Stuudy On Oxidative Stress And Surfactant Protein Expression In Lung Tissue Of Piglets Under Long-term Ammonia Exposure

In recent years,with the epidemic of classical swine fever in Africa,rapid iterations have taken place in the breeding industry,and free-range pigs have been continuously replaced by more intensive large-scale breeding sites.Due to the contradiction between ventilation and heat preservation,in large-scale pig farms,nursing piglets may be exposed to higher concentrations of ammonia for a long time,which will affect the health of piglets.Previous studies of our group have found that the nasal mucosa,trachea and lung tissues of piglets changed to varying degrees after short-term exposure to 80ppm（56mg/m³）ammonia for 12 days.In this study,a lower concentration of ammonia（20ppm or 15mg/m³）lower than the upper limit of the national standard and a higher concentration of ammonia（50ppm or 38mg/m³）higher than the upper limit of the national standard were used to stimulate 42-day-old weaned piglets for 30 days,while the control group did not add ammonia.At the end of the experiment,the lung tissue samples of three groups of piglets were collected and observed by tissue section,HE staining and transmission electron microscope,and to further explore the molecular mechanism of ammonia exposure injury of alveolar epithelial cells and affecting the expression of pulmonary surfactant protein in piglets.The results are summarized as follows:1.HE staining and transmission electron microscopic observation of lung tissue of piglets exposed to different concentrations of ammoniaHE staining showed that the alveoli of piglets shrank and the alveolar septum thickened after 30 days of 20ppm ammonia exposure.The alveolar septum was significantly thickened and the number of alveoli decreased significantly after 30 days of 50ppm ammonia exposure.Furthermore,through transmission electron microscope observation of lung tissue,it was found that after 20ppm ammonia exposure,the nuclear morphology of AECII（type II alveolar epithelial cells）became irregular,and the nuclear condensed,LB（lamellar bodies were emptied obviously.After 50ppm ammonia exposure,the nuclear atrophy of AECII was further intensified,a large number of concentrated particles appeared in the nucleus,and the phenomenon of LB emptying was obvious.Since nuclear concentration is a sign of irreducible cell injury,suggesting cell death（necrosis）in vivo,we infer that ammonia exposure to 20ppm and 50ppm can cause varying degrees of damage to AECII in lung tissue,which may cause necrosis of alveolar epithelial cells,and then induce interstitial cell proliferation,resulting in thickening of alveolar septum.2.Detection of oxidative stress signal pathway in lung tissue of piglets exposed to different concentrations of ammoniaIn order to further explore the molecular mechanism of alveolar epithelial cell injury induced by ammonia exposure,the related indexes and signal pathways of oxidative stress were further detected.Firstly,total antioxidants（T-AOC）and DNA damage marker 8-OHd G in lung tissue of piglets were determined by ELISA kit.The results showed that T-AOC decreased significantly under ammonia exposure of 20ppm and 50ppm,and 8-OHd G increased significantly after ammonia exposure of 50ppm,indicating a significant increase of reactive oxygen species in lung tissue of piglets,Oxidative damage of lung tissue induced by High concentration ammonia exposure.Then,we selected antioxidant genes SOD₂,GPX-3,HSP70 and HSPB7 for gene quantitative detection.The results of q PCR showed that the mRNA levels of the above antioxidant genes were significantly down-regulated after ammonia exposure in20ppm group,and only the mRNA level of HSPB7 gene was significantly down-regulated after ammonia exposure in 50ppm group,but the expression of other genes had no significant change.Furthermore,the mRNA and protein levels of NRF2,the core factor of oxidative stress signaling pathway,NQO1,HO-1,the downstream antioxidant enzyme gene,and p38MAPK,the three upstream regulatory factors of NRF2,were tested.The results showed that the mRNA and protein levels of NRF2,NQO1 and HO-1 genes were significantly up-regulated after ammonia exposure in 20ppm and 50ppm.）;PKC protein was significantly up-regulated after ammonia exposure in 20ppm.The above results suggest that ammonia can induce the production of reactive oxygen species,which can activate the PKC-NRF2-HO-1 oxidative stress signal pathway for antioxidant response at lower concentrations,but at high concentrations,too much reactive oxygen species can not be effectively eliminated,resulting in increased DNA damage.3.Expression of genes related to pulmonary inflammatory response in piglets exposed to different concentrations of ammoniaAs oxidative damage is often accompanied by inflammatory reaction,we selected pro-inflammatory genes IL-6,NF-kappa B（p65）and anti-inflammatory genes IL-10and IL-13 to detect the gene expression level to verify whether inflammation occurred in the lung tissue of piglets.The results of q PCR detection showed that compared with the control group,pro-inflammatory factor IL-6 was significantly up-regulated at 30days after 20ppm ammonia exposure,NF-kappa B（p65）gene was significantly down-regulated after ammonia exposure,and anti-inflammatory factors IL-10 and IL-13were significantly down-regulated after 20ppm and 50ppm ammonia exposure.The protein of NF-kappa B（p65）was quantified.The results of WB showed that NF-kappa B（p65）protein was significantly up-regulated in 20ppm group and significantly down-regulated in 50ppm group.These results suggest that early inflammatory reaction may occur in lung tissue exposed to 20ppm ammonia,mainly pro-inflammatory reaction,while late inflammatory reaction may occur in 50ppm ammonia exposure,that is,tissue damage and repair.4.Results of study on gene expression of pulmonary surfactant protein under different concentrations of ammonia exposureThe above results suggest that ammonia exposure can induce the damage of AECII in alveolar epithelial cells,and one of the important functions of AECII is to synthesize and secrete surfactant proteins to maintain alveolar surface tension and participate in immune defense.Therefore,we detected the gene expression of two main pulmonary surfactant proteins,SP-A1 and SP-D,in lung tissue.The results of q PCR and WB showed that after ammonia exposure in 20ppm,compared with the control group,the mRNA and protein expression patterns of SP-A1 and SP-D genes were the same,and the mRNA expression of SP-A1 gene was significantly up-regulated after ammonia exposure of 50ppm,while the expression of SP-D gene did not change significantly,but the expression of SP-A1 and SP-D protein decreased significantly（P<0.01）.Through the establishment of ammonia-stimulated A549 cell model,the cell injury and the expression of surfactant protein gene were further studied.The results showed that compared with the control group,both low concentration（20mmol/L）and high concentration（40mmol/L）of NH₄Cl could induce apoptosis and cytotoxicity of A549 cells,and high concentration of NH₄Cl could significantly increase the damage of DNA and lipid oxidation.Immunofluorescence colocalization assay was used to detect the protein transport process of SP-A1 and SP-D.It was found that the protein transport was blocked after A549 cells were stimulated with high concentration of NH₄Cl for 36 hours.It is suggested that high concentration of ammonia exposure may affect the secretion of surfactant proteins by blocking protein transport.Conclusion and significance:this study found that long-term exposure of ammonia to the respiratory tract could induce oxidative stress and inflammation in the lungs of piglets,leading to alveolar epithelial cell necrosis and pulmonary interstitial thickening.The surfactant proteins with immune function and reduced alveolar surface tension were induced by low concentration ammonia exposure and inhibited by high concentration ammonia exposure.The results can provide a theoretical basis for further analysis of the mechanism of ammonia toxicity.