The Mechanism Study Of Chloroquine On Anti-inflamatory Effect By Inhibiting NLRP3 Iflammasome

Background and Objectives:Sepsis is a clinical syndrome caused by infection.Main clinical manifestations of sepsis is immune disorder caused by multiple organ dysfunction.Sepsis is one of the primary factors of clinical critically ill patient death,but there is no reliable and effective prevention and control drugs used in treatment.Researches have confirmed that excessive inflammatory reaction is leading cause of sepsis and multiple organ dysfunction.Therefore,antagonism of key link of inflammatory response is important direction of drugs development for sepsis the treatment.Inflammasome is a new type of inflammation regulate platform discovered in recent years,play a crucial regulatory role in inflammation.The NLRP3 inflammasome is the most widely studied one of inlfammasome family.Recently found that the activation of NLRP3 closely associated with sepsis.Studies show that many kinds of PAMPs induced by sepsis can activate the NLRP3 inflammasome,such as LPS and PGN.NLRP3 inflammasome induced IL-1 family cytokine mature to release,and induced immune cells pyrotosis,and play a crucial roles in sepsis when it had been activated excessively.NLRP3 inflammasome activation followed two steps,prime transcription and protein complex assembly.Pathogen-associated molecular patterns(PAMPs)were recognized by pattern recognition receptor(PPRs)to activate intracellular downstream signal transduction pathways to prime NLRP3 transcription,and was recognized as the first signal(signal 1),such as LPS.LPS was recognized by Toll-like receptor 4(TLR4)to activate NF-?B and MAPK signal transduction pathway to prime pro-inflammatory cytokines and NLRP3 transcription.Other activators promoted protein complex to assemble,were recognized as the second signal(signal 2),such as ATP.The mechanism of Signal 2 promoted protein complex to assemble is unclear,but potassium efflux model get more recognition in recently.Potassium efflux by ATP mediated NLRP3,ASC and caspase-1 assembled into protein complex to activate downstream of NLRP inflammasome.Chloroquine are widely used as anti-malarial drugs in clinical firstly,in recent years,also have application in autoimmune diseases recently,such as rheumatoid arthritis and systemic lupus erythematosus.We reported that chloroquine had protective effects on sepsis mice,and is closely related with anti-inflammatory effect in TLR4 pathway.In the follow-up study,we also found that chloroquine restrain the inflammation effects by influence the protein ubiquitin system.These results prompt chloroquine might have multiple inflammatory mechanisms.Wether the protective effect of Chloroquine on sepsis mice associated with activation of NLRP3 inflammasome,Specific molecular mechanism was related to signal 1 and signal 2.There was no related research on this.So,this study first to clear the effect of chloroquine on NLRP3 activation in sepsis mice.And then studied regulation of protein transcription and protein complex assembly in macrophage model in vitro.Eventually we finger out regulation function and molecular mechanism of chloroquine on NLRP3 inflammasome activation,provide experimental basis for expanding chloroquine indications,as well as provide new ideas on sepsis drug research based on the inhibition of inflammasome.Methods:1.Chloroquine inhibited NLRP3 inflammasome and its protection of sepsis mice in vivo research1.1 sepsis mice mode were set up by tail vein injection of endotoxin.Seven days survival rate was record after giving 30 mg/kg or 10 mg/kg dose chloroquine ip.Serum AST and ALT concentration were detected in LPS attacked mice peripheral blood at time point of 6 h and 24 h.Organ damage were detected by histopathological observation after tissues HE staining in liver,spleen,lungs at the same timepoint.1.2 Peripheral blood and Peritoneal lavage fluid were collected respectively at 6 h,12 h and 24 h after LPS attacked.IL-1 beta and IL – 18 released in these samples were detected by ELISA.At the same time,the iver,spleen and lung tissue homogenate was collected for IL-1 beta,IL – 18,NLPR3 expression and activation of caspase-1.2.The mechainism research of chloroquine on inhibition of NLRP3 inflammasome2.1 F4/80 positive cells rate was determined by confocal and flow cytometry instrument in bone marrow derived macrophages(BMDMs)in vitro.The IL-1 beta and IL-18 release were detected in supernatant of cells be treated with different dose chloroquine.ASC,pro-caspase-1 and caspase-1 p10 expression level in cell lysis and supernatant were detected by western blot(WB).LDH release rate was detected at different time points from ATP.The effect of chloroquine on IL-1? release induced by other NLRP3 agonist was detected by ELISA.2.2 IL-1? and IL – 18 precursor in cell lysis and their mature form in supernatant were detected by ELISA.The effect of chloroquine on LPS primed transcription of IL-1?and IL-18 were determined by RT-q PCR in mRNA transcritption.Further detected the influence of chloroquine on NLRP3 gene transcription level and protein expression level.The effect of chloroquine on LPS primed nf-kappa B p65(NF-?B p65)activation levels,I kappa alpha(I?B ?)phosphorylation and I?B ? degradation was determined by WB.Then NF-?B p65 nuclear translocation were observation by confocal.ERK,P38 and JNK phosphorylation were detected by WB to determine the effect of chloroquine on MAPK pathway.2.3 IL-1? released were detected by ELISA when change the onset time or remove of chloroquine.The effect of chloroquine on LPS primed “ASC specks” formation were determined by confocal to calculate the percentage by ASC specks positive cells.Intracellular potassium ion concentration was detected to determine the ffect of chloroquine on potassium enfflux which is crucial for NLRP3 assmebly.Differentially expressed genes in chloroquine or LPS treated macrophage was determined by Affymetrix gene expression profile chip test.Potassium ion metabolism related genes were determined by GO enrichment analysis.After siRNA interferenced ATP1B3 expression,the NLRP3 inflammasome activation and downstream effects was detected to confirm that ATP1B3 participate in inhibition of NLRP3 inflammasome of chloroquine.Results:1.Chloroquine inhibited NLRP3 inflammasome and its protection of sepsis mice in vivo research1.1 Survival rate results show that chloroquine significantly improved sepsis mice survival rate(P=0.0155).Lung of mice treated with LPS show visible diffuse hemorrhage,edema,with scattered inflammatory cells invasion,these syndrome were alleviated by chloroquine.Serum ALT and AST were increased dramatically after LPS treated,and were decreased by chloroquine.These results show that chloroquine has protective effect on thesepsis model mice.1.2 In serum,IL-1?and IL-18 release increased dramatically in LPS treated mice,and were inhibited by chloroquine at 6h,12 h and 24 h.At the same time,similar results was found in peritoneal lavage fluid.Total IL-1 beta and IL-18 levels significantly raised in the liver and lung tissue of LPS treated mice,and has been inhibited by chloroquine.Caspase-1 was activated by LPS,and was decreased by chloroquine.And,NLRP3 expression rised in LPS mode mice,decreased in chloroquine mice.These results suggested that chloroquine inhibited NLRP3 inflammasome activation and downstream of NLRP3 inflammasome in sepsis model mice.2.The mechainism research of chloroquine on inhibition of NLRP3 inflammasome2.1 CQ decreased the release of IL-18 and IL-1? in LPS and ATP primed BMDMs in dose-dependently manner.Caspase-1 activation was improved by ATP in LPS primed BMDMs,inhibited by chloroquine without changed pro-caspase-1 and ASC expression.LDH release improved quickly by ATP in LPS primed BMDMs within two hours,chloroquine decreased LDH release significantly.Chloroquine also inhibted NLRP3 inflammasome activation by nigercin.Similar phenomenon was observed in murine peritoneal macrophages.These show that chloroquine inhibted NLRP3 inflammasome activation by ATP in LPS primed macrophage.2.2 Pretreated with chloroquine decreased the mRNA of pro-IL-1? and pro-IL-18 which were increased dramatically by LPS.LPS activated NLRP3 transcription and expression,which was inhibited by chloroquine.Chloroquine significantly inhibited the rise effect on NF-?B p65(ser536)and I?B ? phosphorylation mediated by LPS.Chloroquine also decrease I?B ? degradation induced by LPS.NF-?B p65 translocation were detected by confocal,chloroquine dampened NF-?B p65 induced by LPS.Chloroquine inhibited activation of MAPK signaling pathways induced by LPS,leaded to decrease in ERK,JNK and p38 phosphorylation level.These results show that chloroquine inhibited signal 1 activated NLRP3 transcription by decrease NF-?B and MAPK signaling pathways.2.3 Chloroquine inhibited IL-1? release even added at signal 2 activated period,as well as removed pre-added chloroquine by wash.High concentrations of potassium significantly enhance the inhibiting effect of chloroquine on IL-1? release.ATP decreasedIntracellular potassium ion concentration in LPS primed BMDMs,and chloroquine inhibted potassium efflux induced by ATP to improve Intracellular potassium ion concentration.This result show that chloroquine inhibited signal 2 activtated NLRP3 inflammasome.Differentially expressed genes related with Potassium metabolism in Affymetrix expression profile chip show that ATP1B3 which was decreased by LPS treatmen was improved significantly by chloroquine treatment.After si RNA interference effectively ATP1B3 protein expression in cells,IL-1? release increased dramatically by ATP in LPS primed DMDMs.Both IL-1? release and ASC specks are decreased by chloroquine pretreatment,but reverse effect happened by interference ATP1B3 expression.These results show that chloroquine up-regulate ATP1B3 expression,inhibited potassium efflux,and dampened NLRP3 inflammasome assembly.Conclusions:1.Chloroquine played a protective role in sepsis animal models,inhibited NLRP3 inflammasome activation,inhibited caspase-1 activation,and IL-1 family cytokines release in vivo.2.Chloroquin inhibited NLRP3 inflammasome activation induced by ATP in LPS primed macrophage,thus blocked IL-18 and IL-1? mature and release,and dampen caspase-1 induced pyrotosis.3.Chloroquine inhibited NLRP3 inflammasome activation via affecting on transcription and assembly of NLRP3 protein complex.Chloroquine inhibited components of NLRP3 inflammasome transcription priming by signal 1 via blocking MAPK and NF-kB pathway.Chloroquine negative regulated NLRP3 inflammasome complex assembled via up-regulated ATP1B3 and inhibited potassium efflux.