| ObjectiveThis study aimed to explore the association among the exposures of ambient air pollution,the characteristics of gut microbiota,and the changes in epigenetic modification in asthmatic children by panel study and check the different biomarkers of gut microbiota and epigenetic modification by strata of the atopy of asthmatics.Methods1.Data collection:From September 2016 to November 2017,physician diagnosed asthmatic children from Children’s Hospital Capital institute of Pediatrics and pupils without asthma from a primary school in Chaoyang District of Beijing were enrolled to participate in a prospective longitudinal panel study.All participants should live in Beijing for more than six months and the age of the participants should be five to fourteen years old.The status of lung function and the fractional exhaled nitric oxide(FeNO)of asthmatics were measured in the hospital at the enrollment.2.Assessment of air pollution exposures:Black Carbon(BC)exposures for the participants was estimated by the measurements of ambient air BC at the designated spots in Chaoyang District of Beijing,Information of PM10,PM2.5,NO2 and O3 were obtained from China National Environmental Monitoring Centre.3.Changes of epigenetic modification:Nebulized saline was administered to collect the induced sputum specimen of all participants by the recommended method.The acetylation levels of histone H3 and H4 of induced sputum were measured with ELLSA.Multilevel models and Multiple linear regression models were used to analyze the different effects of air pollution exposures on the level of histone acetylation in atopic asthma,non-atopic asthma,and controls.4.16SrDNA sequencing of intestinal microbiome:Faeces of participants were collected and the microbiome DNA was isolated for the analysis of 16SrDNA sequencing and microgenome sequencing of intestinal microorganism.Double terminal sequencing(Paired-End)strategy for 16SrDNA sequencing in V4 region was performed with the Illumina HiSeq2500 sequencing platform to observe the difference in the diversity and constitution of microbiota by group.5.Metagenome sequencing of intestinal microorganism:Faeces specimen were selected from those specimens for 16SrDNA sequencing for the metagenome sequencing with Illumina HiSeq4000 sequencing platform by Illumina PE150 double terminal sequencing(Paired-End).The differences in the functional genes of intestinal microflora in children among atopic asthmatics,non-atopic asthmatics,and the controls were annotated and the differences in the functional genes and metabolic pathways of intestinal microflora in the three groups were analyzed with R packages.6.Statistical analysis:R version 3.4.4 was used to ananlysis the results.T test,the one-way analysis of variance(ANOVA),multiple linear regression analysis,and stratified analysis were used to analyze the differences in measurements of the participants when they were first enrolled in the program.Tow-way ANOVA with repeated measures and multilevel regression model were used to test the changes of measurements.The differences of intestinal microorganism groups were analyzed by Adnois,LefSE methods.Mantal Testmethods was used to analysis the effects of BC,PM10,NO2 and O3 on acetylation level of histone(H3&H4)and the diversity of intestinal microflora.Results1.Measurements of histone acetylation:Two-way ANOVA test with repeated measures showed that the acetylation of histone H3 was significant different in atopic asthma,non-atopic asthma and control children(P<0.01).Mulitlevel regrression analysis showed that the differences of histone acetylation were signiciantly associated with the status of atopy and ambient air BC exposure levels(P<0.05).Stratified analysis showed that BC exposure was significantly correlated with H3 acetylation level in children with atopic asthma(P=0.009)2.16SrDNA sequencing of intestinal microbiome:V4 region of the 16SrDNA of 47 faece specimen were sequenced with the Illumina HiSeq2500 sequencing platform.The effective tags got were 76393.9 ± 7730 and the number of clusterings were 625.2±269 OTUs.The dominant bacteria of intestinal microflora were Firmicutes,Bacteroidetes,Actinobacteria,Proteobacteria,and Verrucomicrobia.AMOVA analysis was used to test the difference in the constitution of intestinal microflora.And the constitutions of microflora were significantly different by group of participants(F=3.46,P<0.001).The number of species was marginal correlated with BC exposure(P=0.068),significant correlated with PM10(P=0.014)and O3(P=0.025)exposure in children with asthma.3.Metagenome sequencing of intestinal microbiome:16 selected faece specimen were sent for metagenomic sequencing.A total of 923775 predicted genes were obtained and 617878(66.89%)genes were able to match KEGG database.The abundance of functional genes in children with atopic asthma and non-atopic asthma was marginally significant at the level of KEGG Orthology(P=0.089).As the gene annotation results,the numbers of different KEGG Pathway of intestinal microflora were 44(atopic asthma vs controls),51(non-atopic asthma vs Control)and 56(atopic asthma vs non-atopic asthma).4.Association of BC exposure,acetylation of histone,and intestinal microbiome:VPA analysis showed that environmental factors had greater influence on intestinal microbiome than genetic and epigenetic factors.Mantel test showed that air pollution(BC)exposure levels might be associated with the diversity of intestinal microbiome(r=0.048,P=0.138)and the acetylation of H3 was significantly associated with the intestinal microbiota(r=0.133,P=0.048).ConclusionEpigenetic modifications and the diversity or constitutions of intestinal microbiome were quite different between atopic and non-atopic asthmatics.BC may regulate the excitation mechanism of asthma with different phenotypes through Epigenetic modifications and intestinal microbiome.Acetylation of H3 might be one of the effect biomarkers of BC exposure for atopic asthmatics. |
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