Ambient air pollutions caused by rapid industrial development are crucial environmental factors that threaten human health. Airborne particulate matters (PMs) with less than 10 μm (PM₁₀) are important causes of skin damage (1). The skin is the first protective barrier in humans and in direct contact with a variety of air pollutants (2). PM₁₀ can penetrate deep into the skin through hair follicles and inflamed sites, causing skin inflammation (3). Continuous skin exposure to environmental pollutants may cause skin damage, such as acne, psoriasis, and atopic dermatitis (AD) (4). Exposure to PM stimulates excessive generation of reactive oxygen species (ROS) (5). ROS are essential for maintaining cellular homeostasis, regulation, and defense; however, an imbalance between the levels of antioxidants and oxidants (free radicals or reactive species), leading to excessive ROS accumulation, can cause various diseases, including cancer, inflammation, and aging (6).

The human body has an antioxidant defense system to prevent oxidative damage from free radicals. This system includes free radical scavenging, metal chelation, and enzymatic activities to neutralize reactive species. Antioxidants like catalase, superoxide dismutase (SOD), and thioredoxin (TXN) are well-researched for their role in preventing and treating diseases related to oxidative damage (7).

PM₁₀-induced cellular ROS can cause inflammation in HaCaT cells, leading to the activation of excessive AD-related inflammatory markers, including interleukin (IL)-6, IL-8, IL-1α, CC chemokine ligand (CCL)17, CCL27, and cyclooxygenase-2 (COX-2), resulting in a wide range of tissue damage and human diseases (8). In particular, IL-6 plays a significant role in inflammation and can contribute to skin diseases such as psoriasis, AD, etc. (9). Therefore, a potential antioxidant reagent to protect from PM₁₀-induced skin oxidation, inflammation may be a desirable candidate for developing anti-dust drugs or cosmeceuticals. Specialized pro-resolving mediators (SPMs) produced by neutrophils and macrophages in humans, resolve inflammation in trace amounts and protect against infection (10). Recent studies have indicated that SPMs effectively and stereo-selectively facilitate the resolution of inflammation, tissue repair, and regeneration (11).

Maresin 1 (MaR1) is a newly discovered naturally occurring anti-inflammatory and pro-resolution oxylipin. It is synthesized by human macrophages from docosahexaenoic acid and has been shown to affect immune cells in various ways. Recent studies have revealed that MaR1 reduces neutrophil migration and cytokine production by activating CD^8＋ T cells, CD^4＋ T helper (Th1) cells, and Th17 cells. In response to environmental factors and external pathogens, macrophages and DCs mitigate inflammatory responses through phagocytosis to resolve inflammation. Additionally, MaR1 plays a crucial role in promoting phagocytic activity in macrophages and enhancing anti-inflammatory effects (12).

In addition, 7S,14S-dihydroxydocosahexaenoic acid (7S-epimer of maresin; 7S MaR1), one of the SPMs synthesized from docosahexaenoic acid, is a potential therapeutic approach for downregulating inflammatory mediators for skin inflammation. Hence, we investigated whether 7S MaR1 attenuates skin inflammation by downregulating pro-inflammatory cytokines. The comprehensive understanding of anti-inflammatory and antioxidant effects of 7S MaR1 in HaCaT cells has not been investigated. Herein, we tested whether the pro-inflammatory cytokine IL-6 is inhibited by 7S MaR1.

In our study, we demonstrated that the mechanism of the anti-inflammatory effects of 7S MaR1 via ROS/p38/ERK/NF-κB pathways in PM₁₀-exposed HaCaT cells.

The effects of 7S MaR1 on IL-6 expression in PM₁₀-exposed HaCaT cells

The molecular structure of 7S MaR1 is shown in Fig. 1A. The MTS assay was used to investigate the cytotoxicity of 7S MaR1 in HaCaT cells in the absence or addition of PM₁₀. There was no cytotoxicity observed at concentrations up to 40 μM (Supplementary Fig. 1). To evaluate whether 7S MaR1 inhibited the gene expressions of AD-related pro-inflammatory cytokines, such as IL-6, IL-8, IL-1α, CCL-17, CCL-27, and COX-2 in PM₁₀-exposed HaCaT cells, the cells were treated with 7S MaR1 (20 and 40 μM, 1 h) and then exposed for 24 h to PM₁₀ at the indicated concentration. RT-PCR and qRT-PCR analyses showed that PM₁₀ increased in IL-6 mRNA level in HaCaT cells, and as shown in Fig. 1B, C, 7S MaR1 decreased this level in a dose-dependent manner. On the other hand, 7S MaR1 had no effects on the expression of IL-8, IL-1α, CCL-17, or CCL-27 (data not shown). Moreover, 7S MaR1 did not inhibit COX-2 mRNA expression level in PM₁₀-stimulated HaCaT cells (data not shown). Hence, we focused on IL-6 expression in PM₁₀-exposed HaCaT cells. ELISA analysis revealed that 7S MaR1 significantly suppressed secreted protein IL-6 level in PM₁₀-exposed HaCaT cells (Fig. 1D). We evaluated whether 7S MaR1 attenuated the PM₁₀-induced promoter activity of IL-6 in HaCaT cells. As Fig. 1E shows, 7S MaR1 attenuated PM₁₀-induced IL-6 promoter activity.

7S MaR1 modulated PM₁₀-induced ROS accumulation by recovering ROS scavengers in HaCaT cells

To confirm PM₁₀-induced intracellular ROS accumulation, RT-PCR analyses were performed to evaluate the ROS scavenger gene expression. As shown in Fig. 2A, PM₁₀ decreased the ROS scavenger genes (catalase, SOD-2 and thioredoxin 1 (TXN-1) expression at a high concentration (400 μg/ml) of PM₁₀. As shown in Fig. 2B, 7S MaR1 recovered the levels of ROS scavengers, including catalase, SOD-2 and TXN-1. To examine the potential of 7S MaR1 in reducing PM₁₀-upregulated generation of ROS in HaCaT cells, the cells were pre-incubated with 7S MaR1 (40 μM, 1 h) or N-acetyl cysteine (NAC; 50 mM, 1 h), and exposed to PM₁₀. As shown in Fig. 2C, the assessment of ROS levels demonstrated that both 7S MaR1 and NAC reduced the PM₁₀-induced increase in intracellular ROS production in HaCaT cells. Cotreatment with 7S MaR1 and NAC reduced the intracellular ROS levels to lesser than that of the negative control. We also examined immunofluorescence images to clarify whether 7S MaR1 attenuated intracellular ROS accumulation. HaCaT cells were pre-incubated with 7S MaR1 or NAC and then exposed to PM₁₀. The analysis of fluorescence images revealed that intracellular ROS accumulation induced by PM₁₀ was modulated by 7S MaR1 and NAC (Fig. 2D).

Molecular docking analyses revealed that 7S MaR1 inhibited PM₁₀-induced phosphorylation of MAPKs (p38 and ERK) in HaCaT cells

We performed docking analyses to investigate whether 7S MaR1 binds to MEK1 binding site. Our observations confirmed that 7S MaR1 binds to MEK1 target sites (Fig. 3A) with a high affinity of −7.0 kcal/mol. 7S MaR1 formed hydrogen bonds with the residues Gln153, Ser194, and Gly77, with bond lengths of 2.56 Å, 3.01 Å, and 2.88 Å, respectively. 7S MaR1 also formed additional hydrophobic bonds with Cys207, Leu197, Leu74, Ala95, and Val82. As shown in Fig. 3B, previous studies predicted the top five druggable target sites, suggesting that these binding sites significantly enhance the binding of compounds with drug-like properties to their residues in the pocket. Among them, site 1 includes the activation loop regions where ERK connects to MEK (13). Overall, the results suggest that 7S MaR1 binds to MEK1, blocking its activation.

We also performed docking analyses to explore the binding of 7S MaR1 to the ATP-binding site in p38, to see if it could downregulate IL-6 via the p38-mediated pathway. 7S MaR1 demonstrated a binding affinity of −6.3 kcal/mol to this site (Fig. 3C). It formed hydrogen bonds with His107, Gly110, Ala51, and Leu104 within the ATP site, with bond lengths of 2.67 Å, 2.85 Å, 2.36 Å, and 2.80 Å, respectively. Additionally, 7S MaR1 had hydrophobic interactions with Val38 and Met109 and formed a carbon-hydrogen bond with Thr106. Its binding position was similar to SB203580, a specific p38 MAPK inhibitor, with both occupying the ATP-binding pockets of p38 (Supplementary Fig. 2). To determine whether 7S MaR1 reduced IL-6 levels in PM₁₀-exposed HaCaT cells via the p38 pathway, the cells were pre-incubated with SB203580 (10 μM, 1 h) or 7S MaR1 (40 μM, 1 h) before PM₁₀ exposure (400 μg/ml, 24 h). Treatment with SB203580 alone or with 7S MaR1 downregulated IL-6 levels. These findings suggest that 7S MaR1 inhibits the PM₁₀-induced increase in IL-6 through the p38 pathway (Fig. 3D). To elucidate potential role of 7S MaR1 in modulating ERK activation, a critical pathway in cellular signaling, the effects of 7S MaR1 on PM₁₀-induced phosphorylation of MAPK was investigated using western blotting. HaCaT cells were pre-incubated with 7S MaR1 (40 μM, 1 h) and exposed to PM₁₀ (400 μg/ml, 30 min). As shown in Fig. 3E, 7S MaR1 attenuated PM₁₀-induced phosphorylation levels of p38 and ERK. However, phosphorylated JNK levels were not significantly affected by 7S MaR1 (data not shown). We further investigated the relationship between PM₁₀-induced ROS production and phosphorylation of p38 and ERK and whether 7S MaR1 could inhibit this phosphorylation. HaCaT cells were pre-incubated for 1 h with NAC (50 mM) or 7S MaR1 (40 μM) and then exposed to PM₁₀ (400 μg/ml, 30 min). These results showed that 7S MaR1 and NAC reduced PM₁₀-induced phosphorylation of p38 and ERK (Fig. 3E, F). These results indicate that PM₁₀-induced phosphorylation of p38 and ERK is mediated by ROS and regulated by 7S MaR1.

7S MaR1 inhibited NF-κB translocation in PM₁₀-exposed HaCaT cells

To identify the NF-κB translocation, the cells were pre-incubated with 7S MaR1 (40 μM, 1 h) prior to PM₁₀ exposure (400 μg/ml, 30 min). The translocation was examined using immunofluorescence staining. As shown in Fig. 4A, PM₁₀ markedly increased NF-κB p50 translocation, whereas 7S MaR1 inhibited the translocations. The luciferase assay for NF-κB activity also demonstrated that 7S MaR1 reduced PM₁₀-induced NF-κB activity (Fig. 4B). Bay11-7082, known as an NF-κB inhibitor, was utilized to examine whether the induction of IL-6 protein was mediated by NF-κB. Bay11-7082 and 7S MaR1 inhibited expression of IL-6. In addition, cotreatment of 7S MaR1 and Bay11-7082 synergistically reduced protein expression of IL-6 in PM₁₀-exposed HaCaT cells (Fig. 4C). Collectively, these results indicated that the NF-κB signaling pathway was activated by PM₁₀ plus 7S MaR1 inhibited PM₁₀-stimulated NF-κB translocation and its activity.

PM₁₀, a key health issue worldwide, negatively affects the skin (14). While the skin is directly exposed to various toxicants in the air, the harmful effects of air pollutants on the skin are still being investigated (15). PM₁₀-induced ROS generation significantly influences the MAPK and NF-κB signaling pathway. In addition, ROS may be involved in the upregulation of IL-6 expression which has been linked to the development of skin disorders (AD, psoriasis, etc.) (16). Antioxidant modulators to reduce ROS accumulation in the human body include ROS scavenger genes (catalase, SOD-2, and TXN-1), which remove harmful free radicals protecting cells from the oxidation of biomolecules (17). As shown in Fig. 2B, 7S MaR1 plays a critical role in restoring ROS scavenging ability by recovering the expression of these ROS scavenger genes.

p38 and ERK MAPK signaling pathways in the skin are triggered in response to external stimuli such as environmental contaminants (18). MEK, an intermediate protein in the ERK pathway, is responsible for phosphorylation of ERK, which then phosphorylates a variety of downstream targets involved in the barrier function of the epidermis (19, 20). Previous studies have predicted the druggable pockets of the MEK, where the substrate ERK binds (13). Our study demonstrates that 7S MaR1 binds to important amino acid residues participating in the MEK phosphorylation in the binding pocket (Fig. 3A, B).

To investigate whether 7S MaR1 also inhibits IL-6 levels by regulating the p38 pathway, we performed a docking study and IL-6 ELISA assay (Fig. 3C, D). The N- and C-termini of p38 are linked by a hinge, forming a structural framework that encloses a deep crevice responsible for binding the ATP binding pocket. There are two types of classifications for inhibitors of kinase: type I and type II. Type I inhibitors, also known as competitive inhibitors, bind to the ATP-binding region of p38 MAPK. The key amino acid residues in the ATP binding site are Met109 and Gly110. Type I inhibitors can cause a ligand-induced peptide flipping between Met109 and Gly110, allowing a double hydrogen-bond interaction with the hinge region to develop. Therefore, this interaction holds great significance in the context of p38 MAPK selective inhibitors, as it effectively occupies the adjacent hydrophobic pocket of p38 MAPK (21, 22). By competing with ATP binding, pyridinyl imidazole, known as SB203580, has been effective in studying the role of p38 kinase in regulating transcription and translation in response to diverse stress (23). As mentioned above, the SB203580 obstructs the activation of p38 MAPK by forming hydrophobic interactions with the specific amino acids of the ATP binding site.

This study found that the –OH groups of 7S MaR1 interact hydrophobically with the amino acid residues Gly110, His107, Ala51, and Leu104 (Supplementary Fig. 1). Additionally, 7S MaR1 participates in hydrophobic interactions with Met109, Val38, and Thr106, including carbon-hydrogen bonds with Leu108 and Lys53. Hence, 7S MaR1 can bind to the amino acid residues similar to those targeted by SB203580, a known specific p38 inhibitor. This binding effectively inhibits the activation of p38 MAPK, suggesting that 7s MaR1 can be a therapeutic agent that functions as a p38 MAPK inhibitor.

We assessed whether 7S MaR1 affected the MAPK signaling pathway mediated by ROS generation. As shown in Fig. 3E, F, NAC and 7S MaR1 inhibited PM₁₀-induced phosphorylation levels of p38 and ERK.

NF-κB is widely recognized for its ability to activate the pro-inflammatory cytokine IL-6 expression (24). Upon stimulation, NF-κB is dissociated from the complexes and entered the nucleus. The activation of NF-κB promotes the diverse pro-inflammatory cytokines expression (25). Fig. 4A-C demonstrated the inhibitory effect of 7S MaR1 on the NF-κB p50 translocation, as well as its ability to suppress PM₁₀-induced NF-κB activity. Our findings add to the evidence that activating NF-κB in PM₁₀-exposed HaCaT cells causes an increase in the level of IL-6. As shown in Fig. 4C, the NF-κB inhibitor Bay11-7082 and 7S MaR1 suppressed IL-6 protein levels. Moreover, 7S MaR1 inhibited the transcriptional activity of IL-6 gene promoter (Fig. 1E).

In this study, we investigated the potential of 7S MaR1 in mitigating PM₁₀-induced skin inflammation. Based on our findings, we verified that exposure to PM₁₀ led to the upregulation of IL-6 expression through p38/ERK/NF-κB pathways and increased the transcriptional activity of IL-6 promoter in HaCaT cells. Hence, it is important to develop a therapeutic agent that can resolve inflammation.

Overall, the antioxidative and anti-inflammatory effects of 7S MaR1 involve the modulation of PM₁₀-induced transcriptional activity of IL-6 promoter, as well as the inhibition of p38/ERK/NF-κB signaling pathways (Fig. 5). These impacts are accomplished by recovering genes associated with scavenging reactive oxygen species (ROS) in HaCaT cells.

Cell culture

HaCaT cells were cultured and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were incubated in a 5% CO2 and at 37°C.

Raw materials and reagents

The ERM-certified reference material ERM-CZ100 (PM₁₀-like) was purchased from Merck (Darmstadt, Germany), suspended in phosphate-buffered saline (PBS), and homogenized through sonication for 60 min to achieve a stock concentration of 50 mg/ml. 7S MaR1 was purified as recently reported (26) and obtained from Prof. Deok-Kun Oh (Konkuk University, Seoul, Korea).

RNA extraction and gene expression analysis

HaCaT cells were pre-treated for 1 h with 7S MaR1 (20 and 40 μM) followed by 24 h treatment with PM₁₀ (400 μg/ml). The harvested cells were lysed using the easy-BLUE™ Total RNA extraction kit (iNtRon, Seoul, Korea). For RT-PCR, RNA (1 μg) was reverse-transcribed to cDNA using M-MuLV reverse transcriptase (New England Biolabs, Ipswich, MA, USA). The synthesized cDNA was used real-time polymerase chain reaction (RT-PCR) and qRT-PCR amplification. The expression of all target genes was normalized to that of the housekeeping gene GAPDH. The primers sequences used are listed in Supplementary Table 1.

ELISA

HaCaT cells were pre-treated with 7S MaR1 (40 μM) or Bay 11-7082 (10 μM) for 1 h, followed by 24 h treatment with PM₁₀ (400 μg/ml). The level of IL-6 in the cell supernatant was determined by ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Immunofluorescence

The cells were fixed with 4% formaldehyde for 15 min. After blocking with 1% BSA in PBS, the cells were then incubated overnight at 4°C with NF-κB p50 primary antibody (Cell Signaling Technology Inc, Danvers, MA, USA, 1:200 dilution). Subsequently, the cells were incubated with FITC-labeled goat anti-mouse IgG secondary antibodies (Merck Millipore, Germany, 1:400 dilution). The cells were stained with DAPI (Sigma-Aldrich, USA, 1:1000 dilution). Fluorescence images were obtained using a BX61-32FDIC upright fluorescence microscope (Olympus, Tokyo, Japan) equipped with a 400× objective lens.

Western blotting

HaCaT cells were pre-treated for 1 h with 7S MaR1 (20 and 40 μM) or NAC (20 mM), followed by 24 h treatment with PM₁₀ (400 μg/ml). The cells were lysed in RIPA buffer to obtain whole protein extract. The proteins (30 μg) were separated on 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk for 1 h and incubated with primary antibodies against MAPKs, including phospho-ERK, phospho-JNK, and phospho-p38, for 1 h at room temperature. After incubation, the membranes were incubated with the mouse-IgGκ light chain binding protein conjugated to horseradish peroxidase (HRP) (m-IgGκ BP-HRP) or goat anti-rabbit IgG-heavy and light chain antibody conjugated to HRP for 2 h at room temperature. Finally, the protein bands were visualized using and ECL reagents (Advansta, CA, USA). The densitometric graphs from three independent experiments were performed using ImageJ software. The respective band intensities were normalized to those of GAPDH.

Luciferase assay

For the determination of IL-6 and NF-κB luciferase activity, plasmids containing IL-6 and NF-κB were constructed. The pGL3-IL-6 promoter was prepared as previously reported (27). The cells were transfected with luciferase-expressing NF-κB luciferase reporter vector or IL-6 promoter expression vector for 24 h and pre-incubated with 7S MaR1 for 1 h and treated with PM₁₀ (400 μg/ml) for 24 h.

Evaluation of ROS level in PM₁₀-treated HaCaT cells

HaCaT cells were pre-treated with 7S MaR1 or the ROS scavenger NAC and 2ʹ,7ʹ-Dichloro fluorescein diacetate (DCF-DA) for 1 h, and then exposed for 10 min to PM₁₀ (400 μg/ml). ROS levels were evaluated using a Cyclone Phosphor Imager (PerkinElmer, Waltham, MA, USA).

Molecular docking

A docking study of 7S MaR1 with the MEK and p38 was performed using AutoDock VINA v1.2.0 (28). The crystal structures of MEK1 (PDB code: 1S9J) and p38 (PDB code: 1A9U) were used in the docking experiments. Molecular graphics for the best binding model were generated using Biovia Discovery Studio visualizer v21.1.0. For obtaining a simple and informative representation of the potential intermolecular interactions, the LIGPLOT program was used.

Statistical analysis

Data are presented as mean ± SD. GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. One-way analysis of variance (ANOVA) followed by Tukey’s HSD test was performed. Statistical significance was set at P < 0.05.

7S MaR1 was supplied by Prof. Deok-Kun Oh (Konkuk University, Seoul, Korea). We would like to thank Editage (www.editage.co.kr) for their assistance with English language editing (No. KOUNI-4812). This research was supported by the National Research Foundation of Korea, South Korea (NRF-2021R1A2C3003414, NRF-2021R1A6A1A03038890 and RS-2024-00451912).

The authors have no conflicting interests.