Airborne Fine Particulate Matter-induced Over-expression Of Inflammatory Mediators In Human Bronchial Epithelial Cells And Underlying Mechanisms

Background Haze pollution characterized by excessive PM_2.5 concentration is occurring frequently in some area in china.PM_2.5 exposure can cause serious damage to people’s respiratory system and cardiovascular system.Human bronchial epithelial cells are not only the body’s first line of defense against inhalation of environmental toxicants,but also participate in the development of respiratory tract inflammation by secreting inflammatory factors.This experiment will explore the expression of PM_2.5-induced human bronchial epithelial cells（BEAS-2B）inflammatory factors and related mechanisms,and provides theoretical basis and important clues for further development of interventions in PM_2.5-induced respiratory toxicity.Objective Explore the expression and mechanism of inflammatory factors in BEAS-2B cells caused by PM_2.5 exposure.Methods Part One Natural exposure to haze:Under different haze weather conditions,the BEAS-2B cells planted in Transwell culture were placed in the cell exposure chamber,and the outdoor air was passed through particulate interceptor（0.3μm,1μm,5μm,without interception）.The gas flow rate was controlled at 30 mL/min,the temperature was 37°C,and the exposure time was 4 h.Meanwhile,normal cultured BEAS-2B cells were used as a control group;after the exposure,the cell culture medium was collected,and the enzyme-linked immune sorbent assay（ELISA）was used to measure the levels of interleukin（IL）-1β,IL-6,IL-8.Part Two（1）BEAS-2B cells were cultured into 12-well plates at a density of100,000/well,and different concentrations of PM_2.5 suspensions（0,20,50,100,150μg/mL）exposed to BEAS-2B cells for 2 h,then 2,7-Dichlorodi-hydrofluorescein diacetate（DCFH-DA）fluorescent probe labeled cells,flow cytometry to detect the fluorescence intensity of cells,fluorescence intensity and reactive oxygen species（ROS）levels are positively correlated,and ROS（oxidative stress marker）levels reflect the oxidative stress state of cells;after 6h exposure,the cell culture supernatant was collected and ELISA method was used to detect the levels of IL-1β,IL-6 and IL-8 in BEAS-2B cells,collect the supernatant by centrifugation,and detect the phosphorylation level of EGFR（Y1068）by Western blot,（2）in addition,pretreatment with the antioxidant N-acetyl-L-cysteine（NAC,5mM）for 6h,or inhibitor（PD153035,5nM）pretreated the cells for 2 h.After that,the cells were exposed to 150μg/mL PM_2.5 suspension,the levels of IL-1β、IL-6、IL-8 and ROS、EGFR caused by PM_2.5.5 exposure to BEAS-2B cells was observed;Results Part One Natural exposure to haze:This experiment was conducted in accordance with the Air Quality Index（AQI）standards.The air quality was divided into four stages:good,light,moderate,and severe pollution.（1）Comparison between different interception exposure groups found that the expression levels of IL-1β,IL-6,and IL-8 in the 0.3μm,1μm,and 5μm interception exposure groups were significantly lower than those in the non-interceptive exposure group（P<0.05）.The expressions of IL-1β,IL-6 and IL-8 showed an increasing trend with the increase of the pore size of the intercepted exposure group.（2）Under the pollution conditions of the same particle size,as the pollution level increased,the expression of inflammatory factors IL-1β,IL-6,and IL-8 caused by BEAS-2B cells exposed to haze showed an upward trend（P<0.05）.（3）In the case of severe pollution,the contribution of particulate matter of different particle sizes to the expression level of IL-1βis below 0.3μm>above 5μm>1μm-5μm>0.3μm-1μm;The contribution of particulate matter of different particle sizes to the expression level of IL-6、IL-8 is above 5μm>below 0.3μm>1μm-5μm>0.3μm-1μm.Part Two PM_2.5 suspension:the results showed that（1）the expression of IL-6,IL-8,IL-1βin BEAS-2B cells were positively correlated with PM_2.5 exposure concentrations（0,20,50,100,150μg/mL）;（2）There is a significant positive correlation between ROS levels in BEAS-2B cells and PM_2.5 exposure concentration;inhibition of ROS levels can significantly reduce the levels of IL-1β,IL-6,IL-8 in BEAS-2B cells caused by PM_2.5 exposure（P<0.05）;（3）There is a significant positive correlation between the activation level of EGFR in BEAS-2B cells and the PM_2.5 exposure concentration;inhibition of EGFR activation can significantly reduce the expression levels of IL-6 and IL-8 in BEAS-2B cells caused by PM_2.5 exposure（P<0.05）;（4）Inhibition of intracellular ROS levels can significantly reduce EGFR activation levels caused by PM_2.5 exposure（P<0.05）;the intracellular ROS level was not significantly change after inhibiting EGFR activation levels（P>0.05）.Conclusions 1.The inflammatory response of BEAS-2B cells caused by the exposure of particles with different particle sizes is particle size dependent.The expression of inflammatory mediators caused by the exposure of below 0.3μm and above 5μm particles is higher than caused by the exposure of 0.3μm-1μm and 1μm-5μm particles.Under the condition of the same size particle pollution,the inflammatory response caused by higher concentration haze exposure is more obvious;2.PM_2.5 exposure induced over-expression of pro-inflammatory mediators in BEAS-2B cells,which is regulated by oxidative stress and EGFR.Moreover,oxidative stress can modulate PM_2.5-induced activation of EGFR.