The Mechanisms Underlying Abdominal Infections-induced Hyperbilirubinemia And NLRP3 Inflammasome-targeted Therapy

Severe sepsis is a multifactorial,progressive,and life-threatening clinical syndrome that results from innate immune response to infections.Abdominal infections-induced sepsis constitutes a major part.Despite advanced progress made in current therapies,such as fluid resuscitation,source control,and anti-microbial treatment,are insufficient for sepsis management.The sepsis-associated mortality and morbidity rates remain unacceptably high,with poorer outcomes for septic shock with organ dysfunctions.Liver injury is an early event,as reflected by hyperbilirubinemia within hours after sepsis.Unconjugated bilirubin is the breakdown product of hemoglobin.Within hepatocytes,it becomes conjugated bilirubin and flows into bile across the canalicular membrane transporters,or into blood across the basolateral membrane transporters.The hepatocyte transporters may be involved in the development of hyperbilirubinemia.It is established that IL-1? and IL-18 impair hepatocyte transporters directly.These cytokines have a highly preserved way of activation by inflammasomes,particularly the NLRP3 inflammasome.NLRP3 is involved in the recognition of exogenous and host ligands,then activating downstream caspase-1.Activated caspase-1 cleaves pro-IL-1? and pro-IL-18 to their active forms.It is noteworthy that triggering of NLRP3 inflammasome results in neutrophils infiltration in the organs.Hepatic hypoperfusion may also contribute to the liver injury.The microcirculation rather than the macrocirculation is implicated as the underlying culprit.As a novel technique.laser speckle contrast imaging(LSCI)has gained extensive attention.It allows continuous and real-time recordings of microcirculation with a larger areaUp until now,there is not yet consensus on the precise mechanisms underlying sepsis-induced hyperbilirubinemia.The dynamic alterations of hepatic microcirculation are obscure.The relationship between NLRP3 and sepsis-induced hyperbilirubinemia remains poorly understood.The therapeutic value of inhibiting NLRP3 is unknown.In this study,we first explored associations between hyperbilirubinemia and prognosis in patients with abdominal infections.Afterwards,we further investigated dynamic alterations of hepatic microcirculation during the sepsis by using laser speckle contrast imaging.Finally,we aimed to determine whether gene silencing of NLRP3 protects against sepsis-induced hyperbilirubinemia.Our experiments can be divided into the following three parts:PART I:Direct bilirubin as a prognostic biomarker in enteric fistula patients complicated with sepsisObjective:The objective of this study was to evaluate the predictive value of serial bilirubin determinations for mortality in enteric fistula(EF)patients complicated with sepsis.Methods:From January 1st.2012 to January 13rd.2013.a prospective study enrolling 162 patients was performed.Patients were divided into the survivors group(n=119)and non-survivors group(n=43)according to 28-day outcomes.Laboratory variables on day 0.day 3 and day 7 after admission were recorded.DB0 was defined as serum direct bilirubin(DB)value in admission,while ?DB3 as the changes from DB3 to DB0.The definition applied to other parameters.The results were validated in an independent cohort of 116 patients.Results:Compared with survivors,non-survivors had significantly higher DB7(23.1±10.6 vs.11.2±1.1,P<0.001)and procalcitonin(PCT7)(5.2±2.8 vs.1.7±0.3 P=0.006).ROC analysis showed that DB7>12.8 ?mol/L and ?DB7>7.3 ?mol/L were reliable predictors(DB7:86.4%sensitivity,88.6%specificity(area under the curve(AUC):0.881,P<0.001;?DB7:84.4%sensitivity,85.1%specificity,AUC:0.865,P<0.001)for mortality.The combination form(DB7>12.8 ?mol/L+?PCT7<5.3 ng/ml)had greatest predictive value(AUC:0.894,P<0.001).Their predictive values were confirmed in the validation cohort.Conclusions:Serum direct bilirubin was a reliable predictor for mortality in enteric fistula patients,which should be paid close attention in the critical care.PART ?:Laser speckle contrast imaging for measurement of hepatic microcirculation during the sepsisObjective:Sepsis is a fatal systemic inflammatory response syndrome caused by severe infection.The aim of this study was to measure hepatic microcirculation during the sepsis with laser speckle contrast imaging(LSCI),as well as investigating the underlying mechanisms.Methods:Sepsis was induced by cecal ligation and puncture.Rats were divided into the sham group and sepsis group.The hepatic microcirculation was monitored with LSCI.In addition,hepatic endothelial function(expression of cell adhesion molecules,coagulation and vascular permeability)and neutrophils accumulation in the liver were compared between the two groups.Results:During the sepsis,hepatic microcirculation decreased dramatically(290.3±70.1 LSPU(laser speckle perfusion units)at baseline vs.230.4 ± 60.7 LSPU at 12 h vs.125.2±25.4 LSPU at 48 h,P b 0.001).The rats developed hyperbilirubinemia since 6 h.In the early phase of sepsis,the accumulation of neutrophils and formation of microthrombus increased rapidly.In the late phase,hepatic neutrophils accumulation was already at its maximum level.Meanwhile,the endothelial coagulation status shifted from procoagulation to anticoagulation.The vascular leakage was involved in the microcirculation dysfunction since 12 h after sepsisConclusions:Hepatic microcirculation dysfunction occurs early during the sepsis and is associated with liver injury.This microcirculation dysfunction is due to neutrophil-endothelium interactions,microthrombus formation and vascular leakagePART ?:Gene silencing of NLRP3 protects against the sepsis-induced hyper-bile acidaemia and hyperbilirubinemia in a rat modelObjective:The role of NOD-like receptor family(NLRP3)has been confirmed in various inflammatory diseases.The association between NLRP3 and hyperbileacidaemia during the sepsis remains unclear.We aimed to investigate whether NLRP3 silencing protects against the sepsis-induced hyperbileacidaemia and hyperbilirubinemia.Methods:Sepsis was induced by caecum ligation and puncture(CLP).Gene silencing of NLRP3 was performed by injecting rats with NLRP3 short hairpin RNA plasmids(NLRP3 shRNA)48 h before surgery.Rats were divided into four groups:group 1 sham;group 2:sepsis;group 3:NLRP3 shRNA+sepsis(called the 'NLRP3 shRNA'group);and group 4:scrambled shRNA+sepsis(called the 'scrambled shRNA'group).The serum levels of bile acids and bilirubin,hepatic expression of hepatocyte membrane transporters,hepatic cytokine levels and behaviours of immune cells were compared among the groups.Results:Hepatic NLRP3 expression was increased dramatically during the sepsis,but was suppressed by pretreatment with NLRP3 shRNA.Compared with rats in the sepsis and the scrambled shRNA groups,rats in the NLRP3 shRNA group exhibited significantly decreased serum levels of glycine and taurine conjugated-bile acids,significantly decreased serum levels of conjugated bilirubin,with rehabilitated expression of hepatocyte transporters,suppressed hepatic cytokine levels,decreased hepatic neutrophils infiltration and attenuated macrophages pyroptosis.Conclusions:Gene silencing of NLRP3 ameliorates sepsis-induced hyper-bileacidaemia and hyperbilirubinemia by rehabilitating hepatocyte transporter expression,reducing hepatic cytokine levels,neutrophil infiltration and macrophages pyroptosis.NLRP3 may be a pivotal target for sepsis management.