| Ammonia is a harmful environmental pollutant and one of the components of haze.Ammonia can disturb the normal physiological activities in the body,and exogenous ammonia exposure can induce various diseases and seriously endanger the health of animals and people.Many studies have shown that ammonia is associated with an increased incidence of respiratory diseases,necroptosis is the main form of external stimuli leading to cell death,this form of cell death plays an important role in respiratory diseases.With the gradual deepening of the mechanism of action of non-coding RNA in the body,regulating the expression of downstream genes through the competitive binding of LncRNA to micro RNA is extremely important in exploring the mechanisms of disease development,at present,research on the regulation of human disease progression by Ce RNA has gradually become a hot spot,However,the mechanism of Ce RNA on avian tracheal injury has rarely been reported.Therefore,this experiment uses broilers as research animals,continuously introducing ammonia gas into the environmental control chamber（0-3weeks 20 mg/m3,4-6 weeks 45 mg/m3）,establishing an ammonia exposure model and an LMH cell ammonium chloride exposure model based on,scanning electron microscopy,transmission electron microscopy,transcriptomics detection,immunoblotting,real-time fluorescent quantitative PCR,dual luciferase reporter gene system,flow cytometry,bioinformatics methods,etc.,to observe the morphological changes of tracheae and detect LncRNA and m RNA expression profiles,immune-inflammatory related factors,antioxidant genes,and programmed necrosisrelated genes,Analysis of the relationship between LncRNA-107053293 and mi R-148a-3p-FAF1.The test results are as follows:（1）Ammonia exposure can cause partial loss of inner membrane structure in chicken,tracheal wall,Tracheal cilia,Incomplete alignment,irregular ciliated cell structure,Local inflammatory cells and lymphocytic infiltration.Ultrastructural observation of tracheal epithelial ciliated cell nuclear chromatin condensation,Mitochondrial vacuolation,cell collapse.It indicates that ammonia exposure can cause inflammatory injury and programmed necrosis of chicken tracheal tissue.（2）Ammonia exposure can affect changes in chicken tracheal transcriptomics,differential expression of 81 LncRNA and 1007 m RNA（P < 0.05）,48 LncRNAs and 433 m RNAs were upregulated,33 LncRNAs and 574 m RNAs were down-regulated（P < 0.05）.GO and KEGG enrichment were applied to differentially expressed m RNA,and it was found that genes with significant differences were mainly enriched in primary immunodeficiency,immune inflammatory response,oxidative stress and cell death.Ammonia exposure can cause the decrease of Gpx,CAT,SOD,T-AOC and four ATPase activities and GSH content in chicken tracheae,while H2O2 and MDA levels rise（P < 0.05）,the expression of CYP450 and heat shock protein is increased,and that of antioxidant genes is decreased（P < 0.05）,and the expression levels of proinflammatory cytokines IL-4,IL-6,IL-17,IL-1β,FOXP3 and TNF-α are significantly elevated.While there is a decrease in IFN-γ expression（P < 0.05）,the above results verify the accuracy of transcriptomics.（3）LncRNA-107053293 was down-regulated（P < 0.05）and the expression of mi R-148a-3p was increased in ammonia-exposed chicken tracheal tissues（P < 0.05）.The dual luciferase reporter system assay further confirmed that LncRNA-107053293 was able to target binding.mi R-148a-3p,and FAF1 was a targeted downstream gene of mi R-148a-3p,LncRNA-107053293 is capable of competitively adsorbing mi R-148a-3p to regulate FAR1 expression.（4）Ammonia exposure up-regulated the expression levels of I-κB-β,i NOS and COX-2 m RNA and protein in the NF-κB pathway via LncRNA-107053293/mi R-148a-3p-FAF1 pathway（P < 0.05）,and down-regulated I-κB-α m RNA and protein expression levels（P < 0.05）.Activation of the NF-κB pathway further triggered the NLRP3-MLKL pathway,causing an increase in the m RNA and protein levels of NLRP3-MLKL,which ultimately led to programmed necrosis of chicken tracheal cells.In summary,ammonia exposure can cause programmed necrosis of cells,and affect the transcriptomics of chicken tracheal tissue,leading to decreased antioxidant capacity and immune function.LncRNA-107053293 can competitively adsorb mi R-148a-3p to regulate FAF1 expression,and ammonia exposure induces NF-κB/NLRP3-MLKL signaling pathway through LncRNA-107053293/mi R-148a-3p-FAF1 to induce programmed cell necrosis.The results of this test indicate that ammonia exposure can cause programmed necrosis of the trachea and elucidate the mechanism of Ce RNA.It provides a basis for the research of ammonia toxicology and provides a possible treatment basis for diseases related to ammonia poisoning... |
