The ethanol extract of pear pomace was prepared according to previous protocols [24]. Briefly, pear pomace, the cake left after extracting the juice, was extracted with 50% ethanol, filtered, freeze-dried, and stored in a −80°C freezer. The yield obtained was 27.5%

Five-week-old male NC/Nga mice were purchased from Central Laboratory Animal Inc. (Seoul, Korea), and were housed individually in ventilated cages under conventional conditions with a 12 h light/dark cycle at 22 ± 2°C room temperature and 50 ± 10% relative humidity during the experimental period. Mice had free access to water and were fed an AIN-93G control diet. All experiments were approved by the Laboratory Animal Care and Use Committee of Mokpo National University (MNU-IACUC-2016-005).

DNCB was an efficient tool to induce AD in NC/Nga mice within a short time period topical application. We slightly modified the AD protocol according to published papers [252627]. After 2 week of acclimatization, the dorsal hair of NC/Nga mice was removed using an electric shaver (hair clipper model 6000AD; Thrive Japan, Kawasaki, Japan) and depilatory cream (Veet; Reckitt, Slough, UK). To induce AD-like skin lesions, the mice were divided into 2 groups (normal control [Cont] group and AD group); DNCB (Sigma-Aldrich, St. Louis, MO, USA) was applied to the AD group. The mice were sensitized with 200 uL of 1% DNCB solution (dissolved in a 3:1 mixture of acetone and olive oil) twice over a week. Then, the dorsal skin was challenged with 150 uL of 0.4% DNCB solution 3 times per week for 7 weeks. Three weeks after the challenge, the AD group was further divided into 3 groups: AD control (AD-C), AD + PPE 200 (PPE 200 mg/kg body weight), and AD + PPE 400 (PPE 400 mg/kg body weight). PPE extract was dissolved in distilled water and administered daily by oral gavage for 4 weeks. Distilled water was administered to the Cont and AD-C groups. Fig. 1A illustrates the schematic diagram of AD induction and treatment with PPE.

To investigate the scratching behavior of DNCB-induced AD in NC/Nga mice, the mice were placed individually in clear cages. Seven weeks after sensitization, the mice were treated with PPE upon 0.4% DNCB treatment. For 20 min after DNCB treatment, the total number of mice scratching themselves only with the hind paw was counted; the scores ranged from 0 to 4: 0 (no scratching), 2 (scratching for less than 1.5 sec), and 4 (scratching for more than 1.5 sec). The clinical severity score for dermatitis was evaluated macroscopically on the day of the necropsy using a specific scoring procedure. The skin clinical severity score was expressed as the sum of the individual scores for the 4 symptoms of erythema/hemorrhage, dryness/scaling, edema, and excoriation/erosion. For each skin symptom, 0 was defined as no symptoms, 1 as mild symptom, 2 as moderate symptoms, and 3 as severe symptoms [2528]. To determine the ear thickness, both the left and right ears of the mice were removed, and the thickness was measured using a digital caliper (Mitutoyo Corporation, Kawasaki, Japan), and calculated as the mean thickness value of both sides. Additionally, the spleen was dissected for weighing.

Blood samples were collected, separated immediately by centrifugation, and stored at −70°C. Serum IgE levels were determined using the mouse IgE ELISA kit (Fujifilm Wako Shibayagi Corporation, Shibukawa, Japan) following the manufacturer’s instructions.

To measure the epidermal inflammation and infiltration of mast cells, dorsal skin was obtained and fixed in 10% formalin. The samples were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) or toluidine blue (Sigma-Aldrich). The stained sections were observed under a microscope at 100× magnification.

Murine cell line RAW 264.7 macrophages (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL, Grand Island, NY, USA) which was supplemented with a 10% fetal bovine serum (Gibco-BRL) and 1% penicillin (Gibco-BRL). The cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. At 80% of confluence, the cells were subdivided for future experiments. To examine the cytotoxicity of PPE, the cells were seeded in a 96-well plate at a density of 10 × 10³ cells/well and were subsequently treated with either phosphate-buffed saline (PBS), 100, or 250 ug/mL of PPE. After 24 h of incubation, 2 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich) was added to each well and the cells were incubated further for another 4 h. The medium was then removed, and the formazan precipitate was solubilized in dimethyl sulfoxide (Sigma-Aldrich). Absorbance was measured at 540 nm using microplate reader (Multiskan EX; Thermo Fisher Scientific, Waltham, MA, USA). The relative cell viability was calculated as the percentage of the control cells.

The nitrite concentration in the culture medium was measured as an indicator of NO production. RAW 264.7 macrophages were seeded at a density of 20 × 10⁴ cells/well in a 24-well plate and pretreated with PBS or PPE for 1 h before stimulation with 1 ug/mL of LPS (Sigma-Aldrich). After 24 h incubation, the culture supernatant was collected and centrifuged at 2,400 g for 10 min. The cell culture medium was combined with an equal volume of Griess reagent (Sigma-Aldrich) in a 96-well plate and incubated for 15 min at room temperature. Then, the absorbance was measured at 540 nm using microplate reader.

Mouse skin samples and cells were homogenized in ice-cold radioimmune precipitation assay buffer (Thermo Fisher Scientific) with a 1% protease inhibitor cocktail and a phosphatase inhibitor. The protein concentrations were determined by the Bradford (Sigma-Aldrich) method and equal amounts of protein (40 μg each) were separated on 10% SDS-PAGE gels and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). The membrane was blocked for 1 h at 4°C with 5% skim milk in TBS-T buffer (10 mM Tris-HCl [pH 6.8], 100 mM NaCl, and 0.1% Tween 20). The membranes were incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit or goat anti-mouse [1:1,000]) Antibodies targeting receptor for advanced glycation end products (RAGE; #6996), p-extracellular signal-regulated kinase (ERK) 1/2 (#4370), ERK1/2 (#4695), NF-κB p65 (#8242), inducible nitrite oxide synthase (iNOS; #2982), and cyclooxygenase (COX)-2 (#12282) were purchased from Cell Signaling Technology (Beverly, MA, USA). B-actin antibody (#47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Bands were detected using chemiluminescent substrate (IMGENEX, San Diego, CA, USA), and protein expression was quantified using UVP (imaging system for the chemiluminescent Western blot; UVP, Upland, CA, USA) and Vision Works TMLS (analysis software; UVP).

Statistical analyses were performed using the SPSS program (version SPSS 23; IBM, Chicago, IL, USA). The results are presented as the mean ± SE of 3 independent experiments. Differences between the groups were evaluated using one-way analysis of variance. Multiple comparisons were performed by Duncan’s multiple range test. A P-value of < 0.05 was considered statistically significant.

To induce AD-like skin lesions, NC/Nga mice were sensitized with a high dose of DNCB solution for 1 week. After sensitization, a low dose of DNCB was applied on the dorsal skin of the mice with or without PPE treatment (200 or 400 mg/kg body weight) (Fig. 1A). As shown in Fig. 1B, no significant differences in body weight were observed among the groups at the end of the experimental period, indicating that PPE could be used for in vivo applications safely with no visual side effects. Importantly, serum IgE levels in the AD-C group were notably higher by 76-fold (72.55 ug/mL) compared to 0.94 ug/mL in the Cont group. The high-dose PPE treatment reduced serum IgE levels to about 37.2% (45.53 ug/mL) compared to the AD-C group (Fig. 1C). Investigations were carried out to evaluate the clinical symptoms, scratching behaviors and skin clinic severity score. The application of DNCB remarkably increased scratching behavior; PPE at 200 and 400 mg/kg decreased scratching behaviors by 23.6% and 40%, respectively, versus the AD-C group (Fig. 1D). Additionally, PPE treatment markedly improved skin clinical severity score relative to the AD-C group (Fig. 1E). In previous studies by Ko et al. [29] and Shin et al. [30], NC/Nga mice treated with DNCB showed an enlarged spleen, the organ responsible for maintaining systemic immune responses. Spleen weight in the AD-C group markedly increased compared to that in the Cont group, whereas PPE treatment lowered spleen weight (Fig. 1F). Furthermore, the thickness of the skin and ear was also suppressed by PPE treatment (Fig. 1G and H).

To investigate the histological changes in the dorsal skin of NC/Nga mice, skin samples were stained with H&E and toluidine blue. A thicker epidermis was observed in the dorsal skin of AD-C specimens compared to specimens from the Cont group, whereas specimens from the PPE groups exhibited decreases in epidermal thickness (Fig. 2A). The regulation of mast cells in the allergic response is an essential strategy in autoimmune diseases such as AD [31]. The AD-C group showed significantly higher numbers of mast cells in the dorsal skin than the Cont group. Nevertheless, the infiltration of mast cells diminished with PPE supplementation in AD-induced NC/Nga mice (Fig. 2B). Taken together, these results indicated that the administration of PPE attenuated AD-like lesions in DNCB-induced NC/Nga mice.

Next, we detected inflammation-related protein expression in the dorsal skin, as shown in Fig. 3. The protein levels of RAGE, a marker of inflammation, were elevated upon DNCB treatment, whereas PPE reversed those changes in the dorsal skin of AD induced NC/Nga mice. The application of DNCB drastically increased the p-ERK/ERK ratio relative to the Cont group. However, PPE dose-dependently reduced the p-ERK/ERK ratio compared to the AD-C group; moreover, PPE 400 effectively returned the p-ERK/ERK levels to those of the Cont level. In accordance with the p-ERK/ERK results, the protein expression of NF-κB showed a significant drop in the dorsal skin of the PPE groups (Fig. 3). Compared to the elevation in iNOS protein in the AD-C group, PPE treatment considerably suppressed iNOS expression (Fig. 3). Taken together, these results indicated that PPE treatment could help to prevent DNCB-induced AD in NC/Nga mice.

Due to the anti-inflammation effect of PPE in vivo, we studied whether PPE could inhibit the inflammatory response upon LPS treatment in the RAW 264.7 macrophages. We first measured cell cytotoxicity in the RAW 264.7 cells using MTT assay. The cell viability was not different among the groups. To determine the inhibitory effect of PPE on NO production, RAW 264.7 cells were pre-treated with PPE. Stimulation with LPS remarkably aggravated NO production compared to the control; pretreatment of PPE ameliorated these increases in a dose-dependent manner (Fig. 4B). The elevated NO production in LPS treated cells was consistent with the increased expression of iNOS and COX-2 proteins (Fig. 4C). Conversely, treatment with a high dose of PPE dramatically reduced iNOS and COX-2 protein expression compared to the LPS control in RAW 264.7 macrophages. Consistent with the in vivo study results, LPS significantly raised the p-ERK/ERK ratio (Fig. 4D) and PPE remarkably reduced the p-ERK/ERK ratio in LPS-stimulated cells (Fig. 4D). Furthermore, the expression of NF-κB/p65 was decreased in the PPE treated groups. These results collectively suggested that PPE could potently suppress the inflammatory response in RAW 264.7 macrophages.