NLRP3 Plays A Significant Role In Acute Lung Injury Model In Mice Challenged With Seawater Exposure And Drug Intervention Study

BackgroundDrowning is a major cause of unintentional injury death.Seawater drowning occurs frequently in agricultural and industrial production,military action and everyday life at sea.Acute lung injury(ALI)is a serious body injury induced by seawater aspiration.Recently more and more evidence shows that pattern recognition receptors play a key role in non-infectious lung diseases such as chronic obstructive pulmonary disease,pneumoconiosis and asthma.NOD-like receptor protein 3(NLRP3),an important member of the NOD like receptors,stimulate the activation of caspase-1 and promote the release of IL-1β.NLRP3/caspase-1/IL-1β involved in endotoxin-induced acute lung injury and pulmonary fibrosis.The role of NLRP3 in seawater drowning remained unknown.Whether dexamethasone attenuate the acute lung injury caused by seawater drowning via inhibiting NLRP3/caspase-1/IL-1β is unclear.Objective1.To determine the expression of NLRP3 in mouse lung of pulmonary edema induced by seawater drowning.2.To investigate the therapeutic effect of dexamethasone in acute lung injury caused by seawater drowning via NLRP3/caspase-1/IL-1β signaling pathway.Method:1.Animal experiment1)After the C57 WT mice being anesthetised,seawater(4ml/kg)or saline was instilled into both lungs.Histological sections of mice lungs were obtained from 0h,1h,2h,4h and dexamethasone treatment group to evaluate the pathology.TNF-α,IL-6 and IL-1β content in lungs from 0h,1h,2h,4h and dexamethasone treatment groups were measured by ELISA method.Expression of NLRP3,caspase-1 and IL-1β protein in lungs harvested at indicated time were evaluated by western blot.Expression of NLRP3 m RNA,caspase-1m RNA and IL-1β m RNA in lung were also analyzed by real-time PCR were exposed to seawater by tracheal instilling.2)NLRP3 KO mice and WT mice were exposed to seawater(4ml/kg)by intratracheal instillation.Lung tissue were harvested 4 hours later for further analysis to evaluate the degree of lung injury.TNF-α,IL-6 and IL-1β content in lungs were determined by ELISA method and expression of NLRP3,caspase-1 and IL-1β were evaluated by western blot.Expression of NLRP3 m RNA,caspase-1 m RNA and IL-1β m RNA were analyzed by real-time PCR.2.Cell experiments1)The primary epithelial cells isolated from C57 WT mouse lungs were treated with seawater in vitro to SW-ARDS cell models.Cells were grouped into control,seawater stimulation 3h and 6h groups.Specimens were collected at indicated time points.NLRP3 protein,caspase-1 protein and IL-1β protein in lung were analysed by western blot analysis.2)The primary epithelial cells were grouped into control,dexamethasone intervention3 h and 6h groups.Primary epithelial cells were treated with dexamethasone at 25%concentration after exposure to seawater or DMSO.Expression of NLRP3 protein caspase-1 protein and IL-1β protein in lung were determined by western blot.Results:1.Animal experimentAfter seawater instillation,the pathological changes of lung tissue gradually increased over time and the pathological damage in SW 4h group was most serious.The pathological score of lung tissue in NLRP3 KO group was significantly decreased compared with SW4 h group(5.200±0.3742 vs.9.900±0.7071,P=0.0014).The pathological score in DXM group was decreased compared with SW 4h group(6.000±0.3162 vs.9.900±0.7071,P=0.0047).The expression levels of TNF-α,IL-6 and IL-1β in lung tissue using ELISA increased over time,while those in lung tissue of NLRP3 KO group were significantly decreased compared with SW 4h group(P values were0.0001,<0.0001,0.0015,<0.0001,respectively),and the inflammatory factors of lung tissue in DXM group were also significantly lower SW 4h group decreased(P values were0.0020,<0.0001,0.0040,respectively).The expression of NLRP3 m RNA,caspase-1m RNA and IL-1β m RNA and protein level were remarkably higher in seawater group than in control group by RT-PCR and western blot.The protein and m RNA expression of NLRP3,caspase-1 and IL-1β in the lung tissue of the NLRP3 KO group were significantly reduced.Compared with WT mice treated with seawater,Co-administration of dexamethasone with seawater decreased the m RNA and protein levels of NLRP3,caspase-1 and IL-1β.2.Cell experimentsUp-regulation in NLRP3,caspase-1 and IL-1β in the primary epithelial cells was detected by immunoblotting analysis.The levels of NLRP3,caspase-1 and IL-1β protein decreased in the dexamethasone intervention group.Conclusion:1.Acute lung injury induced by seawater drowning activated the NLRP3/caspase-1/IL-1βsignaling pathway.2.NLRP3 gene deficiency alleviated acute lung injury induced by seawater drowning.3.Dexamethasone alleviated the acute lung injury after seawater drowning by downregulating the expression of NLRP3/caspase-1/IL-1β signal pathway.