The Effects Of Exposure To Atmospheric Compound Pollutants During Pregnancy On Asthma In Offspring Mice

BackgroundThe influence of air pollution on human health is the result of the combined action of a variety of pollutants.However,domestic and foreign studies on the health effects of air pollution usually focus on the independent effects of a single pollutant,which may ignore the effects of compound pollutants on health.At present,some reliable conclusions have been obtained on the relationship between single air pollutant during pregnancy and offspring asthma,but the study on the combined effects of air pollutants during pregnancy on offspring asthma is still a blank.In addition,fetal systems develop in stages during pregnancy.Exposure to harmful factors at different stages of pregnancy may have different effects on offspring.At present,there have been a few epidemiological studies on the sensitivity window of air pollution exposure during pregnancy to offspring asthma,but no experimental studies have verified the sensitivity window of air pollution.Therefore,the purpose of this study was to explore the effects of exposure to atmospheric compound pollutants during pregnancy on offspring asthma and the sensitivity window.Research objectiveTo explore the relationship between exposure to atmospheric compound pollutants in different stages of pregnancy and offspring asthma,and to find the sensitive window of atmospheric compound pollution affecting offspring asthma,so as to provide a scientific basis for determining the incidence pattern of population asthma caused by atmospheric compound pollution.Research method1.Experimental animal groupingSPF ICR mice were kept in a cage at 22:00 every day at a ratio of 1:2 males and females.Vaginal plug was examined at 6:30 the next day,and the date on which vaginal plug was first found was recorded as the first day of pregnancy(GD1).The experimental grouping was divided into two stages:pregnancy and offspring.(1)Pregnant mice were randomly divided into 8 groups(4 exposure groups and 4 control groups)according to the pregnancy order.They were the exposure groups(Pollutant group)of atmospheric compound pollutants in the early(GD1-6),second(GD7-12),third(GD13-18)and whole trimester(GD1-18)of pregnancy and the corresponding air control groups(Air group),respectively,so as to construct the exposure model of atmospheric compound pollutants in pregnancy.(2)The offspring of pregnant mice in each group were randomly divided into 2 groups:Ovalbumin(OVA)group and normal saline(NS)group to establish offspring asthma model.This study included 16 groups.2.Exposure to atmospheric compound pollutants in pregnant miceExposure dose of pregnant mice:Based on the annual average value of the concentration of air pollutants in Jinan in 2017,and according to the body-surface area conversion coefficient between human and mouse and individual difference,the exposure dose of atmospheric compound pollutants during pregnancy was set.Exposure methods:(1)Gases:The exposure of sulfur dioxide(SO2),carbon monoxide(CO),nitrogen dioxide(NO2),ozone(O3)by dynamic inhalation was carried out in two steps.① First,the compound exposure of SO2 and CO was carried out.The time of exposure was 1 h/d.Pregnant mice in the control group were exposed to clean air.②After the compound exposure of SO2 and CO was finished and the air in the poisoning chamber was fully replaced,the compound exposure of NO2 and O3 was carried out.The time of exposure was 1 h/d.Pregnant mice in the control group were exposed to clean air.(2)Particulate matter:The exposure of fine particulate matter(PM2.5)and inhalable particles(PM10)was carried out using trachea dripping.Pregnant mice in control group were treated with tracheal dripping of same volume of PBS.3.Sensitization and challenge of offspring miceThe offspring mice were sensitized by intraperitoneal injection of 100 μl OVA solution at postnatal day 9,11 and 13,and challenged by OVA(3%)at postnatal day 22-28 using a dynamic poisoning device.The offspring mice of control group were sensitized and challenged with normal saline.4.Detection of observation indicators(1)The weight,abortion rate and litter size of pregnant mice were recorded during the experiment.(2)Emms WBP system(whole body plethysmography)was used to detect airway reactivity of offspring mice.(3)The total white blood cells(WBC)and different cells in bronchoalveolar lavage fluid(BALF)of offspring mice were counted by animal haematology analyzer.(4)HE staining and PAS staining were performed on lung tissue sections of offspring mice to observe the inflammatory infiltration and goblet cell hyperplasia in the lungs.(5)The expression level of OVA-specific immunoglobulin(Ig)E in serum of offspring mice was determined by enzyme-linked immunosorbent assay.(6)The expression levels of interleukin(IL)-4,IL-5,interferon(IFN)-γ,IL-17A,IL-10 and Tumor necrosis factor(TNF)-α in BALF of offspring mice were detected by electrochemiluminescence.5.Statistical analysisMeasurement data conforming to normal distribution were represented by mean± standard deviation(x±s).When comparing two groups of measurement data,the homogeneity of variance test was first carried out.If the variances were homogeneous,the t-test of independent samples was used.If the variances were not homogeneous,the independent samples t’-test with corrected degrees of freedom was used.When multiple groups of measurement data were compared,homogeneity of variance test was first carried out.If homogeneity of variance was observed,one-way ANOVA was used for testing,and post-hoc Tukey test was used for inter-group comparison.If variances were not homogeneous,Welch’s ANOVA test and Post-Hoc Dunnett’s T3 test were used for inter-group comparison.The classification data were compared using Fisher’s exact test.α=0.05Research results1.Results of establishment of asthma model in offspring miceAfter sensitization and challenge(each Air/OVA group),the Penh value of the offspring mice of the pregnant mice in control group that exposed during pregnancy was significantly higher(P<0.05)than that of the control group(Air/NS groups).The total number of WBC and the number of different cells in BALF were significantly increased(P<0.05).In the Air/OVA group,there was obvious inflammatory cell infiltration around the bronchus and goblet cell proliferation in the airway epithelium,the levels of OVA-specific IgE in serum were significantly increased(P<0.05),and the levels of pro-inflammatory cytokine TNF-α,helper T cell(Th)2 cytokines IL-4 and IL-5,Th1 cytokine IFN-γ,Th17 cytokine IL-17A,and Regulatory T cell cytokine IL-10,IL-4/IFN-y ratio and IL-17A/IL-10 ratio in BALF were significantly increased(P<0.05).2.The effects of exposure to atmospheric compound pollutants during pregnancy on offspring asthma and identification of sensitive windowAirway hyperreactivity:Penh value of offspring mice in Pollutant GD7-12/OVA group were significantly higher than that in the Air GD7-12/OVA group(P<0.05).Compared with the corresponding Air/OVA groups,the Penh values of Pollutant GD1-6/OVA group,Pollutant GE13-18/OVA group and Pollutant GE1-18/OVA group showed no statistically significant difference.The white blood cells count in BALF:Compared with Air GD7-12/OVA group,the total number of WBC and the number of eosinophils in BALF of offspring mice significantly increased in Pollutant GD7-12/OVA group(P<0.05).There were no significant differences in the total number of WBC and cells of each classification in BALF of Pollutant GD16/OVA group,Pollutant GD13-18/OVA group and Pollutant GD1-18/OVA group compared with the corresponding Air/OVA groups.Lung histopathology:(1)HE staining showed significantly increased inflammatory infiltration in lung tissue of offspring mice in the Pollutant GD7-12/OVA group compared with the Air GD7-12/OVA group.The HE staining results of Pollutant GD1-6/OVA group,Pollutant Gd13-18/OVA group and PollutantGD1-18/OVA group showed no significant difference compared with the corresponding Air/OVA groups.(2)PAS staining showed that the proliferation of airway epithelial goblet cells of offspring mice was significantly increased in Pollutant GD1-6/OVA group and Pollutant GD7-12/OVA group compared with the corresponding Air/OVA group.The degree of airway epithelial goblet cell proliferation in Pollutant GD13-18/OVA group and Pollutant GD1-18/OVA group was not significantly different from that in the corresponding Air/OVA groups.Levels of OVA-specific IgE in serum:Compared with the corresponding Air/OVA group,OVA-specific IgE levels in serum of offspring mice in Pollutant GD7-12/OVA group and Pollutant GD1-18/OVAgroup were significantly increased(P<0.05).There was no statistically significant difference in OVA-specific IgE levels in Pollutant GD1-6/OVA group and Pollutant GD13-18/OVA group compared to the corresponding Air/OVA groups.Cytokine levels in BALF:(1)The results of TNF-α showed that the levels of TNF-α in BALF of offspring mice in Pollutant GD1-6/OVA group and Pollutant GD7-12/OVA group were significantly higher than that of the corresponding Air/OVA group(P<0.05).The levels of TNF-α in BALF of offspring mice in Pollutant GD13-18/OVA group and Pollutant GD1-18/OVA group were not significantly different from that of the corresponding Air/OVA groups.(2)The results of IFN-y,IL-4 and IL-5 showed that the IFN-γ levels in BALF of offspring mice in Pollutant GD1-6/OVA group,Pollutant GD7-12/OVA group,Pollutant GD13-18/OVA group and Pollutant GD1-18/OVA group were not significantly different from the corresponding Air/OVA groups.The levels of IL-4,IL-5 and IL-4/IFN-y ratio in BALF of offspring mice in Pollutant GD7-12/OVA group were significantly increased compared with the corresponding Air/OVA groups(P<0.05),while the levels of IL-4,IL-5 and IL-4/IFN-y ratio in BALF of Pollutant GD1-6/OVA group,Pollutant Gd13-is/OVA group和 Pollutant GD1-18/OVA group were not significantly changed.(3)The results of IL-17A and IL-10 showed that there were no significant differences in the level of IL-17A,IL-10 and IL-17A/IL-10 ratio in BALF of offspring mice in Pollutant GD1-6/OVA group,Pollutant GD7-12/OVA group,Pollutant GD13-18/OVA group and Pollutant GD1-18/OVA group compared with the corresponding Air/OVA groups.3.Maternal and reproductive toxicity of exposure to atmospheric compound pollutants during pregnancyMaternal toxicity:Compared with the air control group,there was no significant difference in body weight of pregnant mice after exposure to atmospheric compound pollutants in first trimester.The body weight of pregnant mice exposed to atmospheric compound pollutants in the second,third trimesters and whole pregnancy was lower than that of the corresponding air control groups(P<0.05).Reproductive toxicity:There were no significant differences in litter size and abortion rate between the pollutants exposure groups and the corresponding control groups.Research conclusions1.Exposure to atmospheric compound pollutants during pregnancy could aggravate the severity of asthma in offspring mice.2.The second trimester of pregnancy is a sensitive window for exposure to atmospheric compound pollutants to aggravate asthma in offspring mice.3.Exposure to atmospheric compound pollutants in the second trimester may mediated the aggravation of offspring asthma by enhancing Th2 immune response.