s2fieldsofstudy: Biology Medicine

text:
Panax Notoginseng Saponins Regulate Transforming Growth Factor-β1 through MAPK and Snail/TWIST1 Signaling Pathway to Inhibit Epithelial-Mesenchymal Transition of Pulmonary Fibrosis in A549 Cells

Panaxnotoginseng saponins (PNS) is one of the active components of traditional Chinese medicine Panax notoginseng which has the function of reducing oxygen consumption, expansion of the cerebrovascular system, and is antithrombotic. PNS also plays a role in the treatment of pulmonary fibrosis. In this study, we found that PNS suppresses fibroblast-like changes in A549 cells through epithelial-mesenchymal transition (EMT). PNS promoted E-cadherin (E-cad) in epithelial cells and decreased Fibronectin (FN) and Vimentin (Vim) expression in myofibroblasts in a dose-dependent manner. Further mechanism studies have shown that PNS inhibits the EMT process by regulating p38, JNK, and Erk signaling factors in the MAPK signaling pathway and then blocking Snail and TWIST1 transcription factors from entering the nucleus. This indicates that PNS can regulate epithelial-mesenchymal transition through MAPK and the Snail/TWIST1 signaling pathway, thereby exerting its antipulmonary fibrosis effect.

Introduction
Pulmonary fibrosis (PF) is a group of noninfectious and noncancerous respiratory diseases and is the final pathological manifestation of many interstitial lung diseases. PF has extensive extracellular matrix deposition, myofibroblast accumulation, and lung remodeling [1,2]. Although many studies have focused on patients with PF, the underlying cause of the disease remains unclear. Patients are therefore often diagnosed with idiopathic interstitial pneumonia (IIP), the most common of which is idiopathic pulmonary fibrosis (IPF) [3,4]. PF usually manifests as exertional dyspnea. With the development of the disease, PF patients will eventually die of respiratory failure. Common clinical symptoms of PF, including fatigue, dry cough, and dyspnea, seriously affect the overall quality of life of the patient [5]. Since there is no treatment to reverse pulmonary fibrosis, slowing disease progression, relieving symptoms, and improving quality of life are the main goals of intervention. e drugs used in the treatment of PF mainly include immunosuppressants, corticosteroids, and antifibrosis drugs, such as Pirfenidone and Nintedanib [6,7]. However, due to its complex etiology and unclear pathogenesis [8], the efficacy of existing drugs is still not satisfactory. Finding effective new drugs for PF has become a clinical demand and a hot research topic [9].
Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their functions and properties under certain physiological or pathological conditions and transform into mesenchymal cells. Its main biological functions are to make fibroblasts and repair tissue damage caused by wounds and inflammation [10]. EMT plays an important role in embryonic development, chronic inflammation, tissue remodeling, cancer metastasis, and various fibrotic diseases. During EMT, epithelial cells lose their attachment to the basement membrane and acquire a migratory and invasive phenotype. Evidence suggests that EMT promotes the development of pulmonary fibrosis [11]. Among the various stimuli involved in pulmonary fibrosis, transforming Growth Factor-β1 (TGF-β1) is one of the important factors. It has been proved that TGF-α1 can induce EMT and induce signal transduction through classical pathway involving downstream phosphorylation of Smad 2/3 or other pathways, such as Wnt/β-catenin signal pathway [12].
Panax notoginseng saponins (PNS) are effective medicinal components extracted from Panax notoginseng, including a variety of saponin active substances, trace elements, proteins, rich vitamins, and polysaccharides. PNS has the effects of reducing oxygen consumption, dilating cerebral vessels, and it is antithrombotic, anticoagulant, and antifibrotic [13,14]. Studies on PNS on bleomycin-induced rat IPF have shown that PNS can improve and delay bleomycin-induced pulmonary fibrosis in rats [15]. Additional studies have reported that PNS could improve cardiopulmonary function and inhibit the progression of pulmonary fibrosis through the NF-κB pathway [16].
Mitogen-activated protein kinases (MAPK), also known as serine/threonine protein kinases, can be activated by different extracellular stimuli such as cytokines, neurotransmitters, and hormones. Cell stress and cell adhesion can activate the MAPK signaling pathway, phosphorylate nuclear reverse transcription and regulate the corresponding genes involved in tissue fibrosis. Experiments confirmed that the MAPK signaling pathway is activated in TGF-β1-induced pulmonary fibrosis signal transduction [17,18]. MAPK signaling can inhibit the activity of GSK-3β and block the degradation of Snail transcription factor, resulting in its increased accumulation in the nucleus. It was shown that the MAPK pathway can increase the level of Snail, thereby reducing the expression of E-cadherin and promoting the occurrence of EMT [19]. erefore, our research group intends to establish the A549 cell model to determine whether PNS can inhibit EMT through the MAPK signaling pathway and to further reveal the role and mechanism of PNS in inhibiting pulmonary fibrosis.

Cell Culture and Grouping
2.2.1. Cell Culture. Epithelial cells of human lung cancer A549 cell line were purchased from the National Experimental Cell Resource Sharing Platform (Beijing, China). A549 cells were cultured with F-12 medium (containing 100 IU/mL penicillin, 100 mg/L streptomycin and 10% fetal bovine serum) at 37°C in a 5% CO2 incubator. After reaching 80% cell confluence, digested, passaged or related experiments were performed using 0.25% Trypsin.

Establishment of Models and
Grouping. A549 cells cultured in vitro were randomized into the control, TGF-β1 group and treatment group. e control group was A549 cells + F-12 medium. TGF-β1 group was A549 cells + F-12 medium + 10 ng/ml TGF-β1. e treatment groups were A549 cells + F-12 medium + PNS concentration with different gradients of 62.5 μg/ml, 125 μg/ml, and 250 μg/ml. e cells were cultured for 72 h.

MTT Cell Viability
Assay. Cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well and incubated overnight. Cells were treated with PNS and PNS + TGF-β1 for 72 h. MTT reagent (5 mg/ml) was added to each well and incubated for an additional 4 hours at 37°C. However 100 μL of lysis buffer was added to each well and incubated overnight at room temperature. Finally, the microplate reader was set to detect the optical density (OD) value at 570 nm. e experiments were performed thrice, each in triplicate.

Cell Morphology Observation Experiment.
e cells in each well of the 6-well plate were observed and imaged in a visual field magnified 200x using an inverted microscope. Five samples were randomly selected for each group to take photos of, and five random visual fields were counted for each sample for cell morphology detection. Based on a previously established quantitative analysis of cell morphology, the effect of PNS on the morphological structure of A549 cells was tested. Cell roundness was quantified using the image Pro-plus 6.0 software.

Transwell Chemotaxis Assay.
e cells were digested with trypsin (0.25%), and the supernatant was discarded. e cell suspension was added without FBS medium to the transformation chamber (100-200 μl/well). e chamber was placed in medium supplemented with 20% FBS. e medium was poured into the cross-well chamber and the bottom of the chamber was wiped with a cotton swab. Placed 4% paraformaldehyde (500 μl/well) into the converter chamber, stained with 0.1% crystal violet solution, and soaked for 30 min. Slices were rinsed twice with distilled water.
e chamber was placed on the slices and the 2 Evidence-Based Complementary and Alternative Medicine migrating cells were visualized by an inverted microscope under the condition of 50x magnification. After incubation at 72 h, cells were stained with crystal violet solution and counted under a microscope for migration. Imagination in a visual field is magnified 100x using an inverted microscope.
2.6. Quantitative Real-Time PCR. One-step extraction of total RNA after drug intervention using Trizol. e purity of the RNA was identified and then transcribed backwards onto cDNA. e cDNA was used as a template, and primers of the target genes (FN, Vim, E-cad, Snail, and TWIST1) and internal reference gene (β-actin) were added into the reaction system. e reaction conditions were set for PCR amplification of the target gene.
ere was a linear relationship between the CT value of the template and the number of initial copies of the template. Among them, FN, Vim, E-cad, Snail, TWIST1, β-actin, and mRNA full-length PubMed sequence were used as templates, and primers for PCR were designed using Oligo software. A7500 real-time fluorescent quantitative PCR system ( ermo Fisher, Waltham, MA, US) was used.
e primer sequences are listed in Table 1.

Western Blot Analysis.
e A549 cells were washed twice with PBS, an appropriate amount of cell lysate was added, ice bathed for 30 min, and the supernatant was collected after high-speed centrifugation. e protein concentration was determined by the BCA method, adjusted to the standard concentration, and 10-20 μL samples were taken, respectively. Perform SDS-PAGE electrophoresis and transfer to the PVDF membrane by the semidry transfer method. 5% nonfat milk powder was blocked for 1 h, and primary antibodies were added respectively at the following dilution ratios: β-actin, FN, Vim, E-cad, Snail, TWIST1, p-p38, p-JNK, p-Erk, incubated overnight in a 4°C freezer, and then used as antibody Rabbit IgG secondary antibody was incubated at 37°C for 1 h. e membrane was washed with PBST buffer for 10 min × 3 times, and the protein expression on the membrane was observed by the electrophoresis gel imaging analysis system (Tanon, Pujiang High-tech Park, SH, China).

Immunofluorescence
Detection. A549 cells were seeded on sterile cover glasses placed on six well slabs. e cells were fixed with 4% paraformaldehyde formaldehyde, infiltrated with 0.1% Triton X-100 for 10 minutes and blocked with 1% bovine serum albumin for 10 minutes. Cells were placed at room temperature and incubated with anti-E-cad antibody, anti-Vim antibody, anti-FN antibody, antiphospho-p38 antibody, antiphospho-JNK antibody, and antiphospho-Erk antibody overnight at 4°C. Cells were washed with PBS and incubated with goat anti-rabbit IgG (A-11010) at room temperature in the dark. Nuclei were counterstained with Hoechst 33342 for 5 min. Cells were fixed with antifade solution and visualized by fluorescence microscopy at 500x magnification.
2.9. Statistical Analysis. All statistical analysis was performed utilizing SPSS programming (SPSS 19.0). Comparisons between multiple groups were performed by oneway ANOVA. In the case of homogeneous variance, the LSD method was used to compare the data. When variance between groups was not homogeneous, data were compared using Dunnett's T3 test. P < 0.05 was a statistical difference.

PNS Did Not Significantly Inhibit the A549 Cells.
After different concentrations of PNS and PNS + TGF-β1 were treated for 72 h, MTT results showed that 31.25∼500 μg/ml PNS and PNS + TGF-β1 did not significantly inhibit the A549 cells ( Figure 1).

PNS Attenuated the Performance of Pulmonary Fibrosis in the A549 Cell Model.
e intervention effect of PNS on the TGF-β1-induced pulmonary fibrosis model was observed under an inverted microscope. As shown in Figure 2, TGF-β1 showed a significant change in cell morphology from paving stone to spindle shape with a significant decrease in the circularity compared to the control group. Cell circularity was significantly increased in the 125 μg/ml and 250 μg/ml PNS Treatment groups compared to the TGF-β1 group. e above experimental results show that PNS can effectively improve and alleviate TGF-β1-induced cell fibrosis-like changes while preserving the morphological structure of epithelial cells.

PNS Inhibits Chemotactic Recruitment of Fibrotic Cells.
e effect of PNS on the chemotactic activity of A549 cells was measured by transwell. e chemotaxis of cells in the TGF-β1 group was higher than that in the control group, as shown in Figure 3, indicating that TGF-β1 could facilitate the chemotactic recruitment of A549 cells. However, after PNS treatment at 125 μg/ml and 250 μg/ml concentrations, the cell chemotaxis index was greatly reduced. It was demonstrated that PNS could attenuate the chemotactic activity of TGF-β1 on A549 cells, thereby blocking the progression of pulmonary fibrosis.

PNS Can Inhibit the EMT Process by Promoting Epithelial Cell Markers and Reducing the Expression of Mesenchymal Cell Markers.
e role and mechanism of PNS in the TGF-β1-induced pulmonary fibrosis cell model were investigated by measuring E-cad, FN and Vim. As shown in Figures 4(a) and 4(b), compared with the control group, the mRNA and protein expressions of FN and Vim in the TGF-β1 group were increased, while the mRNA and protein expressions of E-cad in the TGF-β1 group were decreased, indicating that TGF-β1 can significantly mediate the EMT process. Notably, PNS treatment increased the expression of the epithelial cell markers E-cad mRNA and protein, and decreased the expression levels of mesenchymal cell markers FN and Vim mRNA and protein. e results showed that PNS inhibited the occurrence of EMT in a concentration-dependent Evidence-Based Complementary and Alternative Medicine manner, so the 250 μg/ml treatment group was selected to continue the next experiment. As shown in Figure 4(c), the E-cad fluorescence in the TGF-β1 group was significantly weakened, and the E-cad fluorescence expression was enhanced after adding PNS treatment. In addition, the fluorescence of FN and Vim increased significantly in the TGF-β1 group, and the fluorescence expression of FN and Vim decreased after adding PNS treatment.

PNS Prevents EMT by Inhibiting TGF-β1-Induced MAPK
Signaling Pathway. In order to observe the relationship   Evidence-Based Complementary and Alternative Medicine between the induction time of TGF-β1 and the expression intensity of the MAKP signaling pathway, we measured the expression levels of p38, p-p38, JNK, p-JNK, Erk, and p-Erk at different times. As shown in Figure 5(a), it was found that p-p38, p-JNK, and p-Erk protein increased most significantly after 72 hours of TGF-β1 stimulation, indicating that 72 hours was the best time for TGF-β1 to induce the MAPK pathway expression. Figure 5(b) shows that TGF-β1 can increase p-p38, p-JNK, and p-Erk protein. e addition of PNS treatment decreased p-p38, p-JNK, and p-Erk proteins. We confirmed the above results using the immunofluorescence assay. e results in Figure 5(c) indicated that the fluorescence of p-p38, p-JNK, and p-Erk was significantly enhanced in the TGF-β1 group, and the fluorescence expression was weakened after adding PNS treatment. To ensure the association between the MAPK signaling pathway and EMT, inhibitors of three signaling factors were added: p38, Erk, and JNK. As shown in Figure 5(d), E-cad was decreased by TGF-β1 and restored by MAPK inhibitor. Mesenchymal markers FN and Vim are increased by TGF-β1 and decreased by MAPK inhibitor. TGF-β1-induced MAPK phosphorylation signaling factors p38, Erk, and JNK were significantly inhibited by the addition of PNS treatment. Treatment with three MAPK signaling factors p38, Erk, and JNK inhibitors was found to be the same as PNS treatment. Prove that PNS can prevent the occurrence of the EMT process through the MAPK signaling pathway.

PNS Can Prevent the Expression of Snail and TWIST1
Induced by TGF-β1 While Regulating Snail, and TWIST1 Can Improve the EMT Induced by TGF-β1 in A549 Cells. Snail and TWIST1 are classic transcription inhibitors that induce EMT, which regulate the expression of E-cad and Vim.
rough this mechanism, they induce the occurrence of EMT in the epithelial-mesenchymal transition. As shown in Figures 6(a) and 6(b), qPCR and Western blotting were performed to examine whether TGF-β1 promotes Snail and TWIST1 expression and whether PNS eliminates EMT processes through snail and TWIST1. In these results, Snail and TWIST1 transcripts were upregulated by TGF-β1 and inhibited by PNS, SB203580, PD98059, and SP600125. e results in Figure 6(c) demonstrate that PNS, SB203580, PD98059, and SP600125 blocked the action of TGF-β1, thereby inhibiting the migration and accumulation of Snail and TWIST1 from the cytoplasm to the nucleus. ese data suggest that Snail and TWIST1 are key transcription factors for PNS blockade of EMT.

Discussion
It was considered many years ago that the pathogenesis of pulmonary fibrosis is the result of progressive inflammation and cell damage leading to the activation and proliferation of lung interstitial cells [20,21]. However, with the development of medicine, more and more research confirms that inflammation and chronic fibrosis are not necessarily related [22]. Recent research suggests the EMT phenomenon is very closely related to pulmonary fibrosis. EMT reduces E-cad in epithelial cells, increases FN, and Vim in fibroblasts, and promotes fibrotic remodeling [23][24][25]. Our results suggest that TGF-β1 stimulation significantly altered cell morphology from pebbly to spindly and promoted chemotactic recruitment in A549 cells. However, chemotactic recruitment of A549 cells was attenuated after PNS treatment and maintained their original morphology, and PNS increased E-cad expression and decreased FN and Vim expression. Our findings are essentially in line with previous studies, but this study has further explored its mechanism. We studied the Evidence-Based Complementary and Alternative Medicine effects of PNS on the MAPK signaling pathway and related transcription factors Snail and TWIST1 to explore the deep mechanism of PNS on Pulmonary fibrosis [26,27]. Overall, this data indicated that PNS could effectively improve and treat pulmonary fibrosis through the MAPK signaling pathway and related transcription factors in an in vitro model.
EMT is a process in which cells with an epithelial phenotype differentiate into a mesenchymal phenotype, and it is involved in physiological processes such as human embryonic development and wound repair. It also plays a key role in human organ fibrosis and tumor pathogenesis. e process of EMT is inseparable from TGF-β1. During TGF-β1-induced EMT, epithelial cells lose cell-cell connections, induce causing them to isolate from surrounding cells and acquire mesenchymal-like characteristics. TGF-β1, a major fibroblast promoter of IPF progression, induces  Figure 4: By detecting EMT-related PNS, it was found that PNS could effectively inhibit the occurrence of TGF-β1-induced EMT. e gene and protein of E-cad, FN, and Vim were detected by qPCR and Western blotting, respectively (a)-(b). * P < 0.05. * * P < 0.01. * * * P < 0.001 vs. Control group. ＃ P < 0.05. ＃＃ P < 0.01. ＃＃＃ P < 0.001 vs. TGF-β1 group. (c) en immunofluorescence technology was selected to further detect the fluorescence expression levels of EMT signal molecules E-cad, Vim, and FN (magnification, ×500).
differentiation of lung fibroblasts into myofibroblasts, produces high levels of collagen, and results in loss of lung elasticity and function [28]. TGF-β1 can promote or inhibit the growth of epithelial cells and can also participate in the differentiation of alveolar epithelial cells and the activation of fibroblasts [29]. Some researchers have also found that TGF-β1 contributes to the disturbance of the lung microenvironment, which leads to the accumulation of a large number of fibroblasts and the deposition of extracellular matrix, accompanied by inflammation and damage to the lung structure, eventually leading to pulmonary fibrosis [30]. TGF-β1 could mediate EMT through the classical pathway, bind to the TGF-β membrane receptor, and then activate the receptor serine/threonine kinase [29]. Subsequently, Smad is phosphorylated and transferred from the cytoplasm to the nucleus, leading to the activation of the Snail family and the inhibition of E-cad, which ultimately mediates EMT. In addition, TGF-β1 also mediates EMT through nonclassical pathways and phosphorylates downstream protein kinase B (AKT) through phosphoinositide-3-kinase (P13K) and MAPK pathways (JNK, p38, Erk), resulting in loss of phosphorylation. Live glycogen synthase kinase 3 (GSK-3B) inhibits Snail phosphorylation and enucleation degradation and increases the level of Snail in the nucleus. Fundamental components of the MAPK signaling pathway are a highly conserved tertiary enzymatic cascade from yeast to humans, including MAP kinase kinase kinase (MKKK), MAP kinase kinase (MKK), and MAPK. ese three kinases can be activated in turn and together regulate a variety of important cellular physiological and pathological processes, including cell growth and differentiation [31]. Evidence suggests that EMT is regulated by multiple transcription factors, such as Snail and TWIST1. Snail is an important transcriptional inhibitor regulating E-cad expression in epithelial-mesenchymal transition. e high expression of Snail is a necessary condition for the occurrence of EMT in pulmonary fibrosis. Zhu et al. [32] To study paraquat (PQ) intoxication-induced pulmonary fibrosis and found that hypoxia-inducible factor-1α (HIF-1α) and EMT are linked to the progression of pulmonary fibrosis. e (c) en select immunofluorescence technology to further detect the fluorescence of p-p38, p-Erk, and p-JNK signaling molecules related to the MAPK signaling pathway (magnification, ×500). (d) After adding 3 kinds of MAPK signal pathway inhibitors (SB203580/PD98059/SP600125), Western blotting was used to detect E-cad, FN, and Vim protein related to EMT. * P < 0.05. * * P < 0.01. * * * P < 0.001 vs. Control group. ＃ P < 0.05. ＃＃ P < 0.01. ＃＃＃ P < 0.001 vs. TGF-β1 group.   Evidence-Based Complementary and Alternative Medicine findings suggest that HIF-1α induces EMT by promoting Snail transcription factor, which leads to pulmonary fibrosis. However, TWIST1 is also a critical transcription factor that induces EMT and can regulate the expression of Vim. Mammoto et al. [33] found that vascular remodeling is one of the pathogenesis of pulmonary fibrosis, and they proved that the transcription factor TWIST1 can promote the occurrence of pulmonary fibrosis epithelial-mesenchymal transition. erefore, regulating the expression of TWIST1 is a curative strategy for pulmonary fibrosis. Taken together, ectopic expression of Snail and TWIST1 may be involved in reducing epithelial cells and increasing mesenchymal cells to induce EMT, ultimately leading to pulmonary fibrosis. Our findings suggest that PNS can effectively inhibit the migration of Snail and TWIST1 from the cytoplasm to the nucleus, preventing EMT and further treating pulmonary fibrosis.
Panax notoginseng saponins is one of the main active ingredients of Panax notoginseng, a commonly used blood activating herb in Chinese medicine, and has a variety of pharmacological effects, including enhancing immunity, promoting tissue growth, and antitumor effects [34,35]. In recent years, several research studies have shed light on the significant therapeutic effect of PNS on lung fibrosis. For instance, the mechanism of action of PNS in the treatment of pulmonary fibrosis was studied, and the expression of IL-6 and IL-8 in the PNS group was significantly decreased [16]. ey proved that PNS can reduce AST, LDH, CK, IL-6, and IL-8 in rabbit serum by inhibiting the NF-κB signaling pathway and mitigate pulmonary fibrosis. Liu et al. [15] explored the effect of Panax notoginseng saponins inhalation solution (TIS) on bleomycin-induced IPF in rats, and found that TIS could improve and delay bleomycin-induced IPF in rats. Our results are consistent with previous studies suggesting that TGF-β1 increases FN and Vim expression and decreases E-cad levels. In this study, PNS reversed the progression of pulmonary fibrosis by blocking the expression of transcription factors Snail and TWIST1 through the MAPK signaling pathway and inhibiting epithelial-mesenchymal transition (Figure 7).

Conclusions
Taken together, our study shows that PNS can improve and treat pulmonary fibrosis by modulating EMT occurrence through MAPK and Snail/TWIST1 signaling pathways. Currently, COVID-19 is spreading around the world, and some patients still have pulmonary fibrosis after being cured [36,37]. erefore, finding effective drugs for the treatment of PF has become an urgent need and a research hotspot.

Data Availability
Datasets used and/or analyzed in this study can be obtained from the corresponding authors upon reasonable request.

Authors' Contributions
VietDung Nguyen, Yang Lu, and GuangZhong Zhang designed the study. Experiments were conducted by Viet-Dung Nguyen, Fei Pan, and Qing Zhang. VietDung Nguyen and Fei Pan analyzed the data and wrote the manuscript. Yang Lu and GuangZhong Zhang reviewed and revised the original draft. All authors have read and agreed to take responsibility for the manuscript and to ensure that any part of the work is properly investigated and processed for accuracy and integrity. VietDung Nguyen and Fei Pan made equal contributions to this work and should be considered co-first authors.


Transcriptome Revealed Exposure to the Environmental Ammonia Induced Oxidative Stress and Inflammatory Injury in Spleen of Fattening Pigs

Simple Summary Ammonia is a major environmental pollutant. Previous estimates of ammonia emissions have focused on livestock sources in agricultural systems. Livestock continues to be the main source of ammonia emissions. Exposure to high concentrations of ammonia can cause varying degrees of damage to tissues and organs. However, the damage of ammonia exposure to the spleen of pigs in the fattening pigs is unknown. Therefore, the aim of this study was to explore the mechanism at the gene level of exogenous ammonia-induced spleen toxicity by enzyme-linked immunosorbent assay (ELISA), spleen histomorphological observation, and transcriptome technology. The results showed that ammonia exposure led to oxidative stress, activation of inflammatory pathways, and splenic injury. In addition, the genes that encode histone methyltransferase were found to be significantly upregulated. Therefore, histone methylation may be the epigenetic mechanism of splenic poisoning induced by ammonia. Our findings provide a novel direction for exploring the underlying molecular mechanisms of ammonia toxicity. Abstract Ammonia is one of the major environmental pollutants that seriously threaten human health. Although many studies have shown that ammonia causes oxidative stress and inflammation in spleen tissue, the mechanism of action is still unclear. In this study, the ammonia poisoning model of fattening pigs was successfully established. We examined the morphological changes and antioxidant functions of fattening pig spleen after 30-day exposure to ammonia. Effects of ammonia in the fattening pig spleen were analyzed from the perspective of oxidative stress, inflammation, and histone methylation via transcriptome sequencing technology (RNA-seq) and real-time quantitative PCR validation (qRT-PCR). We obtained 340 differential expression genes (DEGs) by RNA-seq. Compared with the control group, 244 genes were significantly upregulated, and 96 genes were significantly downregulated in the ammonia gas group. Some genes in Gene Ontology (GO) terms were verified and showed significant differences by qRT-PCR. The KEGG pathway revealed significant changes in the MAPK signaling pathway, which is strongly associated with inflammatory injury. To sum up, the results indicated that ammonia induces oxidative stress in pig spleen, activates the MAPK signaling pathway, and causes spleen necrosis and injury. In addition, some differential genes encoding epigenetic factors were found, which may be involved in the response mechanism of spleen tissue oxidative damage. The present study provides a transcriptome database of ammonia-induced spleen poisoning, providing a reference for risk assessment and comparative medicine of ammonia.

Introduction
Ammonia is a toxic, colorless, and corrosive alkaline gas, which is recognized as an atmospheric pollutant [1]. Sources of ammonia include industrial production, automotive exhaust, agriculture, livestock, construction, and soil and marine volatilization [2]. It is noteworthy that the ammonia emission from husbandry accounts for an important share of global ammonia emissions [3]. The excessive ammonia in livestock and poultry houses has an adverse effect on the health of workers and the welfare of animals, causing air pollution through the ventilation system. Therefore, the harm of ammonia emission from the livestock and poultry industry to humans and animals has become a hot issue in the world [4].
Inhalation is the primary route of exposure to ammonia. Ammonia below 25 ppm is harmless to both humans and animals [5]. However, exposure to high concentrations of ammonia in humans can cause the destruction of respiratory tissues, cilia, and the mucosal barrier, leading to inflammation and an increased risk of secondary infections [6]. The spleen is the largest peripheral immune organ, more complex than other lymphoid tissues, and it is crucial to the maintenance of innate immunity and acquired immune function [7]. A recent study shows that ammonia can stimulate an immune response in pufferfish and promote the production of various inflammatory cytokines [8]. However, the mechanism of ammonia-induced splenic injury remains unclear.
Epigenetic modifications are responses to changes in the environment after environmental stimuli (nutrients, toxins, infections, hypoxia) [9]. Histone modification is an important epigenetic modification. Post-translational methylation of histones has been extensively studied in the past few years. Mitogen-activated protein kinase (MAPKs) regulate multiple life processes [10]. P38 MAPK and JNK/SAPK are mainly induced by various environmental stresses. PM2.5 exposure was found to activate MAPK signaling pathways, and phosphorylated expressions of JNK, P38, and ERK were significantly increased [11]. The nuclear factor kappa-B (NF-κ B) signaling network is one of the histone modification pathways that regulate the expression of inflammatory factors. It has been shown to be a downstream consequence of MAPK signaling [12].
Transcriptomes are essential for explaining the function of the genome, the molecular makeup of cells and tissues, and the onset and development of disease [13]. In recent years, pigs have been widely studied in the field of toxicology as a traditional animal model [14] because the life and metabolic system of pigs is similar to that of humans, and pigs allow for repeated sampling [15]. Ammonia is the major noxious gas in pig farms. The impacts of ammonia on livestock and health have been studied relatively well in chickens. However, less research has been conducted on the mechanism of ammonia toxicity to pig spleen.
To study how exogenous ammonia causes spleen damage, we established an ammonia poisoning model of fattening pigs to conduct spleen ultrastructural examination. Simultaneously, we used RNA-seq to analyze the impact of ammonia on spleen tissue and to quantify the variation of expression levels of transcripts under different conditions. We investigated the oxidative stress and inflammatory injury induced by ammonia in the spleen. These research results may provide new ideas and insights for understanding the specific mechanisms of ammonia toxicity.

Experimental Animal Management and Experimental Design
Twelve 125-day-old crossbred pigs with similar body conditions were randomly chosen and indiscriminately distributed into control group (n = 6) and ammonia group (n = 6). All experimental animals were housed in an ambient temperature-controlled room. All environmental parameters were in accordance with the national standard for fattening pig breeding (GB17824. . All pigs made use of feed and water freely.

Ammonia Exposure
For the purpose of simulating the pig houses and ensuring that the ammonia concentration of the control group was maintained below 5 mg/m 3 and the ammonia concentration of the ammonia group was maintained between 75.4 and 76.5 mg/m 3 , the exogenous ammonia was supplied to the ambient temperature-controlled room for a time of 8 h/d. The pigs were adapted to the feeding environment 14 d before the formal experiment, and the ammonia concentration was adjusted 3 d prior to the experiment. The test period lasted for 30 d. After the experiment, the pigs were euthanized, and spleen tissues were taken immediately after blood collection and homogenized under ice conditions. The spleen tissues were taken, washed with sterile saline, and fixed in 10% formalin. Tissue homogenates were carried out for the detection of antioxidant indices, and the rest of the spleen tissues were transferred into DNase/RNase-free cryopreservation tubes and stored at −80 • C for transcriptome and qRT-PCR analysis.

Histological Observation
The spleen tissues were fixed in 10% formalin for at least 24 h and then pruned, cleaned with tap water, dehydrated at different concentrations of alcohol, and embedded in paraffin. The samples were cut into 6 µm thin sections and stained with hematoxylin and eosin (H&E). Finally, the stained slices were sealed with neutral resin. The changes in microstructure were recorded with a multifunctional digital light mirror DMS-651 observation.

Determination of Antioxidant Indexes by ELISA
The spleen tissues were homogenized in 0.9% NaCl solution and centrifuged at 3500 rpm for 10 min at 4 • C. The supernatant was taken, and its antioxidant activity was determined with a specialized assay kit. The contents of glutathione (GSH) (Kit Number: A006-2-1) and malondialdehyde (MDA) (Kit Number: A003-1) and the activity of glutathione peroxidase (GSH-px) (Kit Number: A005) and superoxide dismutase (SOD) (Kit Number: A001-1) in spleen tissue were tested by a spectrophotometric microplate reader (SpectraMax ® ABS00254, Molecular Devices, San Jose, CA, USA). Optical density (OD) values at 405 nm, 532 nm, 412 nm, and 550 nm were measured correspondingly. The quantitative analysis of total protein in the spleen tissue was performed by the Coomassie blue method. All kits were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing Construction Bioengineering Research, Nanjing, China).

RNA-seq and Interpretation of Results
The total RNA was extracted with TRIzol reagent. We purified the total RNA to obtain the mRNA and then decomposed it into small fragments 200-300 bp long. The first-strand cDNA was synthesized as a random hexamer, and then the second-strand cDNA was synthesized by adding buffer, dNTP, RNase H, and DNA polymerase I. Finally, PCR amplification was performed to obtain the final sequencing library. Sequencing data were screened and sorted by the Illumina HiseqTM 6000 sequencer, and the sequencing read length was double-ended 2 * 150 bp (PE150). The entire transcriptome analysis procedure was performed by Lianchuan Biotechnology Co., Ltd. (Hangzhou, China). The study of DEGs was carried out based on the final sequencing library (threshold: |log2foldchange| ≥1, p < 0.05). EdgeR was used for differential expression analysis. The GO database was downloaded for DEG ontology analysis. The KEGG database was used for gene function annotation. The R language was used for a graphical display of the differential expression results, including a heat map, scatter map, and volcano map of DEGs.

Verification of Differentially Expressed Genes
A 0.1 g amount of cryopreserved spleen tissue was weighed. Trizol (Invitrogen, Shanghai, China) was applied to extract total RNA. Firstly, 1 mL of TRIzol was added to the tissue, the homogenate of the tissue was taken out, and 200 µL of chloroform was added for shaking and standing centrifugation. Then, 400 µL of isopropyl alcohol was added to the supernatant. The precipitation was washed with 1 mL of 75% ethanol, and the dried RNA was gently blown with 30 µL of double-distilled water to dissolve. The complementary DNA (cDNA) was synthesized using oligo dT primers and reverse transcriptase II. The cDNA was diluted 5 times in double-distilled water and stored at −80 • C for later use.
Primer Premier 5.0 was used to design specific primers. qRT-PCR was performed using LightCycler ® 96 (Roche, Switzerland). The reaction conditions were as follows: three replicates were performed for each PCR product. The relative mRNA expression levels of each gene were calculated by 2 −∆∆Ct . To corroborate the reliability of the DEGs data, we selected 13 genes associated with oxidative stress, inflammation, histone methylation, and the MAPK signal pathway and used qRT-PCR for investigation. Primers used for qRT-PCR were displayed in Table 1. β-actin was used as a reference gene to analyze the expression trend of the screened genes.

Statistical Analysis
IBM SPSS statistical software (version 25.0, SPSS Inc., Chicago, IL, USA) was used to analyze the experimental data. The data are expressed as mean ± standard deviation. One-way analysis of variance confirmed a significant difference between the ammonia and control groups. p < 0.05 was considered statistically significant.

Splenic Ultrastructural Damage due to Ammonia Exposure
To more intuitively demonstrate the effect of ammonia on the spleen, we evaluated the spleen pathology in the control group and ammonia group of fattening pigs. The changes in the microstructure of pig spleen caused by inhaling ammonia are shown in Figure 1. In the normal spleen, the division of red pulp and white pulp was clear, the shape of splenic nodules was regular, and the number of lymphocytes was abundant. Histopathologically, the size of splenic nodules and the number of lymphocytes were decreased in the ammonia group. Pathological results indicated that ammonia exposure caused structural changes in spleen tissue. The changes in the number of lymphocytes suggested the inflammatory injury in spleen tissue. way analysis of variance confirmed a significant difference between the ammonia and control groups. p < 0.05 was considered statistically significant.

Splenic Ultrastructural Damage due to Ammonia Exposure
To more intuitively demonstrate the effect of ammonia on the spleen, we evaluated the spleen pathology in the control group and ammonia group of fattening pigs. The changes in the microstructure of pig spleen caused by inhaling ammonia are shown in Figure 1. In the normal spleen, the division of red pulp and white pulp was clear, the shape of splenic nodules was regular, and the number of lymphocytes was abundant. Histopathologically, the size of splenic nodules and the number of lymphocytes were decreased in the ammonia group. Pathological results indicated that ammonia exposure caused structural changes in spleen tissue. The changes in the number of lymphocytes suggested the inflammatory injury in spleen tissue.

Effect of Excessive Ammonia on Oxidative Stress Indexes
Oxidative stress could induce the activation of inflammatory signaling cascades. In order to investigate the mechanism of splenic pathological injury caused by excessive ammonia, we detected the activity of GSH-Px and SOD and the content of MDA and GSH. As shown in Figure 2, the GSH content of the ammonia group and the activity of GSH-Px and SOD were significantly reduced (p < 0.05) compared to the control group. Conversely, the MDA content of the ammonia group increased significantly (p < 0.05). Collectively, these data indicated that spleen injury may be induced by the oxidative stress pathway in ammonia groups.

Effect of Excessive Ammonia on Oxidative Stress Indexes
Oxidative stress could induce the activation of inflammatory signaling cascades. In order to investigate the mechanism of splenic pathological injury caused by excessive ammonia, we detected the activity of GSH-Px and SOD and the content of MDA and GSH. As shown in Figure 2, the GSH content of the ammonia group and the activity of GSH-Px and SOD were significantly reduced (p < 0.05) compared to the control group. Conversely, the MDA content of the ammonia group increased significantly (p < 0.05). Collectively, these data indicated that spleen injury may be induced by the oxidative stress pathway in ammonia groups.

Differentially Expressed Gene Screening Analysis
Our preliminary results suggested that ammonia exposure causes pathological damage and oxidative stress in the spleen. To further explore the molecular mechanism of spleen injury, we compared gene expression levels between the two groups via transcrip-

Differentially Expressed Gene Screening Analysis
Our preliminary results suggested that ammonia exposure causes pathological damage and oxidative stress in the spleen. To further explore the molecular mechanism of spleen injury, we compared gene expression levels between the two groups via transcriptome sequencing. Ammonia-induced gene expression changes are shown in Figure 3. A total of 340 differential genes were screened according to log2 FC ≥1 or ≤−1 and p < 0.05, of which 244 genes were significantly increased, and 96 genes were significantly decreased. These results suggested that ammonia poisoning has a complex molecular regulation mechanism, which requires further analysis and clarification.

Ammonia Exposure Activated MAPK Signaling Pathway by KEGG Enrichment Analysis
The MAPK signaling pathway is a classic inflammatory signaling pathway. Excitedly, we obtained significant changes in the MAPK signaling pathway by KEGG enrichment analysis. The enrichment analysis results were represented by scatter plots ( Figure  4). A total of 20 KEGG pathways were obtained, and these pathways pertained to five branches. As shown in Figure 4, DEGs were concentrated in the MAPK signaling pathway. A total of 12 DEGs were enriched in this pathway. It was noteworthy that this pathway was connected with oxidative stress and inflammation.

Ammonia Exposure Activated MAPK Signaling Pathway by KEGG Enrichment Analysis
The MAPK signaling pathway is a classic inflammatory signaling pathway. Excitedly, we obtained significant changes in the MAPK signaling pathway by KEGG enrichment analysis. The enrichment analysis results were represented by scatter plots (Figure 4). A total of 20 KEGG pathways were obtained, and these pathways pertained to five branches. As shown in Figure 4, DEGs were concentrated in the MAPK signaling pathway. A total of 12 DEGs were enriched in this pathway. It was noteworthy that this pathway was connected with oxidative stress and inflammation.

GO Enrichment of DEGs
GO contains three ontologies, which can be described as the biological process, cellular component, and molecular function of genes. The histogram of GO enrichment analysis results reflected the number distribution of differential genes on the three ontologies. DEGs were enriched in 307, 44, and 106 items in biological processes, cell components, and molecular functions, respectively. Among them, the biological process with the highest enrichment degree of differential genes was regulation of transcription and DNA templates, cell components were membrane, and molecular functions were protein binding. We selected the top 20 GO terms for display. A rich factor indicates the degree of GO enrichment. The results of the enrichment bubble diagram are shown in Figure 5B. Among them, histone H3-K4 methylation was the most affected.

GO Enrichment of DEGs
GO contains three ontologies, which can be described as the biological process, cellular component, and molecular function of genes. The histogram of GO enrichment analysis results reflected the number distribution of differential genes on the three ontologies. DEGs were enriched in 307, 44, and 106 items in biological processes, cell components, and molecular functions, respectively. Among them, the biological process with the highest enrichment degree of differential genes was regulation of transcription and DNA templates, cell components were membrane, and molecular functions were protein binding. We selected the top 20 GO terms for display. A rich factor indicates the degree of GO enrichment. The results of the enrichment bubble diagram are shown in Figure 5B. Among them, histone H3-K4 methylation was the most affected.

qRT-PCR Confirmed the Availability of Transcriptome Data
Histone methylation is an epigenetic mechanism by which cells respond to environmental stimuli. Combined with our previous research results, we speculated that the mechanism of ammonia poisoning might be related to histone methylation. To confirm the reliability of transcriptomic results, we performed qRT-PCR on 13 genes associated with histone methylation (KMT2A, KMT2B, KMT2C, KMT2D), inflammation (IL1RL1, IL17RB, IL23R), oxidative stress (UPF1, TRPM6, AOC2), and the MAPK signaling pathway (PRKCG, CACNA2D2, HGF). As shown in Figure 6, with the exception of CACNA2D2, mRNA levels were significantly higher. Transcriptome results showed that KMT2A, KMT2B, KMT2C, KMT2D, IL1RL1, IL17RB, IL23R, UPF1, TRPM6, AOC2, PRKCG, and HGF were upregulated DEGs. CACNA2D2 was downregulated DEGs. Changes in these genes detected by qRT-PCR confirmed the credibility of the transcriptome sequencing results.

qRT-PCR Confirmed the Availability of Transcriptome Data
Histone methylation is an epigenetic mechanism by which cells respond to environmental stimuli. Combined with our previous research results, we speculated that the mechanism of ammonia poisoning might be related to histone methylation. To confirm the reliability of transcriptomic results, we performed qRT-PCR on 13 genes associated with histone methylation (KMT2A, KMT2B, KMT2C, KMT2D), inflammation (IL1RL1, IL17RB, IL23R), oxidative stress (UPF1, TRPM6, AOC2), and the MAPK signaling pathway (PRKCG, CACNA2D2, HGF). As shown in Figure 6, with the exception of CACNA2D2, mRNA levels were significantly higher. Transcriptome results showed that KMT2A, KMT2B, KMT2C, KMT2D, IL1RL1, IL17RB, IL23R, UPF1, TRPM6, AOC2, PRKCG, and HGF were upregulated DEGs. CACNA2D2 was downregulated DEGs. Changes in these genes detected by qRT-PCR confirmed the credibility of the transcriptome sequencing results.

Discussion
The spleen is an important immune lymphoid organ in animals with a variety of immune functions [16]. Many studies have shown that exogenous ammonia exposure causes damage to a variety of tissues and organs, including the spleen [17,18]. In the current study, we analyzed the morphological damage and oxidative damage levels of the spleen in fattening pigs exposed to ammonia. Moreover, we used RNA-seq and qRT-PCR (KMT2A, KTM2B, KMT2C, KMT2D, IL1RL1, IL17RB, IL23R, UPF1, TRPM6, AOC2, PRKCG,  CACNA2D2, HGF) to construct the database of DEGs. Transcriptome profiling revealed that the inflammation and oxidative stress caused by ammonia exposure may be related to histone methylation modification.
An imbalance between reactive oxygen species (ROS) and antioxidant mechanisms leads to oxidative stress [19]. However, exposure to harmful exogenous factors will generate ROS. High concentrations of ROS can adversely modify cellular components such as lipids, proteins, and DNA [20]. Mammalian plasma contains quite active AOC, namely plasma or SAO. As a byproduct of amine oxidation, AOC3 could directly increase reactive oxygen species levels [21]. In this study, AOC2 expression levels increased remarkably. Therefore, we hypothesized that the upregulation of the AOC2 gene in ammonia-induced spleen suggests that its function may be related to oxidative stress. This may provide an important reference for studying the function of the AOC2 gene. UPF1 has been initially identified as a central component of the NMD pathway [22]. It could regulate the expression of related genes through hnRNP E2 to affect ROS production and further induce oxidative stress [23]. The intracellular redox states can significantly alter the gated properties of ion channels. In oxidative stress, TRPM6 could restore its activity by methionine sulfoxide reductase [24]. MDA is the principal and most studied product of polyunsaturated fatty acid peroxidation as a marker of ROS. The measurements of GSH, SOD, and GSH-px could indicate antioxidant responses [25]. In this study, ammonia exposure significantly affected the activities of GSH, GSH-px, SOD, and MDA in spleen tissues. The results demonstrated that ammonia damaged the antioxidant defense system of pigs.
Excessive production of ROS in the mitochondria causes oxidative damage and activates inflammatory signaling cascades [26]. Ammonia exposure has been shown to trigger inflammatory responses and oxidative stress [27]. The current findings were in agreement with many studies. The KEGG pathways obtained by RNA-seq were related to inflammation, such as inflammatory mediator regulation of TRP channels, the MAPK signaling pathway, and the IL−17 signaling pathway. MAPK signaling has been confirmed to mediate inflammatory responses [28]. Previous reports have shown that ROS accumulation activates the NF-κ B/MAPK signaling pathway and increases downstream levels of associated inflammatory cytokines. The increase in ROS in cells activates ERKs, JNKs, or P38 MAPKs, but the mechanism by which ROS activates these kinases is unclear [29]. The PRKCG gene encodes PKC γ. PKC γ directly activates members of the MAPKs family involved in pain and multiple-injury-activated pathways [30]. The CACNA2D2 gene encodes a functionally auxiliary subunit of voltage-gated Ca 2+ channels. Previous studies have found the reduced activation of MAPK cascade signaling in CACNA1C deficient mice [31]. HGF is a cytokine with strong angiogenic activity, which can activate the MAPK signaling pathway and upregulate the expression of iNOS [32]. The results of this study showed that the gene expression levels of PRKCG, CACNA2D2, and HGF were significantly changed in the spleen of pigs exposed to ammonia, suggesting that ammonia exposure activated the MAPK signaling pathway. Consistent with our results, Wang et al. reported that Pb exposure induces necrotic apoptosis of the chicken spleen by activating the MAPK/NF-κ B pathway [33]. The MAPK signaling pathway activates and produces a large number of cytokines that are essential for the functioning of the inflammatory response [34]. Our transcriptome results suggest that IL1RL1, IL17RB, and IL23R pertained to the active regulation of inflammatory responses. IL1RL1, a receptor for interleukin-33 (IL-33), plays a crucial part in type 2 inflammation [35]. Il-17 is an inflammation-promoting cytokine that is produced primarily by activated T cells. It enhances the activation of T cells and stimulates a variety of cells to produce inflammatory mediators, leading to the induction of inflammation [36]. IL23R is expressed in multiple cell types. It has been found to play a key role in cytokine signal transduction and amplification mediated by human macrophages [37].
Histopathological analysis further demonstrated that ammonia could cause inflammatory injury of the spleen in fat pigs.
Histone methylation is a crucial type of epigenetic modification in cells [38]. The effects of oxidative stress on chromatin modification mediate many cellular changes, including regulation of gene expression, apoptosis, and mutations [39]. Histone methylation and acetylation changed significantly after ROS exposure in human bladder tissues [40], which then lead to aberrant gene expression, and may contribute to the process of carcinogenesis. In the present study, GO analysis emphasized that some genes were gathered in "histone methyltransferase activity". KMT2A, KMT2B, KMT2C, and KTM2D are members of the mammalian H3-K4 methyltransferases family, which regulate gene expression by catalyzing methylation on lysine 4 of histone H3 (H3K4) [41]. Transient receptor potential melastatin IDH2 is the major NADPH-producing enzyme in mitochondria. CDH1 ubiquitinates IDH2 and promotes the increased ROS in mitochondria [42]. Transcriptome results showed that the histone lysine methyltransferase 2 (KMT2) gene family changed significantly. Our qRT-PCR results showed that oxidative stress and inflammatory response induced by ammonia exposure were related to histone methylation modification.

Conclusions
In conclusion, the transcriptome map of differential gene expression in the spleen induced by ammonia was established in the present study. Our results suggest that exposure to exogenous ammonia can induce oxidative stress and inflammatory response in spleen tissues. In addition, our study found differences in histone methylase gene expression caused by ammonia exposure for the first time, which may be the epigenetic mechanism of ammonia poisoning of the spleen. The results of this paper provide a new perspective for exploring the mechanism of spleen poisoning induced by ammonia.

Data Availability Statement:
The data presented in this study are available on reasonable request from the corresponding authors.


Respiratory and cardiovascular effects of ambient particulate matter from dust storm and non-dust storm periods in Kuwait

Epidemiological studies demonstrate a positive association between daily changes in concentrations of ambient airborne particulate matter (PM) and adverse respiratory and cardiovascular health effects. However, physicochemical properties of PM can vary greatly across geographical, atmospheric, and temporal conditions and influence the relative toxicity of airborne PM. The purpose of this study was to investigate the adverse pulmonary and cardiovascular health effects of ambient PM collected from discrete sampling sites in Kuwait during dust storm (DS) and non-dust storm (NDS) conditions. Collected dust samples were characterized for their chemical composition using atomic absorption, GC–MS, and HPLC–MS analyses. Male BALB/cJ mice were exposed to 100 µg of either NDS or dust storm (DS) PM in 50 µl of PBS by oropharyngeal aspiration. Lung function was measured and bronchoalveolar lavage was conducted at 1, 7, and 14 days post-exposure. Ischemia–reperfusion injury was performed 24 h after exposures by obstructing the left main coronary artery approximately 4 mm distal to its origin for 20 min, followed by 2 h. of reperfusion. Exposure to either NDS or DS PM resulted in airway hyperresponsiveness to acetylcholine compared to PBS controls. Total protein and cells in BAL fluid were elevated in both dust groups one day after exposure; however, DS PM induced a greater increase in cell numbers than did NDS PM, particularly in neutrophils, eosinophils, and lymphocytes. Representative lung sections exhibited positive staining for mucus in large airways at 7 days which resolved by 14 days in dust storm-exposed mice but persisted in NDS-exposed animals. Our findings suggest that NDS PM may be more effective in producing an adaptive immune response, while the early inflammation induced by DS PM may better resolve. We also observed a prolonged airway mucus response after exposure to NDS PM, suggesting it may produce more asthma-like features than dust storm PM. PM-induced changes to cardiac ischemia–reperfusion injury were not observed in this study. The lack of cardiovascular response may have been due to the limited exposure and single time point used in this study.

Introduction
Over 150 epidemiological studies demonstrate a positive association between daily changes in concentrations of ambient airborne particulate matter (PM) and adverse respiratory and cardiovascular health effects. More than 800,000 excess deaths worldwide each year are attributed to PM. However, physicochemical properties of PM can vary greatly across geographical, atmospheric, and temporal conditions and influence the relative toxicity of PM. Thalib and Al-Taiar reported significant association of dust storm events and increases in hospital admissions for asthma and other respiratory conditions in Kuwait (Thalib and Al-Taiar 2012). We had the opportunity to investigate the acute toxic effects of ambient PM collected from discrete sampling sites in Kuwait during dust storm and NDS conditions on the cardiopulmonary Editorial responsibility: Mohamed F. Yassin.
1st International Conference on Applications of Air Quality in Science and Engineering Purposes 10-12 February 2020, Kuwait.

3
system. The purpose of this study was to investigate adverse health effects of Kuwaiti ambient PM collected during dust storms vs NDS periods and their potential contribution to increased risk of respiratory and cardiac illness.

PM exposure
Male BALB/cJ mice (Jackson Labs, Bar Harbor, ME) at 7 weeks of age were anesthetized with isoflurane and subject to oropharyngeal aspiration of 100 µg of either NDS DS PM in 50 µL of PBS or 50µL of PBS alone n = 18 mice/exposure group. Mice in each exposure group were later euthanized at three separate time points (n = 6 mice/exposure group/ time point).

Lung function
Lung function measurements were made 1, 7, or 14 days post-exposure using the flexiVent system (SCIREQ, Montreal, QC, Canada). Animals (n = 6/exposure group/time point) were anesthetized (~ 400 mg/kg tribromoethanol, i.p.), tracheostomized, and ventilated with room air at 10 ml/ kg and 150 breaths per minute with positive end expiratory pressure of 3 cm H 2 O. Respiratory mechanics were measured using a script to ensure consistent timing of perturbations relative to total lung capacity maneuvers to standardize volume history. Respiratory system resistance (R) was measured at baseline and after increasing concentrations of aerosolized acetylcholine.

Bronchoalveolar lavage
Immediately after lung function measurements, mice were exsanguinated, the left bronchus was clamped, and the right lung was lavaged with four successive aliquots of 26.25 ml/ kg cold Hanks' balanced salt solution (HBSS). Recovered lavage fluid (BALF) was centrifuged at 500×g for 10 min at 4 °C. Supernatant from the first BALF aliquot was used for measurement of total protein. All cells from the same individual were pooled, and total cell counts were made with a hemocytometer. Slides were prepared by cytocentrifugation of 20,000 cells at 600 rpm for 5 min (Cytospin III, Shandon) and stained (Richard-Allan, Kalamazoo, MI). BALF cell differential counts were performed on 300 cells per slide using standard morphological criteria.

Quantitation of protein in BALF
Total protein concentration in BALF (an index of lung permeability and lung injury) was measured using a Bradford protein assay (BioRad, Hercules, CA) according to manufacturer's recommendations and read at 595 nm on a spectrophotometer (Beckman Coulter, Brea, CA). Concentration was calculated from a standard curve.

Measurement of lung collagen
Lavaged right lung lobes were homogenized in RIPA lysis buffer plus protease inhibitor cocktail and PMSF (Sigma-Aldrich, Milwaukee, WI). Lung homogenates were centrifuged at 10,000×g for 10 min at 4 °C. Soluble collagen was measured by adding 500 μl Sircol dye reagent (Biocolor Ltd., Carrickfergus, UK) to 50 μl of lung homogenate supernatant. Samples and standards were agitated for 30 min and then centrifuged at 10,000×g for 10 min to pellet collagen-bound dye. Supernatants were discarded, and pellets were dissolved in 1 ml 0.5 M NaOH. Absorbance was read at 540 nm, and sample concentrations were calculated from a standard curve.

Lung histology
Left lungs were inflated with 10% neutral buffered formalin (Azer Scientific, Morgantown, PA) for 24-72 h before cutting into three sections and undergoing standard processing. Five-μm cross sections were placed in xylene to remove paraffin, hydrated through an ethanol gradient, and stained with Masson's trichrome to allow visualization of collagen and Alcian Blue-periodic acid-Schiff (AB-PAS) for mucoid substances.

Cardiovascular ischemia-reperfusion (I/R) injury
An established protocol was used to measure I/R injury (Cozzi et al. 2006).

Statistical analysis
All data are presented as the mean ± SEM for each group. Two-way analysis of variance (ANOVA) was used to evaluate exposure and temporal effects. Bonferroni post hoc testing was used to determine statistical differences between groups, and p < 0.05 was accepted as statistically significant. SigmaStat 3.0 software (SPSS Science Inc., Chicago, IL) was used for all statistical analyses.

Lung function
Airway resistance increased with increasing concentrations of aerosolized acetylcholine. Exposure to 100 μg of either NDS or DS PM resulted in hyperresponsiveness to the higher concentrations of acetylcholine compared to PBS controls at day 1 (Fig. 1). At days 7 and 14 post-exposure, there were no significant differences among exposure groups (data not shown).

BAL protein and cells
Protein concentrations were significantly elevated in mice exposed to both NDS and DS PM compared to PBS controls one day after exposure, but were not different from PBS controls thereafter (Fig. 2). Similarly, the total number of cells recovered in BAL fluid increased significantly one day after exposure to NDS and DS PM compared to PBS controls, but was not different from controls at later time points (Table 1). Specific cell types differed widely depending on type of PM exposure and time point. Macrophages accounted for approximately 90% of BAL cells in PBS-exposed mice across all time points. Exposure to NDS PM elicited an increase in macrophage numbers at 14 days which was not seen in DS PM-exposed mice. There were no significant differences in macrophage numbers in any exposure group at 1 and 7 days post-exposure. The pattern of epithelial cells mirrored that of BALF protein with significant increases in both PM-exposed groups at one day, but no differences from controls at subsequent time points. Lymphocytes, neutrophils, and eosinophils were elevated in both NDS-and DS-exposed mice at one day, but to a significantly greater extent in DS-exposed mice. Lymphocytes and eosinophils were also elevated at 14 days post-PM exposure, but to a greater extent in NDS-exposed mice.
Histological evaluation of lung sections from mice exposed to Kuwaiti PM showed mild increases in airway mucus content in large airways at 7 days post-exposure (Fig. 3). Mucus production subsided by 14 days postexposure in DS-exposed mice but was still present in NDSexposed mice. Lung collagen content did not differ significantly in any exposure group or time point (data not shown), nor were increases seen in lung collagen histologically.

Conclusion
We found that aspiration of equal masses of Kuwaiti PM collected during NDS and dust storm periods elicited largely similar pulmonary responses, but with some interesting temporal differences. Both types of PM elicited increased airway responsiveness to acetylcholine provocation as well as increases in lung permeability one day after exposure. Additionally, both types of PM induced cellular inflammation in the lungs with increases in lymphocytes, neutrophils, and eosinophils; however, this inflammatory response was greater in DS-exposed compared to NDS-exposed mice one day after exposure. Interestingly, increased numbers of lymphocytes and eosinophils were also seen at 14 days post-exposure, but to a greater extent in NDS-exposed mice at this time point. Furthermore, macrophage numbers were also elevated in NDS-exposed mice at 14 days. These findings suggest that NDS PM may be more effective in producing an adaptive immune response in naïve mice, while the early inflammation induced by DS PM may better resolve.
We also observed a prolonged airway mucus response after exposure to NDS PM, suggesting it may produce more asthma-like features in naïve mice than DS PM. An epidemiological study found that hospitalizations for asthma were associated with dust storm events in Kuwait (Thalib and Al-Taiar 2012). Our data showing greater inflammation in response to DS PM support the epidemiological evidence; however, NDS PM from Kuwait may contribute to the development of asthma in this region, while DS PM may exacerbate existing respiratory conditions such as asthma. Investigation of the differential effects of these different types of PM in mouse model of allergic asthma would better answer such questions. In addition, an analysis of the physical and chemical composition of the PM may assist in explaining the observed differences. The increases in epithelial cell numbers recovered in lavage fluid one day after PM exposure which are in line with the increases in protein content suggest that aspiration of these dusts may induce injury to the epithelial and endothelial barriers in the lungs. Although PM-induced changes to cardiac ischemia-reperfusion injury were not observed in this study, further exploration of cardiovascular effects may be warranted.  Fig. 3 Representative lung sections from mice exposed to PBS, NDS PM, or DS PM. Airways from mice exposed to PBS (top row) did not stain for mucus, while NDS PM-exposed mice exhibited positive staining for mucus in large airways (bronchi) at 7 and 14 days post-exposure. Airways from DS PM-exposed mice stained positive for mucus at 7 days, but this effect had mostly resolved by 14 days. Arrows indicate bronchial epithelium that stained positive for mucus. Alcian Blue-periodic acid-Schiff staining of 5-μm sections. × 100 original magnification