| Ammonia is an important component of odor in pig farms.It can be inhaled into pig’s lungs through mouth and nose,and affects the respiratory health of pigs.In our previous studies,the piglets were exposed to 80 ppm ammonia for 0 d(NC group),4d(D4),8 d(D8),and 12 d(D12)respectively,and it was found that ammonia exposure induced lung fibrosis in piglets.Then,in this study we tried to explore the molecular mechanisms of lung fibrosis in piglets induced by ammonia exposure through immunohistochemistry,immunofluorescence,Q-PCR,and transcriptome analysis.The results could provide a theoretical basis of lung tissue damage that induced by ammonia exposure.The main results of this study are as following:1.Statistical analysis of pulmonary fibrosis in piglets exposed to ammonia for different periods of timeThe degree of pulmonary fibrosis was divided into four grades(F0-F3 from light to heavy).F0 means no fibrosis and F3 means the most serious fibrosis.Subsequently,the degree of pulmonary fibrosis of piglets exposed to ammonia for different periods of time was analyzed on tissue sections by HE stainning.It was found that the D4 group was mainly F2(44%)and F3(56%),and the D8 and D12 groups were mainly F1(45%,56%)and F2(44%,33%),which indicated that the ammonia exposure induced pulmonary fibrosis in the lungs of the piglets,and the fibrosis process was aggravated first and then slowed down with the prolongation of the exposure time.2.The damage of the alveolar epithelial cells in piglets after ammonia exposureTo further explore the molecular mechanism of ammonia-induced pulmonary fibrosis in piglets,immunohistochemical technique was used to examine the effect of ammonia exposure on the alveolar epithelial cells in piglets.The expression of type Ⅰalveolar epithelial cells(AECⅠ)marker protein AQP5 was down-regulated in groupD4,while the expression of type Ⅱalveolar epithelial cells(AECⅡ)marker protein SFTPC was significantly up-regulated in D4 group,indicating that ammonia exposure caused a change in the number of alveolar epithelial cells.It is speculated that AECⅠ may be the target cells for alveolar damage,and AECⅡ,due to its stem cell characteristics,repairs the epithelium through proliferation and differentiation after alveolar epithelial damage.3.Epithelial-mesenchymal transition(EMT)may be involved in progress of ammonia-induced pulmonary fibrosis in pigletsWe detected the expression of the AECⅡ marker protein SFTPC and myofibroblasts marker protein α-SMA by double immunofluorescence labeling technique,respectively.It was found that both epithelial cells and fibroblasts were present in the alveolar epithelium of the D4 group of piglets.We further determined the expression of epithelial cell gap junction protein Cnx43,mesenchymal cell marker protein vimentin,fibroblast marker protein S100A4,and myofibroblast marker protein α-SMA by immunofluorescence/immunohistochemistry techniques,and found that the expression of Cnx43 was down-regulated in D4,while other extracellular matrix-related proteins were up-regulated in the D4 group.The above results indicated that the EMT occurred in the lung tissues of piglets after ammonia exposure,and the EMT phenomenon was most obvious in the D4 group.The expression trend of these proteins was consistent with the process of pulmonary fibrosis,therefore,we speculated that EMT may be involved in the process of ammonia-induced pulmonary fibrosis in piglets.4.Expression changes of pulmonary fibrosis-related genes in piglets after ammonia exposureIn our previous study,we performed transcriptome sequencing in the lung tissues of piglets exposed to 80 ppm ammonia with different periods of time,and found 289 differentially expressed genes(DEGs).Interestingly,the expression of 46 DEGs reached a peak in the D4 group and showed a consistent expression trend withthe preocess of pulmonary fibrosis.Therefore,we speculate that these genes may play important roles in the ammonia-induced pulmonary fibrosis.In this study,through further functional annotation and analysis of the above 46 DEGs,we found that they were mainly related to alveolar epithelial structure and function(BPIFA1,BPIFB1,BPIFB2,LBP,LTF,SCGB3A2,DMBT1,ATP6V1C2,CLDN10,SLC5A1,SLC5A8),extracellular matrix production and degradation(MMP7,FGFBP1,CP,OLFM4,AGR2,DPEP1),transcriptional regulation(BHLHA15,RPL18,DPYS,SSPO,SLC28A3)and inflammation(PI3,SAA1,PGLYRP1,CRISP3,C3,KEL,IGLVs,IGLC1,JCHAIN),in which MMP7,BPIs,IGLVs,SAA1 and PI3 may be closely related to pulmonary fibrosis and EMT.Finally,we used Q-PCR to detect the expression changes of MMP-7,TGF-β1,COL1A1,CTGF,CDH1,AQP5,S100A4,SFTPC and ACTA2 genes in the lung tissues after ammonia exposure.The expression of TGF-β1 was significantly up-regulated in D4(P<0.05)and D8(P<0.01),and COL1A1 was significantly up-regulated in D8(P<0.01).While CTGF(P<0.01),CDH1(P<0.05),S100A4(P<0.05)and ACTA2(P<0.01)was significantly down-regulated in D4 group,and AQP5 and SFTPC were significantly down-regulated in D8 group(P<0.01).Conclusion: The 80 ppm ammonia exposure induced pulmonary fibrosis in piglets,and the degree of fibrosis exhibited a first aggravation followed by a slowing down with prolonged exposure.Epithelial-mesenchymal transition(EMT)may be involved in the ammonia-induced pulmonary fibrosis in piglets. |
