mechanism.High mobility group box 1(HMGB1) protein,originally discovered 30 years ago as a nuclear DNA binding protein,was recently identified as a late-acting mediator of endotoxin lethality.Injection of HMGB1 itself was lethal,and serum levels of HMGB1 increased from 8 to 32 h after the administration of endotoxin,when the tumor necrosis factor(TNF) peak had already occurred.Antibodies to HMGB1 attenuated the mortality associated with endotoxemia,even when the antibodies were administered 2 h after the onset of endotoxemia.HMGB1 was also found to have the capacity to induce cytokines and activate inflammatory cells when it was applied extracellularly. This implicates HMGB1 as a pro inflammatory mediator.Recent investigations reported that serum HMGB1 levels increased in patients with sepsis/endotoxemia, hemorrhagic shock,acute lung injury,rheumatoid arthritis and disseminated intravascular coagulation.It has been demonstrated that HMGB1 is secreted actively by living inflammatory cells such as stimulated macrophages/monocytes,and is released passively from necrotic or damaged cells.Therefore,HMGB1 may be related to infl ammation and necrosis in SAP,and may be an important mediator for multiple organ failure.A recent study has first demonstrated that serum HMGB1 levels were significantly elevated in patients with SAP on admission,and were correlated with the severity of the disease.The HMGB1 levels were higher in patients with organ dysfunction and infection during the clinical course.The HMGB1 levels in nonsurvivors were higher than those in survivors.These results suggest that HMGB1 may play a pivotal role in the pathogenesis of SAP,and that HMGB1 may act as a key mediator for inflammation and organ failure in this disease.In the present study,to clarify the role of HMGB1 in the pathophysiology of SAP,effects of anti-HMGB1 neutralizing antibody were investigated in SAP in mice.Severe acute pancreatitis(SAP) is characterized by the development of systemic inflammatory respond syndrome and multisystem organ failure as well as local pancreatic complications and is still associated with a mortality rate of 15%to 30%, despite continuing improvement in critical care.At present time,therapeutic efforts are limited to supportive treatments because no effective specific medical therapy has been successful.Although the exact mechanism of SAP is still not completely understood, SAP is manifested by inflammation and subsequently by an autodigestive process through the activation of pancreatic enzymes.The pathological sequelae of SAP are mediated by proinflammatory cytokines, including tumor necrosis factor,interleukin(IL),IL-6,and high mobility group box (HMGB) that are released from macrophage,neutrophils,and other cells of the innate immune system.The magnitude and duration of the systemic inflammatory response influence the development of tissue damage,multiple organ failure,and death.Tumor necrosis factor,IL-1,and IL-6 are released early in the development of systemic inflammatory response,but HMGB1 is a late mediator of lethal systemic inflammation in animal models of cytokine-mediated disease initiated by the gram-negative bacterial product endotoxin and released by macrophages only after a delay of 12 to 18 hours during endotoxemia. Anti-HMGB1 antibodies confer significant protection against delayed lethality of established sepsis.The transcription factor,nuclear factor(NF)-κB,is thought to play an important role in immune and inflammatory responses through the regulation of genes encoding proinflammatory cytokines.Ethyl pyruvate(EP),a small molecule that normally is regarded as a key intermediate in the oxidative or anaerobic metabolism of glucose,identified recently by Ulloa and colleagues,inhibits lipopolysaccharide(LPS) induced NF-κB activation in cultured RAW264.7 murine macrophagelike cells and HMGB1 release and TNF gene expression both in vitro and in vivo.Ethyl pyruvate also ameliorates ischemia/reperfusion induced intestinal mucosal injury in rats and ileus induced by bowel manipulation in mice.To date,there has not been a report of an experimental therapeutic agent that inhibits HMGB1 release to modulate systemic inflammation in SAP.Supported by these observations,we design this study to evaluate whether EP could be of benefit in rat model of sodium taurocholate induced SAP.We investigate its role in order to elucidate the pathogenesis of SAP on signal transduction level and provide new therapy strategy for SAP. ·Paper one·The relationship between high mobility group box-1 protein expression and gut mucosal barrier dysfunction during severe acute pancreatitisMethodsForty-eight male health adult wistar rats were divided randomly into groups: Control group,SAP groups.The concentration of plasma lipopolysaccharide(LPS) and the blood level of diamine oxidase(DAO) were determined.The expression of HMGB1 mRNA in intestinal mucosa was detected by reverse transcription polymerase chain reaction(RT-PCR) and the activity of HMGB1 was determined by western blot.ResultsPlasma LPS and the blood level of DAO reached a peak level at 24h(0.852±0.232) EU/ml for Plasma LPS and(1.36±0.22) kU/L for DAO respectively,p<0.01).Elevation of HMGB1 and HMGB1 mRNA expression in intestinal mucosa was found at 6 hour after SAP,HMGB1 and HMGB1 mRNA expression peaked at 24 hours and kept relative high values up to 48 hour compared with normal control group (p<0.01).ConclusionRetrograde infusion of sodium taurocholate via the pancreatic duct produced pancreatic necrosis.The rise of HMGB1 expression might play an important role in the Intestinal mucosal barrier injuries in SAP.HMGB1 as a late mediator involves in the pathogenesis of SAP. ·Paper Two·The Role of ethyl pyruvate against gut mucosal barrier dysfunction during murine severe acute pancreatitisMethodsSAP model was induced by retrograde injection of 5%sodium taurocholate into pancreatic duct.Animals were divided randomly into 3 groups:control group,SAP group,and EP treatment group.EP solution was administered intravenously every 6 hours(40mg/kg once).The mRNA expression of HMGB1 in bowel tissues was detected by reverse transcription-polymerase chain reaction(RT-PCR).The histological examination of the bowel was also performed.ResultsThe levels of D-lactate and LPS were rapidly increased after SAP model was induced,and reached peak at the 24th hour and kept relative high values up to the 48th hour compared with control group.In comparison with those in SAP group,the levels of D-lactate and LPS were markedly lowered in EP group 24 and 48 hours after SAP model was induced(P<0.05).The mRNA expression of HMGB1 in bowel tissues was increased significantly at the 12th hour and maintained to the 24th hour after SAP model was induced,whereas in EP group,HMGB1 mRNA expression was significantly lower than that in SAP group at each time point(P<0.05).The injury of bowel tissues in EP group was milder than that in SAP group.ConclusionEP can decrease the levels of D-lactate and LPS,down-regulate HMGB1 mRNA expression in intestinal tissues of SAP rats,and protect intestine from injury induced by SAP. ·Paper Three·Study on high mobility group box-1 protein inducing endothelial cell activation in vitroMethodsThe cultured human umbilical vascular endothelial cells(HUVECs) strain ECV-304 in vitro were divided into 3 groups:control group,HMGB1 groups,HMGB1 plus ethyl pyruvate group.The quantities of TNF-a secreted in ECV-304 cells were measured by sandwich ELISA,the activation of nuclear factor-κB(NF-κB) was observed by fluorescence microscope and the DNA binding activity of NF-κ3 in endothelial cells was measured with eletrophoretic mobility shift assays(EMSA).ResultIncubation with HMGB1 was found to result in rapid increment of NF-κB in cultured HUVECs.HMGB1 markedly induced the expression of TNF-a from HUVECs.Ethyl pyruvate treatment could decrease the activity of NF-κB and the expression of TNF-a.ConclusionHMGB1 could enhance transient mobilization of NF-κB and markedly induce the release of TNF-a in endothelial cells,which suggested HMGB1 may play an important role in the occurrence and development of sepsis.Ethyl pyruvate is a potent inhibitor of NF-κB activation induced by HMGB1 in endothelial cells,which might explain its beneficial effect in endothelial cells.