Preventative Effects of Antioxidants against PM10 on Serum IgE Concentration, Mast Cell Counts, Inflammatory Cytokines, and Keratinocyte Differentiation Markers in DNCB-Induced Atopic Dermatitis Mouse Model

Particulate matter (PM) can cause oxidative stress, inflammation, and skin aging. We investigated the effects of antioxidants such as dieckol, punicalagin, epigallocatechin gallate (EGCG), resveratrol, and Siegesbeckiae Herba extract (SHE) against PM < 10 μm (PM10) on serum IgE concentration, mast cell counts, inflammatory cytokines, and keratinocyte differentiation markers in a 2,4-Dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model. Seven-week-old BALB/c mice were sensitized with 2% DNCB. Atopic dermatitis-like lesions were induced on the mice with 0.2% DNCB. Antioxidants and PM10 were applied to the mice for 4 weeks. PM10 increased the serum IgE concentration and spleen weight in mice, and all antioxidants downregulated these parameters. Histological examination showed an increase in epidermal thickness and mast cell counts in response to PM10, and all antioxidants showed a decrease. PM10 upregulates the expression of inflammatory cytokines, including interleukin (IL)-1β, IL-4, IL-6, IL-17α, IL-25, IL-31 and thymic stromal lymphopoietin (TSLP) in mice, and all antioxidants inhibited the upregulation of inflammatory cytokines. ELISA showed the same results as real-time PCR. PM10 downregulates the expression of keratinocyte differentiation markers, including loricrin and filaggrin, in mouse keratinocytes and antioxidants prevented the downregulation of the keratinocyte differentiation markers. Conclusively, PM10 aggravated the DNCB-induced mouse model in serum IgE concentration, mast cell counts, inflammatory cytokine, and keratinocyte differentiation markers. In addition, antioxidants modulated changes in the DNCB-induced mouse model caused by PM10.

In this study, we investigated the effects of antioxidants, including dieckol, punicalagin, EGCG, and resveratrol on PM 10 -induced changes in a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) mouse model. In particular, the effects SHE on PM 10 -induced changes in the DNCB-induced atopic dermatitis mouse model were newly investigated.

DNCB-Induced AD Mouse Model
Six-week-old BALB/c mice were purchased from Orient Bio Inc. (Seongnam, Korea) and stabilized for one week. Our animal care and treatment protocols were in accordance with relevant guidelines for Laboratory Animals. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Kyungpook National University (IRB No. KNU 2021-0067).

SHE and Its Solvent Fractions
Leaves (140 g) of dried SHE purchased from Sinsun Herb (Seoul, Korea) were ground and extraction was performed at 90 • C for 1 h. The extracted solution was evaporated, and 9 g of the extract was obtained. It was dispersed in 150 mL of water and partitioned sequentially with an equal volume of methylene chloride (MC), ethyl acetate (EA), and n-butyl alcohol (BA). The organic solvents yielded MC fraction (0.27 g), EA fraction (0.16 g), and BA fraction (0.46 g). After filtration, insoluble material (1.15 g) was removed and then evaporated to obtain a water (WT) fraction (6.64 g).

Sebocyte and ORS Keratinocyte Culture
Sebaceous glands were isolated from occipital hairs produced during hair transplantation and were cultivated in Sebocyte Basal Medium (Cell application, San Diego, CA, USA) with Sebocyte Growth Supplement in a 5% CO 2 incubator at 37 • C. After two weeks of isolation, cells were harvested with 0.25% trypsin/10 mM EDTA in Hank's balanced salt solution (HBSS) and cultured at EpiLife (Gibco BRL, Rockville, MD, USA).
The sebaceous glands and hair bulb were cut off for the isolation of the outer root sheath (ORS) and were immersed in DMEM supplemented with 20% fetal bovine serum. On the third day of culture, the medium was changed to EpiLife (Gibco BRL) medium containing supplement.
After the second passage, sebocytes and ORS keratinocytes were used for these experiments. Informed written consent was obtained. The Medical Ethical Committee of the Kyungpook National University Hospital approved this study (IRB Number KNUH 2021-03-006-001).

Measurement of Serum IgE Concentration and Spleen Weight
All mice were sacrificed using CO 2 and their peripheral blood and spleen were collected. Serum levels of immunoglobulin (IgE) were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, UK) according to the manufacture's instructions. Briefly, after adding 100 µL of standard and samples to each well coated with IgE, they were incubated for 30 min at room temperature. After washing four times, they were incubated with 100 µL of an antibody conjugate for 30 min, and then IgE was detected using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 450 nm after adding Chromogen Substrate Solution. All spleens were removed immediately after sacrifice and weighed.

Measuring Epidermal Thickness and Counting Mast Cells
The excised dorsal skins of the mice were fixed with 10% formaldehyde and embedded in paraffin. The sections of 6 µm thickness were stained with hematoxylin and eosin (H&E) or toluidine blue (TB). Epidermal thickness was measured in H&E-stained images. Mast cell numbers were counted in TB-stained images. A purple color indicates mast cells.

Real-Time Polymerase Chain Reaction (PCR)
Total RNA was isolated from cutaneous dorsal mouse skin treated with PM 10 and antioxidants using a TRIzol reagent (Invitrogen, Waltham, MA, USA), and cDNA was synthesized from 3 µg of total RNA using a cDNA synthesis kit containing ImProm-IITM reverse transcriptase and oligo (dT) primers (Promega, Madison, WI, USA) at 72 • C for 10 min and 42 • C for 90 min. PCR primer sequences are summarized in Table 1. Real-time PCR was performed with 50 ng of cDNA and 10 pM primers in the SYBR Green mixture on the Step One Plus Real-Time PCR Assay (Applied Biosystems, Foster City, CA, USA). The cycling conditions for amplification were performed: 95 • C for 10 min, 40 cycles at 95 • C for 15 s, and 60 • C for 60 s. The PCR products were analysed using Step One Plus Real-Time PCR analysis software (Applied Biosystems).
Absorbance was measured at 450 nm using a spectrophotometer (VersaMax Microplate Reader, MA, USA).

Statistical Analysis
Data are expressed as means ± standard deviation (SD). The experiment was performed independently. Analysis of variance (ANOVA) was used for statistical analysis. p-values < 0.05 were considered statistically significant.

An Increase in the Production of Reactive Oxygen Species by PM 10 in Cultured Sebocytes and ORS Keratinocytes Was Decreased by Antioxidants
PM 10 can damage the skin via the synthesis of reactive oxygen species (ROS) [26,29,30]. First, we investigated whether dieckol, punicalagin, EGCG, resveratrol, or SHE inhibited the production of ROS induced by PM 10 . The production of ROS in cultured sebocytes and ORS keratinocytes increased in 100 µg/mL PM 10 . The increased production of ROS by PM 10 in cultured sebocytes and ORS keratinocytes was inhibited by dieckol, punicalagin, EGCG, resveratrol and SHE ( Figure 1).

Preventative Effects of Antioxidants against the Upregulation of Serum IgE Levels and Spleen
Weights in the PM 10 -Treated, DNCB-Induced AD Mice Serum IgE levels were significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with those after PM 10 treatment only ( Figure 2a). The order of effectiveness was as follows: punicalagin > EGCG = resveratrol > dieckol > SHE. The spleen weights were also significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with those after PM 10 treatment only (Figure 2b). The order of effectiveness was as follows: punicalagin = EGCG > dieckol = resveratrol = SHE.

Preventative Effects of Antioxidants against the Upregulation of Serum IgE Levels and Spleen Weights in the PM10-Treated, DNCB-Induced AD Mice
Serum IgE levels were significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with those after PM10 treatment only ( Figure 2a). The order of effectiveness was as follows: punicalagin > EGCG = resveratrol > dieckol > SHE. The spleen weights were also significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with those after PM10 treatment only (Figure 2b). The order of effectiveness was as follows: punicalagin = EGCG > dieckol = resveratrol = SHE. The serum immunoglobulin E (IgE) levels were significantly decreased after the combined treatment with dieckol, punicalagin, epigallocatechin-3-gallate (EGCG), resveratrol, or Siegesbeckiae Herba extract (SHE) compared with those after PM10 treatment only. The data in the bar graphs represent the mean ± standard deviation (SD) from three independent experiments (* p < 0.05). (b) The spleen weights were also significantly decreased after the combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE compared with those after PM10 treatment only. The data in the bar graphs represent the mean ± SD from three independent experiments (* p < 0.05).

Preventative Effects of Antioxidants against Increased Epidermal Thickness and Mast Cell Counts in the PM10-Treated, DNCB-Induced AD Mice
Epidermal thickness was significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with that after PM10 treatment only (Figure 3a,b). The order of effectiveness was punicalagin > EGCG > dieckol > resveratrol > SHE. The number of mast cells was also significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with that after PM  Preventative effects of antioxidants against the upregulation of ROS in the particulate matter <10 μm (PM10)-treated sebocytes and ORS keratinocytes. An increase in the production of reactive oxygen species by PM10 in cultured (a) sebocytes and (b) ORS keratinocytes was decreased by dieckol, punicalagin, epigallocatechin-3-gallate (EGCG), resveratrol, and Siegesbeckiae Herba extract (SHE). Data are represented as the mean ± SD from three independent experiments (* p < 0.05).

Preventative Effects of Antioxidants against the Upregulation of Serum IgE Levels and Spleen Weights in the PM10-Treated, DNCB-Induced AD Mice
Serum IgE levels were significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with those after PM10 treatment only (Figure 2a). The order of effectiveness was as follows: punicalagin > EGCG = resveratrol > dieckol > SHE. The spleen weights were also significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with those after PM10 treatment only (Figure 2b). The order of effectiveness was as follows: punicalagin = EGCG > dieckol = resveratrol = SHE. The serum immunoglobulin E (IgE) levels were significantly decreased after the combined treatment with dieckol, punicalagin, epigallocatechin-3-gallate (EGCG), resveratrol, or Siegesbeckiae Herba extract (SHE) compared with those after PM10 treatment only. The data in the bar graphs represent the mean ± standard deviation (SD) from three independent experiments (* p < 0.05). (b) The spleen weights were also significantly decreased after the combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE compared with those after PM10 treatment only. The data in the bar graphs represent the mean ± SD from three independent experiments (* p < 0.05).

Preventative Effects of Antioxidants against Increased Epidermal Thickness and Mast Cell Counts in the PM10-Treated, DNCB-Induced AD Mice
Epidermal thickness was significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with that after PM10 treatment only (Figure 3a,b). The order of effectiveness was punicalagin > EGCG > dieckol > resveratrol > SHE. The number of mast cells was also significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with that after PM The data in the bar graphs represent the mean ± standard deviation (SD) from three independent experiments (* p < 0.05). (b) The spleen weights were also significantly decreased after the combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE compared with those after PM 10 treatment only. The data in the bar graphs represent the mean ± SD from three independent experiments (* p < 0.05).

Preventative Effects of Antioxidants against Increased Epidermal Thickness and Mast Cell Counts in the PM 10 -Treated, DNCB-Induced AD Mice
Epidermal thickness was significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with that after PM 10 treatment only (Figure 3a,b). The order of effectiveness was punicalagin > EGCG > dieckol > resveratrol > SHE. The number of mast cells was also significantly decreased after combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE, compared with that after PM treatment only (Figure 3a,c). The order of effectiveness was punicalagin > SHE > dieckol > EGCG > resveratrol.
Antioxidants 2022, 11, x FOR PEER REVIEW 6 of 12 treatment only (Figure 3a,c). The order of effectiveness was punicalagin > SHE > dieckol > EGCG > resveratrol. The data in the bar graphs represent the mean ± standard deviation (SD) from three independent experiments (* p < 0.05). (b) Epidermal thickness was significantly decreased after the combined treatment with dieckol, punicalagin, epigallocatechin-3-gallate (EGCG), resveratrol, or Siegesbeckiae Herba extract (SHE) compared with that after PM10 treatment only. The data in the bar graphs represent the mean ± SD from three independent experiments (* p < 0.05). (c) The number of mast cells was also significantly decreased after the combined treatment with dieckol, punicalagin, EGCG, resveratrol, or SHE compared with that after PM10 treatment only. The data in the bar graphs represent the mean ± SD from three independent experiments (* p < 0.05).

Preventative Effects of Antioxidants on the Upregulation of the Keratinocyte Differentiation Markers in the PM 10 -Treated, DNCB-Induced AD Mice
The downregulation of keratinocyte differentiation markers by PM 10 was prevented by the combination of dieckol, punicalagin, EGCG, resveratrol, or SHE ( Figure 6). The order of effectiveness on loricrin expression was resveratrol = SHE > dieckol = punicalagin = EGCG. The order of effectiveness on filaggrin expression was dieckol > resveratrol = SHE > punicalagin = EGCG. Figure 5. Preventative effects of antioxidants on the upregulation of inflammatory cytokines protein expression in the particulate matter <10 μm (PM10)-treated, 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis mice. The protein expression of interleukin (IL)-1β, IL-4, IL-17α, and thymic stromal lymphopoietin (TSLP) was also significantly decreased after combined treatment with dieckol, punicalagin, epigallocatechin-3-gallate (EGCG), resveratrol, or Siegesbeckiae Herba extract (SHE) compared with that after PM10 treatment only. The data in the bar graphs represent the mean ± standard deviation (SD) of three independent experiments (* p < 0.05).

Preventative Effects of Antioxidants on the Upregulation of the Keratinocyte Differentiation Markers in the PM10-Treated, DNCB-Induced AD Mice
The downregulation of keratinocyte differentiation markers by PM10 was prevented by the combination of dieckol, punicalagin, EGCG, resveratrol, or SHE ( Figure 6). The order of effectiveness on loricrin expression was resveratrol = SHE > dieckol = punicalagin = EGCG. The order of effectiveness on filaggrin expression was dieckol > resveratrol = SHE > punicalagin = EGCG.

Discussion
Air pollution may be related to the development and exacerbation of AD [30][31][32][33]. Woo et al. [34] reported that PM10 exposure induces and aggravates skin inflammation via the differential expression of genes controlling skin barrier integrity and immune response in ovalbumin (OVA)-treated mice. Li et al. [35] proved that PM2.5 promotes the expression of TSLP in PAM212 cells and may aggravate allergic responses through this pathway. Our study also demonstrated that PM10 exposure upregulated the expression of inflammation-related biomarkers and downregulated that of keratinocyte differentiation markers in DNCB-treated mice.
Park et al. [36] established an in vivo model to evaluate PM-induced lung injury in mice. They found that serum IgE levels tended to increase in mice treated with OVA and PM. Cohen et al. [37] showed that there were nominal effects on rat organ weights, including the spleen, as a result of dust exposure at the World Trade Center. Jin et al. [38] suggested that PM may affect mast cell activation in a study using bone-marrow-derived mast

Discussion
Air pollution may be related to the development and exacerbation of AD [30][31][32][33]. Woo et al. [34] reported that PM 10 exposure induces and aggravates skin inflammation via the differential expression of genes controlling skin barrier integrity and immune response in ovalbumin (OVA)-treated mice. Li et al. [35] proved that PM 2.5 promotes the expression of TSLP in PAM212 cells and may aggravate allergic responses through this pathway. Our study also demonstrated that PM 10 exposure upregulated the expression of inflammationrelated biomarkers and downregulated that of keratinocyte differentiation markers in DNCB-treated mice.
Park et al. [36] established an in vivo model to evaluate PM-induced lung injury in mice. They found that serum IgE levels tended to increase in mice treated with OVA and PM. Cohen et al. [37] showed that there were nominal effects on rat organ weights, including the spleen, as a result of dust exposure at the World Trade Center. Jin et al. [38] suggested that PM may affect mast cell activation in a study using bone-marrow-derived mast cells. Kataoka et al. [39] investigated the cytotoxic effects of water-soluble extracts of coarse and fine atmospheric PM on mast cell lines. Wang et al. reported that PM 2.5 promotes the IgE-mediated mast cell activation of ROS [40]. Several groups [41][42][43] reported that air pollution, including PM 10 , is associated with an increased risk of atopic dermatitis. The study showed that cytotoxic effects and PM concentrations were not always correlated. In our study, PM increased serum IgE levels in DNCB-treated mice. In addition, spleen weights and the number of mast cells were increased in DNCB-treated mice.
The association between PM and AD in humans has been introduced. Park et al. [44] investigated the association between PM and AD and other chronic inflammatory dermatoses using data from the Korean Health Insurance Review and Assessment Service. They concluded that PM was associated with AD and other chronic inflammatory skin diseases. Nakhjirgan et al. [45] also showed a statistically significant relationship between the concentration of PM and the exacerbation of sleep disturbance and itching in Iran. He et al. [46] evaluated the association between PM concentrations and outpatient visits for AD in Lanzhou and concluded that PM was closely related to outpatient visits for AD.
Antioxidants have preventative effects against oxidative stress. Our study revealed that a variety of antioxidants affected serum IgE concentration, spleen weight, mast cell counts, inflammatory cytokines, and keratinocyte differentiation markers in a mouse model of DNCB-induced AD. Punicalagin is more effective than other antioxidants, such as dieckol, EGCG, and resveratrol, for serum IgE concentration, spleen weight, and mast cell counts. The newly studied SHE was also effective in the regulation of serum IgE concentration, spleen weight, mast cell counts, inflammatory cytokines, and keratinocyte differentiation markers in a mouse model of DNCB-induced AD.
Shin et al. [26] reported that resveratrol inhibits PM 10 -induced inflammatory responses in human keratinocytes by reducing aryl hydrocarbon receptor activation and reactive oxygen species formation. Seok et al. [22] also reported that PM 10 -induced inflammatory response is restored by punicalagin and EGCG in keratinocytes. Moreover, Ha et al., [27] reported that dieckol attenuates PM 10 -induced PGE2 production. Recently, we reported that SHE and dieckol inhibit the expression of PM-induced inflammatory cytokines and matrix metalloproteinase in sebocytes and keratinocytes [47,48]. Sargassum horneri is a brown alga with antioxidant, anti-inflammatory, and antiallergic effects. Herath et al. [49] sought to determine whether the ethanol extract of S. horneri mitigated the effect of PM exposure on asthma development. Orally administered S. horneri mitigated the expression of Th2 cytokines in lung homogenates of PM-exacerbated asthmatic mice. In addition, S. horneri markedly mitigated the activation of mast cells and IgE levels in the serum. 2,3,5,4 -tetrahydroxystilbene-2-O-β-d-glucoside (THSG), the main compound of Polygonum multiflorum, has various biological activities, including anti-inflammatory and antioxidant activities. Hwang et al. [50] examined the effects of THSG on Th2 immune responses in an OVA-induced asthma animal model. This study confirmed the potential of THSG in asthma treatment through the modulation of inflammatory responses. Natural functional foods, including antioxidative blueberries and black rice, can be an alternative for AD therapy. Hong et al. [51] investigated the effect of fermented blueberry and black rice extracts containing Lactobacillus plantarum MG4221 on the PM-induced expression of proinflammatory cytokines in vitro and in vivo. Fermented blueberry and black rice extracts decreased IL-1β, IL-6, and IL-8 levels in PM-treated HaCaT cells. In addition, their oral administration significantly decreased transepidermal water loss and the production of serum IgE, and increased the protein expression of filaggrin and involucrin in skin tissue. Polyphenolic compounds, including dieckol, punicalagin, EGCG, and resveratrol, are antioxidative and anti-inflammatory agents. However, the antioxidant activity of SHE has not yet been elucidated.

Conclusions
In conclusion, PM 10 upregulated serum IgE concentration and increased spleen weight in the DNCB-induced AD mouse model. In addition, PM 10 enhanced mast cell counts, inflammatory cytokines, including IL-1β, IL-4, IL-6, IL-17α, IL-25, IL-31, and TSLP, and keratinocyte differentiation markers, including loricrin and filaggrin, in the DNCB-induced AD mouse model. Antioxidants, including dieckol, punicalagin, EGCG, resveratrol, and SHE, had preventative effects against PM 10 on serum IgE concentration, spleen weight, mast cell counts, inflammatory cytokines, and keratinocyte differentiation markers in the mouse model of DNCB-induced AD. Further studies are needed to investigate the adverse