Ammonia Inhalation Induces Transcriptome Changes In The Respiratory Tract And Liver Of Piglets

Ammonia is a kind of toxic and harmful gases prevalent in large-scale pig farms.A high level of ammonia is a safety hazard for piglets and workers,and the current impact of ammonia on the health of piglets is still need to be illustrated.In this study,12 piglets of 7 weeks old and 14 kilograms were selected and randomly divided into 4 groups;the 3 experimental groups were stimulated for 4 days（D4）,8 days（D8）and 12（D12）days with t 56 mg/m3 of ammonia.The transcriptome of the 4 period samples was sequenced by RNA-seq to study the transcriptome changes of the lung,nasal mucosa and hepatic after the stimulation of ammonia.Results:1.Identification of differentially expressed genes in three tissues by DESeq with the threshold of q ≤ 0.05.There were 6 comparisons in lung and nasal mucosa: the comparison between NC and 3 treatment groups,the comparison within the 3 treatment groups.The results of DESeq showed that there were 289 and 825 differentially expressed genes in the lung and the nasal mucosa;one comparison in liver（NC with D12）explored 589 differentially expressed genes with 367 up regulated and 222 down regulated.2.The expression patterns of differentially expressed genes in lung and nasal mucosa were constructed and the functional enrichment of each gene was also analyzed.The expression patterns of the differentially expressed genes were constructed and 289 differentially expressed genes in lung were divided into 18 groups.The GO and KEGG enrichment of each cluster gene were analyzed by DAVID software.The results showed that SCGB3A2,MMPs,BPIFA1,IL8,ARG2,COMP ARG2,CLCA1,ELANE,COLs main in cluster 1,cluster 2 and cluster 3 were associated with pulmonary fibrosis and airway mucus secretion.The differentially expressed genes of nasal mucosa were divided into 20 groups by using the same method.Functional analysis showed that CFAPs,CCDCs,TEKTs,DNAHs,LRRCs,SPAG17,COL11A2,ACAN,SNTN in cluster 1,cluster 3,cluster 2 and cluster 6 related to mucosal structure and function;SFTPs,BPIFBs in Cluster 4 related functions of respiration and defense reaction;cluster 7 MASP1,LBP,C4 BPA,IL1B,REG3 G associated with acute inflammation;cluster 8 SOCS3,cluster 4 SFTPs and cluster 7 S100 protein related to mucus secretion;PAI-1,MMPs,KRTs in cluster 8 and cluster 9 may play a role in epithelial repair.3.Pathway enrichment analysis for hepatic DEGs found that the most enrichment（p≤0.05）pathway related to immunity,cancer,liver disease,vascular,amino acid metabolism and circadian rhythm.Protein interaction analysis showed that PIK3 CA,TNF,NFKBIA,THBS1,JUN,HIF1 A and FOS may be important regulators.4.AS analysis results showed that the nasal mucosa genes: CD40,SAT1,ETFB,CLK1,CHTOP and lung genes ETFB,SAT1,CLK1,TNFRSF1 A had a significant differential（FDR≤0.05）alternative splicing between NC group and D12 group;at the same time 2,7 and 18 differentially expressed isoforms（DEIs）were found in the lung,nasal mucosa and liver（FDR≤0.05）respectively.Functional analysis showed that most of these genes were related to immunity,cancer and apoptosis.5.We identified 903 new linc transcripts which belonging to the 753 genes by using the sequencing data,then the linc genes and coding genes were anlyzed by WGCNA.We found that LIPG was highly expressed in the liver D12 group,transferrin was highly expressed in the liver NC group.While the new Linc gene XLOC₀04910 was specifically highly expressed in nasal mucosa D12 group and the 9 genes（upstream and downstream 100 kb）,which are close to XLOC₀04910 are associated with immunity,sugar/hormone metabolism,and hematopoiesis,suggesting that this linc gene may be involved in these processes.Conclusions:Ammonia stimulation caused a large number of up-regulated gene expression in the nasal mucosa,lung and liver tissues of piglets;and the number of differentially expressed genes was highest in 12 days after irritation.These differentially expressed genes are mainly involved in respiratory remodeling,oxidative stress,inflammation and immune response.Meanwhile,alternative splicing of genes and lincRNA may play an important regulatory role in the response to ammonia stimulation of piglets.