Effects Of Different Concentrations Of Ammonia Exposure On Gene Expression Profiles And Metabolic Pathways In Lungs Of Piglets

Ammonia is a toxic gas,mainly produced in the livestock farms,fertilizer plants,and field fertilization.Long-term or high concentrations of ammonia exposure affect the health of humans and animals,leading to the decline of animal productivity,and even death of individuals.Lung is the terminal organ of respiratory system and plays an important role in maintaining body health.Lung is also one of the important target organs of ammonia.Our previous study found that structural lesions of lungs were observed include increasing in extracellular matrix deposition and thickening of alveolar septum after pigs were stimulated with 15 mg/m~3 and 38 mg/m~3 ammonia for30 days.In this study,transcriptome analysis and molecular biology experiments were conducted to further explore the effects of ammonia exposure on gene expression in lungs.At the same time,combined with the metabolomics data obtained in a previous study,the transcriptome and metabolome integrated analysis was conducted to further analyze the toxicological mechanism under long-term exposure of different concentrations of ammonia to lungs from the levels of gene expression and metabolic regulation.The results are summarized as follows:1.Gene set enrichment analysis in lungs of pigs after different concentrations of ammonia exposureBy aligning,counting and annotating,gene expression profiles of pig lungs were obtained after 15 mg/m~3 and 38 mg/m~3 ammonia exposure.All detected genes were enriched in KEGG pathway using gene set enrichment analysis.The results showed that compared with the control group,the expression levels of gene sets in ECM-receptor interaction pathway were lower,while the expression levels of gene sets in lymphocyte-associated pathway,toll-like receptor signaling pathway,proteolysis,apoptosis and DNA replication pathway were higher under 15 mg/m~3 ammonia exposure.The expression levels of gene sets in cell cycle,DNA replication,homologous recombination and aminoacyl-tRNA synthesis pathways were lower under 38 mg/m~3 ammonia exposure.2.Identification and functional analysis of differentially expressed genes in lungs of pigs after different concentrations of ammonia exposureCompared with the control group,a total of 183 differentially expressed genes(DEGs)were identified in the 15 mg/m~3 ammonia group,and these DEGs were mainly related to chemotaxis and activation of inflammatory cells,cellular response to cytokines,defense response,and immune response.A total of 300 DEGs were identified in the 38 mg/m~3 ammonia group,and these DEGs were mainly related to oxidation-reduction reaction,lipid transport,ion transport,and protein digestion and absorption.58 DEGs were common between the two ammonia treatment groups.Among the 58 DEGs,27 genes were up-regulated and 31 genes were down-regulated.The common up-regulated DEGs between the two treatment groups were mainly related to the functions of cytidine deamination,neutrophil activation,antigen binding and lipid transport,while the common down-regulated DEGs were mainly related to the functions of cell motility,cytoskeleton maintenance,immune regulation and amino acid metabolism.3.Identification and verification of hub genes and in key biological process genes in lungs after different concentrations of ammonia exposureIn this study,hub genes of lungs after 15 mg/m~3 ammonia exposure were obtained:ADRA2C,CCR2,CCL8,CCL1,AMCF-Ⅱ,CCR8,TNFRSF9,CTLA4,IFNG,and CD69.These genes were mainly related to leukocyte migration,chemokines and T cell reaction.After 38 mg/m~3 ammonia exposure,the hub genes in lungs were NPY,HRC,APOB,TNC,AMCF-Ⅱ,SSTR1,APLN,P2RY13,CDH2,and PCSK9.These genes were mainly related to signal transduction process.The expression levels of T cell response-related genes were detected,the results showed that exposure to 15 mg/m~3 ammonia induced T cell-mediated proinflammatory response.At the same time,the expression levels of alveolar extracellular matrix component genes were detected,the results showed that the expression of these genes was significantly down-regulated in the 38mg/m~3 ammonia exposure group.4.Integrated analysis of transcriptome and metabolome in lungs after different concentrations of ammonia exposureIntegrated analysis of current transcriptome and previously obtained metabolome was conducted.A total of 13 pathways were enriched in the 15 mg/m~3 ammonia group,and 19 pathways were enriched in the 38 mg/m~3 ammonia group.Compared with the control group,taurine metabolism,tryptophan metabolism,and prolactin signal pathway were affected under an exposure of 15 mg/m~3 ammonia,and the changes of these pathways may be related to inflammation.However,pyruvate metabolism and oxidative phosphorylation pathway were affected under an exposure of 38 mg/m~3ammonia,indicating that 38 mg/m~3 ammonia exposure had a great effect on lung energy metabolism of pigs.In conclusion,this study showed part of the toxic effects of exposure to ammonia at different concentrations on lungs from the levels of gene expression by using transcriptome method and combining with the results of HE staining in the previous study.At the same time,the effect of long-term ammonia exposure on pulmonary metabolism was explored by transcriptome and metabolome integrated analysis.Our results can provide a theoretical basis for the analysis of the lung toxicity caused by long-term exposure to different concentrations of ammonia.