Particulate matter 2.5 promotes inflammation and cellular dysfunction via reactive oxygen species/p38 MAPK pathway in primary rat corneal epithelial cells

Abstract Purpose Numerous studies have linked particulate matter2.5 (PM2.5) to ocular surface diseases, but few studies have been conducted on the biological effect of PM2.5 on the cornea. The objective of this study was to evaluate the harmful effect of PM2.5 on primary rat corneal epithelial cells (RCECs) in vitro and identify the toxic mechanism involved. Materials and methods Primary cultured RCECs were characterized by pan-cytokeratin (CK) staining. In PM2.5-exposed RCECs, cell viability, microarray gene expression, inflammatory cytokine levels, mitochondrial damage, DNA double-strand break, and signalling pathway were investigated. Results Exposure to PM2.5 induced cytotoxicity and morphological changes in RCECs. In addition, PM2.5 markedly up-regulated pro-inflammatory mediators but down-regulated the wound healing-related transforming growth factor-β. Furthermore, PM2.5 promoted mitochondrial reactive oxygen species (ROS) production and mediated cellular damage to mitochondria and DNA, whereas these cellular alterations induced by PM2.5 were markedly suppressed by a potential ROS scavenger. Noteworthy, removal of ROS selectively down-regulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the activation of the nuclear factor-κB (NF-κB) p65 in PM2.5-stimulated cells. Additionally, SB203580, a p38 MAPK inhibitor, markedly suppressed these PM2.5-mediated cellular dysfunctions. Conclusions Taken together, our findings show that PM2.5 can promote the ROS/p38 MAPK/NF-κB signalling pathway and lead to mitochondrial damage and DNA double-strand break, which is ultimately caused inflammation and cytotoxicity in RCECs. These findings indicate that the ROS/p38 MAPK/NF-κB signalling pathway is one mechanism involved in PM2.5-induced ocular surface disorders.


Introduction
Dust refers to particulate matter (PM) that floats in the atmosphere. PM is classified as PM 10 with size less than 10 lm and PM 2.5 with size less than 2.5 lm. Normal dust is not a big problem because it is filtered and discharged from the mucous membrane of the nose and the bronchus 1 . However, fine dust is very small and cannot be filtered out, consequentially it can be absorbed into the cells and tissues body via breathing and skin 1,2 . The concentration of fine dust is increasing day by day, causing serious air pollution 3 . In January 2020, the average PM 2.5 exposure per population in Korea was 25 lg/m 3 , which was the highest among Organization for Economic Cooperation and Development (OECD) countries, and the severity of air pollution due to fine dust in Korea is gradually increasing 3 . In this regard, the World Health Organization has been providing guidance on the concentration of fine dust in the air since 1987 4 , and the International Agency for Research on Cancer designated fine dust as a Class 1 carcinogen in 2013 5 . Accumulated evidences support that fine dust has toxic to biological organisms and that it is involved in the development of various diseases, including cancer, respiratory disease, neurological disorder, skin disease, and cardiovascular disease 2,6-8 . Despite active research into the pathological mechanisms and toxicity caused by fine dust, studies on biological effects of fine dust on eyes and underlying damage mechanisms remain insufficient.
Eye is naturally and directly exposed to the outside, especially to air pollutants including fine dust without any protection 9 . Corneal disease is a major cause of blindness, and the surface of the eye is damaged by various factors 10 . Trauma or infection by foreign objects may induce itchiness and pain of the eye, which may cause various ocular surface disorders, such as allergic keratitis, conjunctivitis, and dry eye syndrome 11 . Accumulated epidemiological studies have shown that people often experience symptoms, such as itching, irritation, and foreign body when they are exposed to severe air pollution for a short term or a long term 9 . In this respect, several studies have reported that air pollution is closely associated with an increase in the number of ophthalmic outpatients with allergic keratitis and conjunctivitis [12][13][14][15] . Torricelli et al. 16 have suggested that exposure to air pollution can break tear film stability and influence tear film osmolarity. Furthermore, decreased tear break time has been observed in subjects who are exposed to PM 2.5 , suggesting that PM 2.5 can result in instability of the tear layer and suppression of tear volume 17 . Recently, a few studies have suggested the pathological mechanism of PM 2.5 -mediated ocular surface damage. Tan et al. 18 have demonstrated that mice show characteristics of dry eye syndrome including detachment of corneal epithelium, destroy of the tear film, and inflammation of lacrimal gland after they are exposed to PM 2.5 . PM 2.5 can induce cytotoxic effects in corneal epithelial cells due to DNA damage, senescence 19 and autophagic cell death caused by increased reactive oxygen species (ROS) 14 . Large amounts of ROS can promote cellular damages and dysfunctions, such as DNA damage, mitochondrial damage, cell death, enzyme inactivation, and amino acid acidification 20 . It is well-known that the generation of ROS by PM 2.5 exposure can cause a variety of cellular lesions 21,22 . In CECs, house dust which contains fine dust can cause inflammation and mitochondrial DNA damages through ROS-mediated cytotoxicity 23 . Furthermore, numerous studies demonstrated that PM triggers inflammation via ROS/mitogen-activated protein kinase (MAPK)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) signalling pathway in bronchial epithelial cells 24,25 . More recently, it has been established that PM 2.5 can promote delayed wound healing via interruption of focal adhesion kinase (FAK) signalling pathway, causing dry dye syndrome in C57BL/6 mice 26 . Although there is a growing interest in the effect of exposure to fine dust on eyes with a few lab-scale experiments and epidemiological studies reporting toxicological effects of PM 2.5 , the underlying pathological mechanism remains unclear and more research studies are needed. Therefore, the aim of this study was to evaluate the biological effects of PM 2.5 on primary rat corneal epithelial cells (RCECs), and underlying mechanisms involved in the pathological effects were also explored.

PM 2.5 preparation
Standard diesel PM 2.5 (SRM 1650 b) was obtained from the National Institute of Standards and Technology (Gaithersburg, MD). The 1650 b diesel PM2.5 was predominantly composed of heterocyclic polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. As described previously 8 , PM 2.5 was dissolved in dimethylsulphoxide (DMSO; Invitrogen-Gibco, Carlsbad, CA) to prepare a 25 mg/mL stock solution, which was diluted in culture medium or normal saline just before use.

Culture of primary rat corneal epithelial cells
Female Sprague-Dawley rats (six weeks old) were obtained from Samtako Bio Korea (Osan, Republic of Korea) and acclimated for one week. This study was approved by the Institutional Animal Care and Use Committee of Dong-eui University (approval No. R2019-005). All procedures were followed in accordance with the guide for the Use of Animals in Ophthalmic and Vision Research. RCECs were isolated from specimens collected after surgical excision using a modified procedure [27][28][29] . In brief, corneal buttons were cut from the eye and cleaned of extraneous tissues. They were then plated flat on a six-well plate with epithelium side up. After 10 min to allow for attachment of the explant, Mg 2þ and Ca 2þ -free Hank's balanced salt solution (Thermo Fisher Scientific, Waltham, MA) containing 1.2 U dispase V R II (Sigma-Aldrich Chemical Co., St. Louis, MO) was added to each well and incubated at 37 C for 10 min. Corneal epithelium was separated from buttons under a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany) and incubated in keratocyte serum-free medium (KSFM; Invitrogen-Gibco) containing 25 mg bovine pituitary extract and 2.5 mg human recombinant epidermal growth factor at 37 C with 95% humidity and 5% CO 2 . The medium was changed every 2 d. After approximately 10 d, the explant was carefully transferred to a new dish. RCECs were subcultured using TrypLE Express (Thermo Fisher Scientific) at a split ratio of 1:3 after small cells reached subconfluence. Passaged cells were cultured in complex medium (1:1 ratio) with KSFM and Dulbecco's Modified Eagle's medium/F12 (Thermo Fisher Scientific) medium supplemented with 10% foetal bovine serum (Thermo Fisher Scientific). Cells between 5th and 10th passages were used for all experiments. Cell morphology was observed under a phase-contrast microscope.

CCK-8 assay
To evaluate the cytotoxicity of PM 2.5 in RCECs, CCK-8 assay (Abcam Inc., Cambridge, UK) was performed according to the manufacturer's instructions as previously described 30  NanoString nCounter V R miRNA assay Microarray gene expression was performed using an nCounter Sprint platform (NanoString Technologies, Inc. Seattle, WA) as described previously 31 . Briefly, RCECs were treated with 200 lg/mL PM 2.5 for 24 h and harvested. Total RNA was isolated from these cells using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was then subjected to quality evaluation and quantitative analysis using an AATI fragment analyser (Agilent Technologies, Santa Clara, CA) and a DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE). After solutionphase hybridization between the target mRNA and reportercapture probe pairs, excess probe was removed. Probe/target complexes were aligned and immobilized in an nCounter cartridge (NCT-120), which was then placed in a digital analyser for image acquisition and data processing. After digital analysis, the raw data were normalized against housekeeping genes. Each gene expression change was expressed as a log-2-fold change value compared to control cells. A heat-map was generated to show the difference in gene expression that as red represents up-regulated genes and green represents down-regulated genes, respectively.

Fluorescence staining
To investigate intracellular ROS levels, cells were stained with 10 lM 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCF-DA; Thermo Fisher Scientific) dye as previously described 33 . In order to investigate mitochondrial ROS levels, cells were stained with 5 lM MitoSOX TM red mitochondrial superoxide indicator (Thermo Fisher Scientific) dye images were then acquired with an EVOS Cell Imaging System (Thermo Fisher Scientific). To evaluate live mitochondrial mass, 100 nM MitoTracker V R Red probe (Thermo Fisher Scientific) was used to stain cells and 4 0 ,6 0 -diamidino-2-phenylindole (DAPI; Sigma-Aldrich Chemical Co.) was used to counterstain nuclei. After 30 min of staining, cells were fixed with 4% formaldehyde and observed using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) at the Core-Facility Centre for Tissue Regeneration, Dong-Eui University (Busan, Republic of Korea).
Immunoblotting Cells were treated with or without 1 mM NAC and 10 lM SB203580 for 1 h, and then treated with 100 lg/mL PM 2.5 for 24 h. Total protein was extracted using Pro-prep protein extraction solution (Intron Biotechnology, Seongnam, Republic of Korea). Nuclear and cytoplasmic proteins were isolated using NE-PER TM Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific). As previously described 35 , equal proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to Immun-Blot V R PVDF membranes (Bio-Rad Laboratories, Hercules, CA). Subsequently, these membranes were probed with specific primary anti-bodies (Supplementary Table S1). The membranes were then exposed enhanced chemiluminescence solution (Thermo Fisher Scientific) and visualized using a Fusion FX Imaging System (Vilber Lourmat, Torcy, France). Densitometric analysis of the bands was performed using the ImageJ V R software version 1.48 (NIH, Bethesda, MD).

Statistical analysis
All experiments were performed at least three times. Data are expressed as mean ± standard deviation (SD). GraphPad Prism version 5.03 (GraphPad Software Inc., La Jolla, CA) was used for all statistical analyses. Significant differences were analysed using analysis of variance (ANOVA) followed by Tukey's test. Probability values of p < 0.05 were considered as statistically significant.

Characterization of RCECs
Some fibroblasts were observed in early-passage (<passage 5) RCECs (Figure 1(A)). There was a heterogenous population of small and large squamous cells. Partially, large cells made up blebs near the tissue were detached from the culture surface. Meanwhile, between the 5th passage and the 10th passage, uniformly small cells with topical cobblestone morphology similar to stable primary corneal epithelial cell lines obtained from human and murine were observed 28,36 . However, cells with passage number more than 15 underwent senescence and showed morphological changes. Therefore, we used stable cells with passage number between 5 and 10 for all experiments. To determine whether cultured RCECs have properties of epithelial cells derived from cornea, cells were stained with pan-CK antibody. Figure  1(B) showed that RCECs strongly expressed pan-CK, a positive marker for corneal epithelial cells 37 . These results confirmed that RCECs used in this study were derived from the cornea.

PM 2.5 promotes cytotoxicity in RCECs
Cytotoxicity of PM 2.5 to primary RCECs was evaluated using CCK-8 assay. As shown in Figure 1(C), PM 2.5 had no cytotoxicity to primary RCECs at concentration up to 50 lg/mL. However, viabilities of cells treated with PM 2.5 at concentration above 100 lg/mL were significantly decreased compared to those of normal cells. After treatment with PM 2.5 at 100 and 200 lg/mL, viabilities of RCECs were approximately 83 and 50%, respectively. In addition, PM 2.5 caused morphological changes including cell membrane collapse and aggregation ( Figure 1(D)). These results suggest that PM 2.5 can induce morphological changes of RCECs, and it is cytotoxic to RCECs at high concentrations.

PM 2.5 increases the expression of inflammationrelated mediators
To evaluate the effect of PM 2.5 on gene expression, NanoString nCounter V R miRNA gene analysis was performed. Results of gene microarray analysis of RCECs exposed to 200 mg/mL PM 2.5 compared to normal cells are shown as heatmaps. Expression of each gene was converted to a log2fold-change value. If the value showed an increase of more than 3-fold, it was marked in red. If it showed a decrease of more than 3-fold, it was marked in green ( Figure 2(A)). Figure  2(B) shows results of quantification based on the log2-fold change values of gene expression. Overall, PM 2.5 -treated cells greatly enhanced the expression of genes involved in the inflammatory response. In addition, changes in the expression of chemokine receptors and chemokine ligands were observed in PM 2.5 -treated cells. The expression of Toll-like receptor (TLR) gene involved in innate immunity was upregulated by PM 2.5 , and the expression of its corresponding immune-related gene was also increased by PM 2.5 . Furthermore, expression levels of genes involved in inflammatory responses, including those in MAPK and nuclear translocation factor NF-jB signalling pathways, were enhanced by PM 2.5 . However, the expression of TGF-b gene known to contribute to wound healing and repair response was markedly down-regulated in PM 2.5treated cells. Based on the altered mRNA expression of the inflammatory factor induced by PM 2.5 , we verified that secretion levels of major inflammatory cytokines including TNF-a, IL-1b, and PGE 2 were upregulated by PM 2.5 . PM 2.5 treatment increased cytokine production in RCECs compared to the control without such treatment. Figure 2(C) shows that TNF-a, IL-1b, and PGE 2 levels were increased by 1.3-fold, 1.75-fold, and 1.36-fold relative to the control, respectively, in cells treated with 100 mg/mL PM 2.5 . Moreover, secretion levels of TNF-a, IL-1b, and PGE 2 were markedly increased 1.68-fold, 2.48-fold, and 2.27-fold, respectively, following treatment with 200 mg/mL of PM 2.5 compared to those in control cells. Results of nCounter mRNA analysis revealed that the secretion level of TGFb was significantly decreased to 0.4-fold of control in 200 lg/mL PM 2.5 -treated cells. These results suggest that PM 2.5 can markedly up-regulate the expression and secretion of pro-inflammatory mediators while down-regulating the mRNA expression and secretion of wound healing-related TGFb in RCECs.

PM 2.5 induces intracellular ROS production and cellular organelle damages
To investigate whether PM 2.5 might affect intracellular ROS production, RCECs were stained with DCF-DA and MitoSOX TM red. Figure 3(A) shows that intracellular and mitochondrial ROS levels are markedly enhanced by PM 2.5 dose-dependently. Next, we evaluated the effect of PM 2.5 on mitochondria and nuclei of RCECs. Many MitoTracker V R Red-positive cells indicating live mitochondria were observed in control cells. However, these cells were substantially decreased in PM 2.5 -treated group (Figure 3(B)). In addition, the expression of cH2AX, a marker of DNA double-strand break 38 , was notably increased by PM 2.5 (Figure 3(C)). These results indicate that PM 2.5 can promote intracellular ROS production and mediate cellular damage by affecting mitochondria and DNA doublestrand break in RCECs.  A and B) Heatmap of candidate gene expression using NanoString nCounter V R miRNA expression assay in PM 2.5 -stimulated RCECs. Cells were treated with or without 200 lg/mL of PM 2.5 for 24 h. Total RNA was isolated and hybridization was performed using a reporter probe and a capture probe. (A) Heatmap representing expressed genes with fold-change cut-off of 3.0 (upregulation and downregulation in red and green, respectively). (B) Expression of each gene was indicated as fold change compared to the control. (C) Levels of cytokines including TNF-a, IL-1b, PGE 2 , and TGF-b1 in supernatants of PM 2.5 -stimulated RCECs. Three independent experiments were performed in duplicate. The data are expressed as the means ± SD (n ¼ 6). Ã p < 0.05; ÃÃ p < 0.01; and ÃÃÃ p < 0.001 between groups. PM 2.5 : particulate matter2.5; RCECs: rat corneal epithelial cells; TNF-a: tumour necrosis factor; IL-1b: interleukin-1b; PGE 2 : prostaglandin E2; TGF-b1: transforming growth factor-b1.

PM 2.5 activates mitogen-activated protein kinase (MAPK) and NF-jB signalling pathway
To explore signalling pathways involved in PM 2.5 -induced inflammation and cellular damages, Western blot analysis, and immunofluorescence staining were performed. Figure  4(A,B) shows that the phosphorylation of p38 MAPK markedly up-regulated by PM 2.5 , and the levels sustained from 1 to 24 h after expose. As shown in Figure 4(C,D), PM 2.5 up-regulated the expression of extracellular signal-regulated kinase (ERK) and the phosphorylation of p38 MAPK, but slightly down-regulated the expression of c-Jun N-terminal kinase (JNK). Meanwhile, the expression and phosphorylation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) did not alter after treatment with PM 2.5 . Furthermore, we determined whether NF-jB signalling pathway was activated by PM 2.5 in RCECs. The result of Western blot analysis showed that the expression of NF-jB p65 in the nucleus fraction was markedly increased by PM 2.5 in a time and a dosedependent manner (Figure 4(E-H)). This result is consistent with the result of immunofluorescence staining suggesting that the phosphorylated form (p)-NF-jB p65 was strikingly expressed in nucleus (Figure 4(I)). These results suggest that PM 2.5 can activate MAPK signalling pathway and markedly induce the translocation of NF-jB p65 into the nucleus from the cytoplasm.

PM 2.5 -induced cellular damage is triggered by ROS in RCECs
Based on the above findings that PM 2.5 induced intracellular ROS production, we next evaluated the role of ROS in PM 2.5mediated cellular dysfunction including cytotoxicity, inflammation, and organelle damages. To determine the effect of blocking ROS in PM 2.5 -stimulated RCECs, cells were pretreated with NAC, a potential ROS scavenger, before exposure to PM 2.5 . As a result of CCK-8 assay, NAC significantly recovered PM 2.5 -induced decrement of cell viability ( Figure  5(A)). Furthermore, pre-treatment with NAC prominently decreased morphological changes such as cell membrane collapse following PM 2.5 ( Figure 5(B)). In addition, PM 2.5 -mediated increases of TNF-a, IL-1b, and PGE 2 levels were significantly suppressed by NAC treatment, leading to similar to those in control cells ( Figure 5(C)). Moreover, removal of ROS by NAC remarkably decreased the population of DCF-DApositive cells and MitoTracker V R Red-positive cells ( Figure 5(D), top and middle panels). Additionally, NAC completely suppressed the expression of cH2AX in PM 2.5 -stimulated RCECs ( Figure 5(D), bottom panels). Noteworthy, blocking ROS with NAC markedly down-regulated the phosphorylation of p38 MAPK without affecting the expression or phosphorylation of ERK and JNK ( Figure 5(E,F)). As shown in Figure 5(G), NAC also repressed the expression of p-NF-jB p65 in the nucleus compared to PM 2.5 -treated cells. These results suggest that removal of ROS by NAC can markedly suppress PM 2.5 -mediated cellular damages including cytotoxicity, inflammation, mitochondrial damage, and DNA double-strand break. More importantly, ROS blocking selectively down-regulated p38 MAPK signal and inactivated NF-jB p65 in PM 2.5 -stimulated cells.

PM 2.5 -induced cellular dysfunction involves ROS/p38 MAPK signalling pathway in RCECs
Since NAC specifically suppressed the phosphorylation of p38 MAPK in PM 2.5 -stimulated RCECs, we next investigate whether p38 MAPK was directly involved in PM 2.5 -mediated cellular dysfunction. As shown in Figure 6(A), SB203580, a selective inhibitor of p38 MAPK, significantly suppressed PM 2.5 -induced cytotoxicity. Furthermore, pre-treatment with SB203580 remarkably decreased mitochondrial damage and levels of pro-inflammatory cytokines following exposure to PM 2.5 ( Figure 6(B,C)). Additionally, p38 MAPK inhibition greatly blocked the expression of p-NF-jB p65 in the nucleus (Figure 6(D)). These results suggest that p38 MAPK signalling pathway is activated after ROS generation in RCECs following PM 2.5 treatment.

Discussion
The front of the eye is protected by tear film that covers the entire eye 39 . Accumulating evidences have shown that PM can aggravate dry eye disease or allergic keratitis and conjunctivitis, causing ocular discomfort and inflammation 7,40,41 . Dry eye syndrome is a representative ocular surface disease caused by a variety of factors including air pollution, changes in visual acuity, and instability of tear film. It is often accompanied by potential ocular surface damage, tear osmotic pressure elevation, and ocular inflammation 40 . Damage to the ocular surface epithelium by various factors including fine dust can trigger an inflammatory response that may lead to further decrease of tear production and worsening of symptoms 42,43 . Herein, we verified that results of this study revealed that RCECs could recognize PM 2.5 as an external harmful substance, thus generating immune responses. PM 2.5 up-regulated the expression of genes involved in inflammation and immune response. Among pro-inflammatory cytokines, TNF-a, IL-1b, and PGE 2 showed significantly increased secretion levels in RECEs after treatment with PM 2.5 . However, PM 2.5 markedly decreased the expression and secretion level of TGF-b known to play a critical role in wound healing 44 . Several studies have also reported that PM 2.5 can induce inflammatory responses in corneal epithelial cells 18,26,29 . Ma et al. 29 have reported that PM 2.5 treatment significantly elevated mRNA and protein levels of interferon Bar graphs indicate the relative band density of the phosphorylated protein/non-phosphorylated protein ratio in western blot analysis. Significant differences compared to the control cells at Ã p < 0.05 and ÃÃÃ p < 0.001. (E and G) Expression levels of NF-jB p65 protein in the cytosol and nucleus. b-actin and lamin B proteins were used as internal controls in the cytoplasm and nuclear, respectively. (E) Cells were treated with 200 lg/mL of PM 2.5 for indicated times. (G) Cells were treated with indicated concentration of PM 2.5 for 24 h. (F and H) Bar graphs indicate the relative band density in western blot analysis. Significant differences compared to the control cells at Ã p < 0.05, ÃÃ p < 0.01, and ÃÃÃ p < 0.001. (I) Immunofluorescence images for p-NF-jB p65 (red) and DAPI (blue). Scale bar: 75 lm. All experiments were performed three independent times. PM 2.5 : particulate matter2.5; MAPK: mitogen-activated protein kinase; NF-jB: nuclear factor-jB; RCECs: rat corneal epithelial cells; PI3K: phosphatidylinositol 3-kinase; Akt: protein kinase B; p-NF-jB: phosphorylated nuclear factor-jB.
gamma (IFN-c), IL-10, IL-17, and IL-21 in human bronchial epithelial cells. They have suggested that increases of cytokines require the presence of macrophages and that PM 2.5 can promote cytotoxic inflammation of T cells in a macrophagedependent manner. Another study has shown that topical expose to PM2.5 increased the expression levels of proinflammatory cytokines, including TNF-a and IL-6, in murine model 45 . In addition, PM 2 can increase oxidative stress and inflammation through the NF-jB/TNF-a pathway 45 . NF-jB can increase transcriptional activities of various pro-inflammatory genes including cytokines and chemokines, thus playing a key role in inflammatory regulation 46 . Numerous studies have shown that the expression of NF-jB p65 protein is increased in corneal tissues of PM 2.5 and PM 10 injected mice showing clinical symptoms similar to dry eye syndrome 18,44 . In this study, PM 2.5 caused inflammatory responses with NF-ᴋ B strongly expressed in the nucleus of PM 2.5 -treated cells. In addition, Western blot analysis revealed that p-NF-jB p65 (G) Effect of NAC on PM 2.5 -induced NF-jB activation. Immunofluorescence images for p-NF-jB p65 (red) and DAPI (blue) are shown. Scale bar: 75 lm. All experiments were performed three independent times. PM 2.5 : particulate matter2.5; ROS: reactive oxygen species; RCECs: rat corneal epithelial cells; NAC: N-acetyl-L-cysteine; DAPI: 4 0 ,6 0 -diamidino-2-phenylindole; cH2AX: phosphorylated histone H2AX; MAPK: mitogen-activated protein kinase; NF-jB: nuclear factor-jB; p-NF-jB: phosphorylated nuclear factor-jB.
ROS by NAC markedly suppressed PM 2.5 -mediated cellular damages including cytotoxicity, inflammation, mitochondrial damage, and DNA double-strand break. These findings demonstrate role of ROS in PM 2.5 -mediated cellular damages in RCECs. Specifically, ROS blocking selectively down-regulated p38 MAPK signal and caused in inactivation of NF-jB p65 in PM 2.5 -stimulated cells. The cascade of MAPKs signalling pathways is involved in a variety of cellular functions including cell proliferation, differentiation, migration, and survival 50 . Among various MAPKs, p38 MAPK is strongly activated by environmental stresses, inflammatory cytokines, and various extracellular stimuli 51 . There has been reported that PM can lead to injury in human lung endothelial cells via the ROS/ p38 MAPK-dependent pathway and that the disruption to the endothelium can be attenuated by NAC 52 . Another study has also reported that SB203580, a selective inhibitor of p38 MAPK, can inhibit the proliferation of vascular smooth muscle in PM 2.5 -mediated atherosclerosis model, indicating that the p38 MAPK signalling pathway plays a critical role in PM 2.5induced pathogenesis 53 . More recently, it has been suggested that PM 2.5 -induced inflammation can activate the ROS/p38 MAPK pathway in human skin keratinocytes 54 . Lee et al. 55 have demonstrated that the ROS/p38 MAPK/protein kinase B pathway is involved in PM 2.5 -induced inflammatory lung injury and vascular hyperpermeability. Thus, PM 2.5 is expected to activate the p38 MAPK signalling pathway with overall effects on various cells and organs. In this study, we found that the p38 MAPK signalling pathway was activated as a down-stream event of ROS under PM 2.5 -stimulated condition in RCECs. Furthermore, blocking the p38 MAPK signalling pathway by SB203580 significantly suppressed PM 2.5 -induced cytotoxicity, mitochondrial damage, and levels of pro-inflammatory cytokines. Therefore, exposure to PM 2.5 can lead to the production of ROS which then activates the p38 MAPK signalling pathway, causing cellular dysfunction in RCECs.
Overall, this study suggests that PM 2.5 can increase intracellular ROS to trigger the activation of the p38 MAPK signalling pathway, ultimately resulting in inflammation through NF-jB translocation into the nucleus of RCECs. Furthermore, PM 2.5 could activate the ROS/p38 MAPK signalling pathway and lead to mitochondrial damage and DNA double-strand break, causing cytotoxicity (Figure 7). Taken together, these findings suggest that the ROS p38 MAPK/NF-jB signalling pathway is one of the mechanisms involved in PM 2.5 -induced ocular surface disorders.