Role Of NLRP3 Inflammasome In Regulating IL-1β And IL-18 Release In Human A549 Cell Lines

Acute lung injury and acute respiratory distress syndrome(ALI/ARDS)are a common clinical critical syndrome.Despite considerable progress has been made in the treatment technology,including lung-protective ventilation and supportive care,the case fatality rate is still high,maintain at about 40%.The pathogenesis of ALI / ARDS has not been fully elucidated,but dysregulated inflammation plays a key role in the development of this complex syndrome.However,the mechanisms of inflammatory mediators production and release are still needed to be solved.IL-1β and IL-18 are important inflammatory cytokines in the development of ALI/ARDS,the present study shows that the release of IL-1β and IL-18 are regulated by NLRP3 inflammasome pathway.NLRP3 inflammasome is a multiple protein complex in the cytoplasm,which consist of nucleotide-binding oligomerization domain-like protein 3(NLRP3)、 apoptosis-associated speck-like protein containing CARD(ASC)and cysteinyl aspartate specific proteinase-1(caspase1).NLRP3 inflammasome can transform into the form of activated from the that of stabile when under the stimulation of a variety of pathogen-associated molecular pattern(PAMP),such as lipopolysaccharide,or damage-associated molecular pattern(DAMPs),such as adenosine triphosphate,which then promotes the release of IL-1β and IL-18.It has been shown that alveolar macrophages,neutrophils and endothelial cells are regulated by NLRP3 inflammmasome pathway,and its activation was involved in the development of acute lung injury.However,whether NLRP3 inflammasome pathway exists in alveolar epithelial cell(AEC)remains pooly understood,and whether it can be activated and contribute to release of inflammatory cytokines-IL-1βand IL-18 under the stimulation of the common activator of NLRP3 inflammasome still remains to be explored.So,in this study,We chose A549 cell lines as the experimental object and to investigate whether NLRP3 inflammasome pathway exists in A549 cell lines,as well as to explore the expression of NLRP3 inflammasome pathway proteins and the secretion of cytokines-IL-1β and IL-18 under the stimulation of lipopolysaccharide(LPS)or lipopolysaccharide(LPS)combined with adenosine triphosphate(ATP),in order to illustrate whether the release of IL-1β and IL-18 were regulated by the NLRP3 inflammasome pathway.Objective:The aim of this study was to investigate the expression of NLRP3 inflammasome pathway proteins and the secretion of cytokines-IL-1β and IL-18 in A549 cell lines under the stimulation of LPS or LPS combined with ATP,preliminary understanding the effect and mechanism of NLRP3 inflammasome pathway in human A549 cell lines.Methods:1.The mRNA and protein level of NLRP3/ASC/caspase-1 in A549 cell linesThe mRNA and protein level of NLRP3/ASC/caspase-1 in A549 cell lines were assessed by reverse transcription polymerase chain reaction(RT-PCR)and Western blot,respectively.2.Effects of LPS on the mRNA and protein expressions of NLRP3 inflammasome pathway in A549 cell linesThe effects of different concentrations(0,1,5,10,50,100μg/ml)and different times(0,3,6,12,24,48h)of LPS stimulation on the mRNA and protein expressions of NLRP3 inflammasome pathway were detected by RT-PCR,Western blot,respectively.According to the experimental results,select the optimal stimulation time and concentration of LPS.3.Effects of LPS combined with ATP on the proteins expression of NLRP3 inflammaome pathway in A549 cell linesAccording to the proteins expression of NLRP3 inflammasome pathway in A549 cell lines under the stimulation of LPS,LPS 10μg/ml was selected to prime the A549 cell lines for 6 hours firstly,and then the different concentrations of ATP(0,0.5,1,2,4 mmol/L)stimulated A549 cell lines 1 hour,the activation of NLRP3 inflammasome pathway of A549 cell lines was detected by western blot.4.Effects of different stimulation conditions on the caspase-1 activity and the secretion of IL-1β and IL-18 in A549 cell linesThe experiment was divided into control group,LPS group,ATP group and LPS combined with ATP group,caspase-1 activity was detected by Caspase1 Activity Assay Kit,and the proteins expression of NLRP3 inflammasome pathway and the secretion of IL-1β and IL-18 in A549 cell lines were determined by western blot and ELISA,respectively,5.Effects of down-regulation NLRP3 on the secretion of IL-1β and IL-18 induced by LPS combined with ATP in A549 cell linesA549 cell lines were exposed to NLRP3-siRNA to down-regulation NLRP3 gene expression,than the secretion of IL-1β and IL-18 induced by LPS combined with ATP in A549 cell lines were determined by ELISA.Results:1.The mRNA and protein expressions of NLRP3,ASC,caspase-1 were observed in human A549 cell lines.2.Compared with the control group,the expression of NLRP3 m RNA,caspase-1mRNA and NLRP3、caspase-1、pro-IL-1β proteins in A549 cell lines were increased(P<0.05)in a time-and dose-dependent manner after stimulated by LPS,but LPS can not induce the expression of caspase-1p20 protein and the secretion of cytokines-IL-1β and IL-18(P>0.05).3.Compared with the control group,ATP had no influence on the expression of NLRP3、caspase-1 p45、caspase-1p20、pro-IL-β proteins and the secretion of cytokines-IL-1β and IL-18(P>0.05).4.Compared with the LPS stimulation group,the expression of caspase-1p20 protein and the secretion of IL-1β and IL-18 were increased in the co-stimulation of LPS and ATP group(P<0.05).The caspase-1 activity of A549 cells was enhanced in the co-stimulation of LPS and ATP group.5.The secretion of IL-1βand IL-18 were inhibited in the co-stimulation of LPS and ATP group after down-regulation the gene expression of NLRP3(P<0.05).Conclusions:1.The NLRP3 inflammasome pathway is present in A549 cell lines.2.LPS could upregulate the expression of NLRP3 mRNA、caspase-1mRNA and NLRP3,caspase-1,pro-IL-1β proteins,but could not activate the NLRP3 inflammasome.ATP could not upregulate the expression of NLRP3 inflammasome pathway proteins,also could not activate the NLRP3 inflammasome.3.LPS combined with ATP stimulated the A549 cells could activate the NLRP3 inflammasome,inducing the expression of caspase-1 p20 protein,release of proinflammatory mediators-IL-1β and IL-18,the enhanced of caspase-1 activity,suggesting that the activation of NLRP3 inflammasome in A549 cell lines may need two different signal stimulations.4.Down-regulation the expression of NLRP3 in A549 cell lines could significantly decrease the release of cytokines-IL-1β and IL-18 under the stimulation of LPS combined with ATP,suggesting that the NLRP3 inflammasome pathway regulated the release of IL-1β and IL-18.In conclusion,this study preliminary observed and explored the expression and regulation mechanism of NLRP3 inflammasome pathway in human A549 cell lines,which will provide some evidence for the further research the role and mechanism of NLRP3 inflammasome on the alveolar epithelial cells during the development of pulmonary inflammatory disease,such as ARDS.