The Effect And Mechanisms On Lipopolysaccharide Induced Macrophage To Release Exosomes Resulting In Acute Lung Injury

Backgroup and Objective:Acute lung injury(ALI)is a type of acute hypoxemic respiratory failure associated with damage of lung alveolar epithelial cell and capillary endothelial cell.ALI could develop into acute respiratory distress syndrome(ARDS),with about the mortality rate of 40%.Recently,the molecular mechanism of ALI remains unclear.Therefore,exploring the mechanism provides a novel pathway to intervene ALI in clinical trial and plays a vital significance on clinical value and social benefit.Recently,much research has focused on extracellular vesicles(EVs)that could mediate the intercellular communication between macrophage and alveolar epithelial cell in ALI,while the undering mechanism is unclear.In addition,our team has found that angiotensin(1-7)[Ang(1-7)],an important component of the angiotensin system,could inhibit the inflammation mediated by NLRP3 inflammasomes;inhibit the macrophage inflammation caused and counteract the organ damage by lipopolysaccharide(LPS).Hence,this study aim to explore the molecular mechanism on the intercellular communication between macrophage and alveolar epithelial cell with exosomes as the core in ALI;and the effect of Ang(1-7)on macrophage-derived exosomes-mediated ALI.Method:1.The extraction and identification of exosomes from mononuclear macrophages(RAW 264.7,RAWs)in mice:gather the supernatant from RAWs respectively under the stimulation of PBS,LPS and LPS plus Ang(1-7)after 24h.And then extract exosomes from supernatant via supercentrifugation;Identification of exosomes by method of NTA analysis,High resolution transmission electron microscope,and Western blot.2.In vivo,C57BL/6 mice were injected with these exosomes through the tail vein.Then detecting the morphological changes of mice lung,analysing the expression of NLRP3/ASC/Cleave-caspase-1 and the cleavage of GSDMD(one of marker proteins of pyroptosis)in tissue.3.In vitro,murine lung epithelial cell line(MLE-12)incubated with these exosomes was detected the secretory level of inflammatory cytokines(IL-1β,TNF-α),the expression of NLRP3/ASC/Caspase-1 and GSDMD.Western blot and qPCR were used to investigate the changes of NLRP3 released from exosomes by LPS-induced RAWs.4.Detection of the level of NLRP3 in exosomes and RAWs.Ang(1-7)was pretreated to RAW before the stimulation of LPS for 1h.5.Mice were injected with these exosomes extracted from RAWs under the stimulation of LPS pretreated with Ang(1-7)for 1h.MLE-12 co-cultured with exosomes released by RAWs under the stimulation of LPS pretreated with Ang(1-7)for 1h.Through the above two methods,detect the morphological changes in lung,analyse the expression of NLRP3/ASC/Cleave-caspase-1 and measure of the GSDMD-N protein.6.Statistical analysis Data were analyzed by SPSS 22.0 and results are presented as mean±SD.Significant differences were identified by AVOVA followed by multiple-comparsion testing(with the least-significant difference tests and Dunnett’s T3 method).P<0.05 were considered statistically significant.Results:1.Exosomes extracted from RAWs supernatant under the stimulation of LPS could cause ALI in model animals:the lung injury scores in the treatment of LPS-Exosome group is higher than other group;the level of W/D ratio of lung in model group is higher than other two group(*P<0.05 vs control;*P<0.05 vs CM-Exosomes);the total cells and inflammation factors(IL-1β、TNF-α、IL-6)in BALF were exceeded than other two group(*P<0.05 vs control;*P<0.05 vs CM-Exosomes).2.Exosomes extracted from RAWs supernatant under the stimulation of LPS could increase the expression of NLRP3/ASC/Cleave-caspase-1 and GSDMD protein.3.Exosomes extracted from RAWs supernatant under the stimulation of LPS were assimilated by MLE-12 resulting in the expression of inflammatory factors(IL-1β、TNF-α),activation of NLRP3 inflammasome and the cleavage of GSDMD.The above changes may be mediated by NLRP3 in exosomes induced by LPS.4.Ang(1-7)could supress the expression of NLRP3 in exosomes and RAWs.5.Ang(1-7)could down-regulate the expression of NLRP3 in exosomes,supress the activation of NLRP3 inflammasome and the cleavage of GSDMD in MLE-12 to relieve ALI.Conclusion:1.LPS-induced and NLRP3 enriched exosomes from RAWs triggers the cleavage of GSDMD via activating NLRP3 inflammasome involved in ALI2.Ang(1-7)could down-regulate the levels of NLRP3 in exosomes,supressed the activation of NLRP3 inflammasome and the expression of GSDMD to relieve ALI.