Ammonia regulates chicken tracheal cell necroptosis via the LncRNA-107053293/MiR-148a-3p/FAF1 axis
Graphical abstract
Introduction
Ammonia (NH3) is a component of haze and typically arises from livestock waste treatment and volatile synthetic fertilizers (Beusen et al., 2008; Singleton et al., 2005), petroleum smelting (Paerl et al., 2014), wildlife reserves for birds, seals, and sea lions and forest fires (Theobald et al., 2006; Uematsu and Mitsuo, 2004). In the global distribution map of ammonia pollution, it is seen that ammonia pollution caused by human activities dominates, and haze with high ammonia concentrations is found in urban areas. NH3 can also form secondary organic aerosols, and as the concentration of ammonia increases, the damage caused by haze to humans and animals also increases (Leith et al., 2009; Bauer et al., 2016). As early as the last century, long-term exposure to an ammonia-containing environment was found to shorten vibrissa or amotio (Oosterhuis, 1951). Exposure to ammonia can also cause the tracheal cilia to produce increased mucus or the cilia to be removed, directly exposing the basal layer of the tracheal cilia. Additionally, exposure to NH3 can also cause chronic respiratory disease and significantly worsen the symptoms of emphysema and asthma (Coltart et al., 2013; Dutton et al., 1959; Warren, 1962). In broilers, high concentrations of NH3 reaching 70 mg/m3 can interfere with the development of immune organs and gut villi, influence the feed conversion ratio, and finally decrease the absorption of nutrients in gut villi and the quality of broilers (Zhang et al., 2015). A decreased body weight is positively related to an increased NH3 concentration (0–75 ppm) (Miles et al., 2004). However, most ammonia in the alveoli inhaled through the respiratory tract is absorbed into the blood, causing the blood ammonia concentration to increase and the state of the central nervous system to be paralyzed after excitement (Schaerdel et al., 1983). Ammonia exposure can also cause fat-soluble changes in the cell membrane of rats which, in turn, can cause hepatic steatosis and myocardial damage (Dasarathy et al., 2017; David and Michael, 2018).
Necroptosis functions in many pathophysiological processes. PM 2.5 exposure induced RIP/MLKL-dependent necroptosis in mouse lungs, and this dependence was also confirmed in the 16 HBE cell line (human tracheal epithelial cell line) (Dornhof et al., 2017; Maxey et al., 2004). In the mouse-ARDS (acute respiratory distress syndrome) model, the ratio of apoptosis to necrosis was affected by the dose of lipopolysaccharide (LPS), where RIPK3 and MLKL were increased with increasing LPS dose, which induced necroptosis; necrostatin-1 (Nec-1) treatment or RIPK3 hereditary deficiency improved symptoms in rat respiratory distress syndrome (Pan et al., 2016). Competing endogenous RNA (ceRNA) generally refers to long-noncoding RNA (lncRNA) and circular RNA (cirRNA), both of which regulate protein-coding RNA via competitive binding with microRNA (miRNA). CeRNA binding to miRNA plays a role in the regulation of gene expression patterns through microRNA response elements (MREs). CeRNA can affect biological processes, signal transduction and cell growth, and ceRNA also regulates necroptosis, which affects proliferation, cell death and disease treatment (Bossi and Figueroa-Bossi, 2016; Su et al., 2019). In liver cancer research, the depletion of Linc00176 could induce necroptosis in HCC (Hepatocellular carcinoma) cells by releasing tumor suppressors miR-9 and miR-185 and targeting Myc (myeloma cell oncogene) activation; this provides an effective target for the treatment of liver cancer (Tran et al., 2018). Furthermore, in the study of ischemia apoplexy treatment, MEG3 acts as a ceRNA and combines with miR-21 in competition with programmed cell death 4 (PDCD4), mediating the necroptosis caused by PDCD4 (Yan et al., 2017). Generally, we know that necroptosis is negatively regulated by Fas-associated death domain protein (FADD). FAF1 is a member of Fas-DISC (Fas-death inducing signaling complex), which functions upstream of caspase 8 and interacts with caspase 8 and FADD; when caspase 8 and FADD are suppressed, FAF1 is artificially increased, inhibiting cell death (Ryu et al., 2003). FAF1 also has a negative effect on NF-κB-p65 entering the karyon to mediate necroptosis (Park et al., 2004; Chin et al., 2019).
In summary, NH3 causes damage to the respiratory tract. CeRNA is involved in the occurrence and development of respiratory system injury and may play a role in regulating cell necroptosis, and the occurrence of bronchiolitis is related to the abnormal expression of lncRNA and miRNA (Dong et al., 2015). Whether the damage caused by ammonia exposure to tracheal cells can be regulated by a ceRNA mechanism is unknown; therefore, the purpose of this study was to explore whether necroptosis is involved in the pathological injury of trachea tissue caused by ammonia exposure. The effect of ammonia exposure on the expression of lncRNA and miRNA was investigated to illuminate which lncRNAs regulate necroptosis by ceRNA and participate in tracheal injury caused by ammonia exposure. Therefore, we established an ammonia-exposed broiler model, observed the effect of ammonia on the morphology of chicken trachea by transmission electron microscopy, detected differentially expressed lncRNAs and miRNAs by transcriptomics sequencing, and screened the target gene FAF1 by using a bioinformatics website. The dual-luciferase reporter gene system verified the targeting relationship between lncRNA-107053293 and miR-148a-3p, miR-148a-3p and FAF1, and changes in the relevant indicators of necroptosis were detected by flow cytometry, RT-PCR, and western blotting. These results further clarify the biological mechanism of chicken trachea damage caused by ammonia and establish a deeper theoretical basis for the study of ammonia toxicology.
Section snippets
Establishment of animal model and groups
All procedures used in our experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. The animals were raised in environmentally controlled chambers, and the grouping and treatment of the experimental animals was as previously published (Shi et al., 2019). The NH3 treatments were 19.5–20.5 mg/m3 (0–3 weeks) and 44.5–45.5 mg/m3 (4–6 weeks) according to the national standard. The temperature, relative humidity and light time in the two
Observations of tracheal cell ultrastructure
Transmission electron microscopy (TEM) was performed to observe the ultrastructure of tracheal cells from group C and group H. In group C, the tracheal cells showed normal morphology, the cell membrane edge was clear and intact, and the mitochondrial size was normal without abnormal structural changes. However, we found many typical features of necrosis in group H, including concentrated ciliated cell nuclei, absent nucleoli, mitochondrial swelling, vacuolization, and even fragmentation in the
Discussion
Exposure to harmful substances can cause damage to tracheal cells (Qu et al., 2019; Zhao et al., 2018; Wang et al., 2018, 2019a). Cell necrosis mainly refers to cell cytoplasmic swelling, membrane rupture and cell lysis (Galluzzi et al., 2011; Chu et al., 2018). For example, exposure to PM 2.5 leads to the damage of cytoplasmic nuclei in the cilia of respiratory bronchioles and increases MLKL expression (Zhao et al., 2019). By studying necrosis in lung cancer cells, it was found that LINC00336
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported by the National Key Research and Development Program of China (No. 2016YFD0500501) and China Agriculture Research System-41-17.
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