Study Of The Damage Effect Of PM2.5 On Airway Epithelial Cells Based On Pyroptosis

Objective Different sterilization methods were used to establish the model of PM2.5exposure injury to airway epithelial cells in vitro,which confirmed the occurrence of a new type of programmed death-cell pyroptosis in airway epithelial cells.Evaluate the effects of PM2.5 on airway epithelial inflammatory factors and pyroptosis,and to predict the correlation between the occurrence and development of asthma and pyroptosis by bioinformatic analysis.Methods PM2.5 was collected from Guangzhou and divided into two groups: ultraviolet sterilization and high-pressure sterilization.The components were analyzed by inductively coupled plasma mass spectrometry and ion chromatography.PM2.5 suspension was prepared and divided into low concentration group(100μg/ ml)and high concentration group(200μg/ml)to stimulate human normal bronchial epithelial BEAS-2B cell line: 1)the effect of PM2.5on cell proliferation was determined by CCK8 method,and the response curves of different concentrations and time were drawn;Cell death was observed by light microscope and flow cytometry;2)Scanning electron microscope(SEM)was used to observe the morphology of pyroptosis: whether there were cell swelling,cell membrane rupture and release of cytoplasmic contents;3)RT-PCR and Western blot were used to detect the expression of pyroptosis pathway NLRP3,caspase-1,-4,-5,GSDMD,IL-1β,IL-18 while inflammation factor TNF-α and IL-10 m RNA was detected;4)The GEO database was mined to screen differentially expressed genes,and the correlation between asthma and pyroptosis was predicted by functional enrichment analysis.Results There was no significant difference in the composition of PM2.5 water-soluble ions,heavy metals,total organic carbon and carbon components between UV sterilization and high-pressure sterilization.Bioinformatic mined NLRP3 as one of the up-regulated genes in asthma.24 hours after different concentration PM2.5 stimulate airway epithelial: 1)compared with the control group,the death of airway epithelial cells was increased in the experimental group under light microscope,CCK8 and flow cytometry;2)Compared with the control group,IL-10 was detected by RT-PCR(P = 0.0024;P=0.0028)、TNF-α(P=0.0034;P=0048);Western blot showed that TLR4,PARP,COX2,p-p38 and Bcl-2 were activated in UV group,while NF-κB was activated in HP group as dominant;3)Scanning electron microscopy showed that the cells in the experimental group were uneven,which was consistent with the morphological changes of pyroptosis;Compared with the control group,the expression of RT-PCR(NLRP3,IL-1β、IL-18,caspase-1,caspase-4,Caspase-5 and GSDMD)was increased,but there was difference in multiple;Western blot showed that the activation of NLRP3 was predominant in UV group,while caspase-1,IL-1β and Cleaved GSDMD were predominant activated in HP group.Conclusion 1)PM2.5 can induce airway epithelial cell death,inhibit cell proliferation and activate inflammatory factor TNF-α IL-10 m RNA and NF-κB inflammatory pathway signaling.PM2.5 can induce NLRP3/caspase-1-mediated pyroptosis,however,there may be some differences in the above results due to different sterilization methods of PM2.5;2)NLRP3,as one of the key up-regulated genes in GSE137268 dataset,is involved in the development of asthma.