The Effects Of Ozone Exposure During Pregnancy On Asthma In Offspring Mice And Its Mechanism

BackgroundOzone(O3)is a highly oxidizing gas with a fishy smell,and is divided into stratosphere O3 and troposphere O3.Tropospheric O3 is a common atmospheric pollutant,mainly produced by nitrogen oxides(NOx)and volatile organic compounds(VOCs)through a photo-chemical reaction.Both epidemiological and laboratory studies have found that O3 can cause respiratory diseases such as cough,asthma,and decreased lung function in exposed individuals.Asthma is a respiratory disease involving multiple inflammatory cells and cytokines,characterized by Airway Hyper Reactivity(AHR)and bronchial inflammation.Since the 20th century,the incidence of asthma has increased rapidly in more than 200 countries and regions in the world.Childhood asthma is a common respiratory disease.Its frequent recurrence will cause serious harm to children’s health and growth,and even cause a series of diseases in adulthood.Exposure to ambient air pollutants during pregnancy and early life can affect fetal and neonatal developmental plasticity,and can change the genetic program or affect epigenetic markers in fetal developmental programming,resulting in permanent functional structure changes,which significantly enhances the offspring’s sensitivity to ambient air pollutants,and induces the occurrence and development of a series of allergic diseases.Moreover,a large number of epidemiological studies have shown that exposure to ambient air pollutants during pregnancy and early life is an important factor leading to the occurrence of infant and childhood asthma.Although studies have shown that O3 exposure can cause a range of respiratory diseases in exposed individuals,few studies have explored the effects and mechanisms of O3 exposure during pregnancy on offspring asthma.Therefore,this study aimed to explore the possible effects and potential mechanisms of O3 exposure during pregnancy on asthma in offspring mice.Research objectiveUsing OVA(Ovalbumin OVA)to sensitize and challenge to induce animal models of asthma,and based on the average concentration of atmospheric O3 in Jinan in 2017,we simulate the average O3 exposure level of the population for O3 exposure in mice during pregnancy,to explore the effects of low-level O3 exposure during pregnancy on asthma in offspring mice.We measure the expression levels of molecules associated with the development of asthma in the offspring of the femal mice at the end of pregnancy and lactation.The potential mechanism of O3 exposure during pregnancy on asthma in offspring mice will be explored from both the offspring mice and the dams.The results will provide a scientific basis for further understanding the mechanism of O3 exposure during pregnancy on asthma in offspring.Research method1.Experimental designThe study design consists of two parts:the first part is the O3 exposure model of pregnant mice,to explore the maternal toxicity,embryo toxicity and possible mechanisms of O3 exposure during pregnancy on asthma in offspring mice.The pregnant mice were randomly divided into four groups according to whether they were exposed to O3 and the end point of observation.The two groups were the O3 exposure group and the two groups were the Air control group.The O3 exposure groups were exposed to O3 during 13-18 days of pregnancy(1h/d,6d).Randomly select one group ending the experiment at the end of pregnancy(marked as OP group,O3 exposure and ending at the end of pregnancy),and the other group ending the experiment at the end of lactation(marked as OL group,O3 exposure and ending at the end of lactation).The two control groups are the end of pregnancy control group(marked as AP group,Air exposure and ending at the end of pregnancy)and the lactation termination control group(marked as AL group,Air exposure and ending at the end of lactation).The related indexes of pregnant mice and post-lactating mice were detected,and the potential mechanisms of the effects of O3 exposure during pregnancy on asthma in the offspring mice were explored from the dams.The second part is a model of asthma in offspring mice,to explore the effects and possible mechanisms of O3 exposure during pregnancy on asthma in offspring mice.The offspring of OL and AL groups in the first part of the study were randomly divided into two groups after lactation.The offspring of OL group were recorded as O3/PBS group and O3/OVA group,and the offspring of AL group were recorded as Air/PBS group and Air/OVA group.The O3/OVA group and Air/OVA group mice were sensitized and stimulated by OVA to construct offspring asthma model.Four groups of offspring mice were tested for asthma-associated indicators and changes in spleen NK cells,and the effects of O3 exposure during pregnancy on asthma in offspring mice and their underlying mechanisms were explored from the offspring.2.Animal groupingMature BALB/c mice were adaptively fed for one week after purchase in SPF-grade animal rooms,and male and female mice were closed at a ratio of 1:2.The day of conception is the first day of gestation(the first day of gestation,GD1).According to the research design,in the first part of the model of female mice,the experimental animals were divided into four groups,namely the OP group and OL group receiving O3 infection and the corresponding AP and AL control groups.In the second part of the offspring mice model,the offspring of the OL group and the AL group of the first part of the female mice model were randomly divided into two groups at the end of lactation.That is,the OL group of offspring mice exposed to O3 during pregnancy were divided into O3/OVA group and O3/PBS group;the AL group of offspring mice controlled by Air during pregnancy were divided into Air/OVA group and Air/PBS group.3.Exposure of pregnant miceO)3 exposure method:pregnant mice are exposed to O3 inhalation through HOPE-MED 8050G dynamic poisoning cabinet;O3 exposure concentration:7.3mg/m3,and exposure time:GD13-GD18 and 1h/d,6 consecutive days.The pregnant mice in the control group breathed normal fresh air in the poisoned cabinet.4.Sensitization and challenge of offspring miceSensitization:the offspring mice were injected intraperitoneally with 0.1mL of sensitizing fluid(50μg Grade V OVA and 2 mg alum adjuvant)on postnatal day(PND)9,11,and 13 days,and the control group was injected with PBS instead,the injection volume is the same as OVA.Challenge:At 4 weeks of age,the offspring mice were inhaled with 3%OVA(Grade Ⅱ OVA),PND22-28 and 30min/d;the control group was inhaled with PBS,and the time is the same as OVA.5.Observation indicators①HEMAVET 950 blood cell analyzer to classify and detect inflammatory cells of bronchoalveolar lavage fluid(BALF)in offspring mice,pregnant mice and post-lactating dams;②HE and PAS staining were used to observe inflammatory cell infiltration and mucus secretion around the bronchus of offspring mice,pregnant mice and post-lactating dams;③Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of IL-4,IFN-y,IL-17 and TNF-α in BALF supernatant of offspring mice;④Serum 8-isoprostanes(8-isoprostanes,8-IP)levels in pregnant mice and post-lactating dams were detected by ELISA;⑤Immunohistochemistry(ICH)was used to determine the expression level of IL-4 protein in the lung tissues of offspring mice;⑥Flow cytometry to detect the proportion and number of CD3ε-CD49b+NK cells in the spleens of offspring mice,pregnant mice and post-lactating dams;⑦The body weight of the pregnant mice and post-lactating dams and the weight of the organs at each observation point;⑧The miscarriage rate of pregnant mice and the number of litters in dams.6.Statistical analysisWhen comparing multiple sets of experimental data,if the homogeneity of variance test is met,one-way ANOVA is used.When the overall analysis of variance is statistically significant,the Tukey method is used for pairwise comparison.If the data do not conform to the homogeneity of variance,non-parametric analysis is used(Kruskal-Wallis).When the two sets of experimental data are compared,if the variances are equal,two independent sample T tests are used;when the variances are not uniform,the Fisher’s test is used.Test level:P<0.05 considers the difference to be statistically significant.7.Quality control①Mice were randomly divided into experimental groups according to body weight;②During the experiment,O3 was generated by the same generator,and OVA and Alum used the same batch;③The exposure of mice and collection of samples were carried out within the same time period;④Blind observation and blind determination were used during the experiment.Research results1.Construction of asthma model in offspring miceCompared with the Air/PBS control group,the number of white blood cells(White blood cell,Wbc),eosinophils(Eosinophil,Eos),lymphocytes(Lymphocyte,Lym),neutrophils(Neutrophil,Neu)and macrophages(Macrophage,Mac)increased significantly and the differences were statistically significant(all P values were less than 0.05).The number of inflammatory cells infiltrating in the circle around the bronchi in the lung tissue,lung tissue bronchial mucus secretion and serum OVA-specific IgE level in the Air/OVA group were significant increased,and the differences were statistically significant(all P values were less than 0.05).Compared with the Air/PBS control group,IL-4/IFN-y in BALF of Air/OVA group was significantly increased,the difference was statistically significant(PAir/OVA vs.Air/PBS<0.05),IL-4/IFN-γ increased by 48.6%.In the experiment,the asthma model of BALB/c offspring mice was successfully constructed by OVA sensitization and challenge.2.Effects of O3 exposure during pregnancy on asthma in offspring mice①The results of classification and count of inflammatory cells in BALF were as follows:compared with the Air/OVA control group,the total number of Wbc,Eos,Lym,Neu,and Mac in BALF in offspring mice of the O3/OVA group increased significantly,and the differences were statistically significant(P<0.001),and percentage of attributable risk for changes in Wbc,Eos,Lym,Neu,and Mac numbers in BALF after O3 exposure were 43.2%,63.6%,57.2%,71.5%and 50.6%respectively.②Results of infiltration and mucus secretion of inflammatory cells in lung tissue:the pathology HE staining results showed that compared with the Air/OVA control group,the infiltration of inflammatory cells around the bronchi in the lung tissue of offspring mice of the O3/OVA group was significantly increased;the semi-quantitative scoring results of HE staining showed that compared with the Air/OVA control group,the semi-quantitative scoring of inflammatory cell infiltration around the bronchi in the lung tissue of offspring mice of O3/OVA group was significantly increased(P<0.001).The pathology PAS staining results showed that compared with the Air/OVA control group,bronchial goblet cell(Gc)proliferation and mucus secretion in the lung tissue of offspring mice of the O3/OVA group were significantly increased;the semi-quantitative scores of PAS staining showed that compared with the Air/OVA group,the semi-quantitative scores of bronchial Gc proliferation and the area/Pbm of Gc in the lung tissue branches of offspring mice of the O3/OVA group were significantly increased(Gc proliferation:P<0.01;the area/Pbm of Gc:P<0.001).③Results of detection of IFN-γ,IL-4,IL-17,TNF-αin BALF and lung tissue IL-4 levels:the Th1 representative factor IFN-γ results showed that there was no significant difference in the level of IFN-y in BALF between offspring mice of the O3/OVA group and the Air/OVA group.The Th2 representative factor IL-4 showed that the IL-4 level in BALF in the O3/OVA group was 21.1±4.97pg/ml,which was significantly higher than that in offspring mice of the Air/OVA group,and the difference was statistically significant(P<0.01),the AR%of IL-4 level change in BALF after exposure was 43.2%;Lung tissue IL-4 immunohistochemical results showed that compared with the Air/OVA control group,the expression of IL-4 around the bronchi increased significantly in the lung tissue of offspring mice of the O3/OVA group;and compared with the Air/OVA group,the average optical density(Average Optical Density,AOD)of IL-4 expression staining around the bronchi in the lung tissue of offspring mice of the O3/OVA group increased significantly,and the difference was statistically significant(P<0.05),after O3 exposure,the AR%of lung IL-4 AOD change was 20.4%.IL-4/IFN-γ(Th2/Th1)results showed that compared with the Air/OVA control group,IL-4/IFN-γ in BALF in offspring mice of the O3/OVA group was significantly increased,and the difference was statistically significant(P<0.01),the AR%of IL-4/IFN-γ level change in BALF after O3 exposure was 26.8%.Th17 representative factor IL-17 showed that the IL-17 level in BALF in offspring mice of the O3/OVA group was 14.6±4.92pg/ml,which was significantly higher than that in the Air/OVA group,and the difference was statistically significant(P<0.01),the AR%of IL-17 level change in BALF after O3 exposure was 84.3%.The results of the pro-inflammatory factor TNF-α showed that the level of TNF-α in BALF in offspring mice of the O3/OVA group was 35.1±7.06pg/ml,which was significantly higher than that in the Air/OVA group,and the difference was statistically significant(P<0.05),the AR%of TNF-α level change in BALF after O3 exposure was 23.6%.④The results showed that the OVA-specific IgE level in the serum of offspring mice of the O3/OVA group was 603±125ng/ml,which was significantly higher than the Air/OVA group,and the difference was statistically significant(P<0.001),the AR%of serum OVA-specific IgE level change after O3 exposure was 55.4%.3.Results of study on the mechanisms of O3 exposure during pregnancy on asthma in offspring mice①Offspring mice:the results of detecting the CD3ε-CD49b+NK cells in the spleen of offspring mice showed that compared with the Air/PBS and Air/OVA control groups,the proportion and number of CD3ε-CD49b+NK cells in the spleen of offspring mice of the O3/PBS and O3/OVA groups were significantly increased,and the difference were statistically significant(P<0.01,P<0.001),the AR%of the CD3ε-CD49b+NK cell number of O3 exposed spleen were 20.0%and 24.1%,respectively.②Dams:the results of the CD3ε-CD49b+NK cells in the spleens showed that the proportion and number of CD3ε-CD49b+NK cells in the spleen of pregnant mice in OP group who terminated the experiment after O3 exposure during pregnancy were significantly lower than that in AP group(P<0.05).Compared with the AL group,the proportion and number of CD3ε-CD49b+NK cells in the spleen of the OL group that terminated the experiment after the exposure of O3 during pregnancy at the end of lactation were significantly still lower than that of the AL group(P<0.05).O3 exposure during pregnancy damaged the CD3ε-CD49b+NK cells of the spleen of the dams.Research conclusions1.OVA sensitization and challenge leads to the imbalance of Th2/Th1 in offspring mice,showing a Th2 dominant response,which may be the mechanism of OVA-induced asthma in young offspring mice.2.O3 exposure during pregnancy aggravates OVA-induced asthma development in offspring mice,which may be directly related to the increase of CD3ε-CD49b+NK cells in offspring mice and indirectly related to maternal immune system damage.