The Effects Of PM_2.5 On Airway Epithelial Cells' Injury And Secretion Of Inflammatory Factors And Barrier Function

BackgroundRecently,epidemiological data revealed that respiratory diseases such as asthma,chronic obstructive pulmonary disease,bronchial lung cancer,interstitial lung disease and other disease incidence increased year by year,which had high correlation with air pollution.Besides,long-term exposuring to air pollutants will also lead to a decrease in lung function.The fine particulate matter massive exposured in a short time increase the emergency hospitalization rate,which had enlarged sharply medical resource demand pressure.The Global Initiative for Chronic Obstructive Lung Disease updates guidelines on 2018,According to the results of a cross-sectional study conducted by Professor Ran Peixin of the Institute of Respiratory Diseases in Guangzhou,China,and a meta analysis of PM2.5,PM10 and COPD by Professor Xu Jinfu of Shanghai Lung Hospital,added new contents about air pollution particles exposed will affect the progression of COPD and some other contents according to a cross-sectional study of professor Ran from China on 2017,which found that PM2.5 and PM10 levels had correlation between prevalence rate of COPD.The guide reminds the necessity of strict control of fine particulate pollution Worldwide.PM2.5(atmospheric particle dynamics with diameter less than 2.5um)because of its small size and long residence time in the air,there is deposition of specific characteristics,there were into human body through respiration easily,which transport and deposition to the bronchioles interfering with alveolar surfactant and lung gas exchange,thereby,endangering human health and leading the respiratory system disease.As the first level of lung tissue contact with the external environment,airway epithelial cells play the primary role of site PM2.5.According to the pathogenicity research of PM2.5,outside experiments reveal the pathogenicity mechanisms of PM2.5 mainly induced by oxidative stress and antioxidant genes following the occurrence and development of airway injury caused by irreversible injury and diseases related to inflammatory injury.Research objectiveAirway epithelial cells are the first barrier of respiratory system and an important target of PM2.5.Does PM2.5 induce airway inflammation through the pathway of airway epithelial cell injury leading to the occurrence and development of bronchial asthma?The aim of this study was to establish an in vitro culture system in which PM2.5 acted on airway epithelial cells(ordinary culture system and transwell culture plate mimic normal airway gas contact system).Th2 cytokines and anion currents were produced around the survival of airway epithelium cells.The injury and function of airway epithelial cells induced by PM2.5 were studied.Research contents1.Through respiratory tract,Does PM2.5 affect the survival of airway epithelial cells?2.Whether PM2.5 induces airway inflammation by afftecting on airway epithelial cells and inducing secretion of Th2 type inflammatory mediators?3.PM2.5 plays an important role in airway epithelial cells,which can destroy airway epithelial cells as an airway barrier function.Method1.Collection of PM2.5 and preparation of suspensions:a large flow atmospheric sampler was used at the Guangzhou Institute of Geochemistry,Guangzhou Institute of Geochemistry,Chinese Academy of Sciences,in November 2017.Preparation of PM2.5 and PM2.5 suspensions collected by fiber filter membrane:ultrasonic shock,washing and centrifugal freeze-drying of fiber filter membrane containing particulates into PM2.5 freeze-dried powder.2.Object:the human airway epithelial cell line(Calu-3,P53)was provided by Sun Yat-sen University.Stimulate airway epithelial cells,observe airway epithelial cell function and related cytokines expression under different action conditions.Part one.The effect of PM2.5 on the survival of airway epithelial cells.Research methods:1).Set up a blank control group,PM2.5 group,PAH group,OVA group,group OVA+PM2.5 control;2).Establish action time group 24h,48h,72h;3).MTT method to detect the relative survival rate of each group cell;4)statistical analysis of the differences between each group.Result:1.Under the light microscope,it was observed that PM2.5 could kill the airway epithelial cells,the high concentration effect was obvious,and it was related to the time of action.2.The relative survival rate of the cells was determined by MTT assay.PM2.5 could-affect the relative survival rate of airway epithelial cells.The high concentration of PM2.5 had a significant effect on the relative survival rate of airway epithelial cells.Conclusion:The effect of PM2.5 on airway epithelial cells was related to the concentration,and the higher the concentration was,the more toxic effect was on the cells,resulting in the death of airway epithelial cells and inhibition of cell proliferation.PM2.5 on airway epithelial cells has had time effect,48h group is the strongest one.Time effect included acute and chronic effect.Part two.PM2.5 increase the level of Th2 type cytokines in airway epithelial cells.Research methods:1)1.Set up blank control group,PM2.5 group,OVA group,group OVA+PM2.5 control;2).Set up action time group 24h,48h,72h;3).Collect cell and supernatant,ELISA and Rt-PCR methods to detect TSLP,IL-33,Eotaxin-3,E-cadherin and protein expression;4)Analysis of the differences between groups.Result:1.ELISA test:there was no difference in the concentration of PM2.5,IL-33,TSLP and Eotaxin-3 between the groups and the time group.2.The expression of TSLP mRNA and Eotaxin-3 mRNA in airway epithelial cells treated with PM2.5 was higher than that in control group.The expression of IL-33 E-cadherin mRNA was lower than that of control group,but there was no significant difference in the expression of IL-33 mRNA and E-cadherin mRNA.Conclusion:The effect of PM2.5 on airway epithelial cells can induce TSLP,Eotaxin-3 inflammatory factor mRNA expression level rise,ELISA detection TSLP,Eotaxin-3 protein expression level is not significantly increased,ELISA detection value has no statistically significant.PM2.5 effect in the airway epithelial cells to increase the level of Th2 type inflammatory factors may be induced,there is the existence of induced airway epithelial cell line Asthma related pathology,physiological may be change.Part three.PM2.5 affects airway barrier function by affecting airway epithelial cells.The materials and objects of the experiment are the same as those before.The transwell culture plate is used to simulate the gas contact surface of the normal airway.Research methods:1).Establishment of blank control group,PM2.5 group,PM2.5+FSK group,FSK group,2).Detection of anion current by short-circuit current method,3).Statistical analysis of differences among groups.Result:In group PM2.5,the anionic currents of airway epithelial cells were significantly inhibited;group PM2.5+FSK were significantly inhibited.Conclusion:PM2.5 inhibits the production of anionic currents and the secretion of surface fluids in airway epithelial cells.