The NGE was prepared by Nongshim Corporation (Seoul, Korea), and contained 88.8% crude protein, 1.9% crude fat, 2.2% crude ash and 3.0% moisture. The total ganglioside content as sialic acid in the extract was 0.09%. The total amino acid and free amino acid contents in the extract were 922.5 and 8.21 mg/g, respectively. NGE, as a water-extracted white powder, was dissolved in water for the in vivo and in vitro experiments. Further information on the NGE production process can be requested from the company (http://nongshim.co.kr).

The HMC-1 human mast cells were purchased from the Korea Cell Line Bank (Seoul, Korea). The cell line was grown in Iscove's modified Dulbecco's medium (Welgene, Daegu, Korea) supplemented with heat-inactivated 10% fetal bovine serum (FBS; Welgene) and 1% Penicillin-Streptomycin solution (1X; Welgene) in a 5% CO₂ incubator at 37°C.

The HMC-1 cells (1×10⁴ cells/well) were plated in 96-well culture plates and incubated for 24 h. Subsequently, the HMC-1 cells were treated with 5 ng/ml PMA and 500 ng/ml ionomycin (Sigma, EMD Millipore, Billerica, MA, USA) in the presence or absence of various concentrations of NGE (5, 25, 100, 250, 500, and 1,000 mg/ml). Following 24 h of incubation, 10 µl of WST solution was added to each well of the plate, and the plate was incubated in the dark at 37°C for an additional 1 h. The optical density was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Versa Max; Molecular Devices LLC, Sunnyvale, CA, USA).

Male, 6-week-old BALB/c mice (20±2 g) were purchased from Orient Bio, Inc. (Sungnam, Korea). The mice were randomized into three groups: Vehicle (saline), DNCB, and DNCB + NGE, each comprising six mice. All mice were maintained in a pathogen-free environment and allowed free access to food and water; they were maintained on a 12-h light/dark cycle at a temperature of 24°C. All procedures performed on the mice were approved by the Animal Care Center of Kyung Hee University [approval no. KHUASP (SE)-16-095]. All methods were performed in accordance with the relevant guidelines and regulations. At the end of the experiment, the mice were sacrificed by CO₂ inhalation, and cardiac blood was collected.

The procedure for the induction of AD-like skin lesions is shown in Fig. 2A. For this purpose, the dorsal skin of the mouse was shaved and coated with 100 µl of 2% DNCB in 1×1 cm patches. At 1 week post-sensitization, the dorsal skin was challenged with 100 µl of 0.2% DNCB solution twice per week (first and fourth day of each week). The NGE was administered orally to the mice for 2 weeks following sensitization with DNCB. Following the final application of NGE, the mice were sacrificed, and immunological and histological assessments were performed. The method was performed as described previously (28).

The thickness of the dorsal skin was measured with digital calipers (Mitutoyo, Kawasaki, Japan) prior to sacrifice. For each mouse, three different sites on the dorsal skin were measured randomly, and the measurements were averaged. The entire spleen was removed from the mice following sacrifice and was weighed.

A section of the skin biopsies was fixed in 4% paraformaldehyde and embedded in frozen section compound (Surgipath FSC22 Clear Leica Biosystems GmbH, Wetzlar, Germany) on dry ice. Skin sections measuring 20 µm in thickness were cut and stained with hematoxylin and eosin (H&E) to visualize inflammatory cells (neutrophils and mononuclear cells) or with toluidine blue to visualize mast cells, and were examined under light microscopy (Olympus Corporation, Tokyo, Japan). The numbers of mast cells and inflammatory cells were counted in 10 regions of high-power fields at ×400 magnification.

The expression of CD4⁺ lymphocytes was detected by immunohistochemical analysis using a mouse monoclonal anti-CD4⁺ antibody (cat. no. SC-7219; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The skin sections were hydrated. Following microwave treatment, the sections were treated with 3% hydrogen peroxide in PBS for 15 min to inhibit the endogenous peroxidase activity of the blood cells. The sections were blocked with 5% bovine serum albumin (Sigma, EMD Millipore) in PBS for 1 h at room temperature. The skin sections were incubated with the mouse monoclonal CD4⁺ antibody (1:100 dilution) overnight at 4°C and subsequently incubated with a biotinylated anti-mouse IgG secondary antibody (1:50 dilution; Vectastain ABC kit; cat. no. PK-6102; Vector Laboratories, Inc., Burlingame, CA, USA) for 1 h at room temperature. The sections were treated with avidin-biotin horseradish peroxidase (HRP) complex (Vectastain ABC kit; cat. no. PK-4000; Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min at 4°C and finally stained with diaminobenzidine tetrachloride (DAB) as a substrate. The slides were mounted with an aqueous mounting solution (Permount; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and cover-slipped. All the sections were analyzed using a ZEISS Scope A1 light microscope (Carl Zeiss AG, Oberkochen, Germany), and images were captured using a digital video camera.

Whole blood samples were collected by cardiac puncture. The blood was placed in Vacutainer™ tubes containing EDTA (BD Biosciences, San Jose, CA, USA) by centrifugation (200 × g, 20 min, 4°C). Anticoagulated blood was processed to determine the hematological parameters of white blood cells (WBCs), lymphocytes, monocytes, eosinophils, basophils and neutrophils in a HEMAVET 950 hematology system (Drew Scientific, Inc., Dallas, TX, USA) in accordance with the manufacturer's recommendations.

The total IgE levels in the serum of the mice were determined by sandwich ELISA using the BD Pharmingen mouse IgE and human ELISA sets (BD Biosciences, San Diego, CA, USA). Briefly, the plates were coated with capture antibody in ELISA coating buffer (Sigma, EMD Millipore) and incubated overnight at 4°C. The plates were then washed with PBS-Tween-20 (0.05%) and subsequently blocked (10% FBS in PBS) for 1 h at 20°C. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates, and the plates were incubated for 2 h at 20°C. Following washing, biotin-conjugated anti-mouse IgE (1:500 dilution) and streptavidin-HRP conjugate (1:250 dilution) were added to the plates, and the plates were incubated for 1 h at 20°C. Finally, tetramethylbenzidine substrate solution was added to the plates for 15 min incubation in the dark, and a 2N H₂SO₄ solution was added to terminate the reaction. The optical densities were measured at 450 nm on an automated ELISA reader (VersaMax; Molecular Devices LLC). The levels of IL-10 and IL-12 in the plasma and the levels of IL-4, IL-13, TNF-α and IL-6 in the cell line were also measured by sandwich ELISA using the BD Pharmingen mouse and human ELISA sets. The subsequent procedure followed the same protocol described above.

The cells were harvested by centrifugation (500 × g, 20 min, 4°C) and the pellet was washed with ice-cold PBS. RNA was isolated from the cells using the Easy-Blue RNA extraction kit (iNtRON Biotech, Sungnam, Korea) according to the manufacturer's protocol. The isolated RNA content was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Total cellular RNA (2 µg) from each sample was reverse transcribed using a cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan). The sqPCR analysis was performed with a 20-µl reaction mixture consisting of DNA template, each gene-specific primer at a concentration of 10 pM, 10X Taq buffer, 2.5 mM dNTP mixture, and 1 unit of Taq DNA polymerase (Takara Bio, Inc.). PCR was performed according to the instructions of the Takara Taq kit as follows: Pre-DNA denaturation at 95°C for 3 min; and 30 cycles of DNA denaturation at 95°C for 45 sec; annealing for 40 sec at 56°C; elongation at 72°C for 50 sec. The PCR reaction was performed using a SimpliAmp Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR products were subsequently separated using agarose gel electrophoresis [5% (w/v)] and were stained with ethidium bromide at room temperature for 5 min. All experiments were performed in triplicate. The relative optical density ratio was calculated using ImageJ software (version 1.42q; National Institutes of Health, Bethesda, MD, USA) with GAPDH as an internal control. The primer sequences used for human and/or mouse IL-4, IL-6, TNF-α and GAPDH are shown in Table I.

All experimental results were expressed as the mean ± standard deviation or the mean ± standard error of the mean of at least three separate tests. P<0.05 was considered to indicate a statistically significant difference and P<0.05, <0.01, and <0.001 have been assigned respective symbols in figures. Statistical significance was determined using a one-way analysis of variance followed by Tukey-Kramer multiple comparisons post-tests to analyse differences between groups. Statistical analyses were performed using PRISM software (version 5.0; GraphPad Software Inc., La Jolla, CA, USA.).

Mast cell mediators, including tryptase and histamine, contribute to the induction of pruritus in AD (29). In the present study, the viability of HMC-1 cells was examined following treatment with various concentrations of NGE (Fig. 1A). NGE did not induce any cytotoxicity in HMC-1 cells over a 24-h period. The PMA + ionomycin-mediated stimulation of HMC-1 cells has been shown to significantly increase the production of cytokines and chemokines (27). The present study examined the role of NGE in the suppression of cytokine expression in HMC-1 cells. NGE cotreatment significantly reduced the agonist-stimulated production of IL-4, IL-13, TNF-α and IL-6 in the HMC-1 cells (Fig. 1B). Further experiments were performed to determine whether NGE regulates the expression of cytokines at the transcriptional level. RT-qPCR analysis showed that the mRNA levels of IL-4, IL-6 and TNF-α were induced by agonist treatment and were decreased by NGE cotreatment in HMC-1 cells (Fig. 1C). In particular, treatment with NGE in a concentration of 250 µg/ml reduced the levels of IL-4 and IL-6 to the control levels. These results suggested that NGE regulates proinflammatory cyto-kine production in HMC-1 cells.

Following the application of DNCB to the skin of mice to induce an acute allergic reaction, NGE was administered orally for 2 weeks at a regular time every day. The level of toxicity and the body weight was measured twice a week to determine the degree of stress caused by the NGE treatment (Fig. 2A). Body weight and food intake were also monitored throughout the study. As a result, it was found that the mice in the vehicle group (n=6) were higher in weight compared with those in the DNCB-treated (n=6) or NGE-treated (n=6) groups. It was also found that the mice in the DNCB-treated group did not exhibit any changes in food intake but exhibited a marginal decrease (15%) in body weight compared with those in the vehicle-treated group (Fig. 2C). To investigate the effects of NGE on DNCB-induced AD-like symptoms in BALB/c mice, skin lesions were examined and the skin thickness and spleen weight were measured. It was found that DNCB induced AD in BALB/c mice (Fig. 2B). In addition, the skin thickness was gradually increased in the DNCB-treated BALB/c mice compared with that in the non-DNCB-treated mice, whereas NGE markedly attenuated the DNCB-induced increase in skin thickness (Fig. 2D). Similarly, the spleen weight of the DNCB-treated BALB/c mice was heavier than that of the mice in the vehicle group, whereas NGE significantly decreased the DNCB-induced increase in spleen weight (Fig. 2E). These finding suggested that NGE alleviated DNCB-induced AD.

To determine whether NGE decreases the infiltration of inflammatory cells (neutrophils and mononuclear cells) into AD skin lesions, H&E staining of skin sections was performed following topical administration of NGE. The infiltration of inflammatory cells into the epidermis and dermis of mice in the DNCB group was observed, whereas topical NGE decreased the infiltration of inflammatory cells into the skin (Fig. 3A). Subsequently, toluidine blue staining was performed for mast cell visualization. The repeated cutaneous application of DNCB increased the dermal mast cell number. However, this effect was significantly suppressed by NGE (Fig. 3B). Immunocytochemistry was performed to measure the intracellular level of CD4⁺ T cells. It was found that DNCB increased the number of CD4⁺ T cells, whereas NGE decreased the number of CD4⁺ T cells in the epidermis and dermis (Fig. 3C). These results indicated that NGE treatment significantly decreased the number of inflammatory cells, mast cells and CD4⁺ T cells, compared with DNCB treatment (Fig. 3D-F).

To investigate whether cutaneous NGE administration can decrease the levels of inflammatory cells, leukocyte levels in cardiovascular blood samples were measured using a HEMAVET 950 hematology system. It is known that WBCs, neutrophils, lymphocytes, monocytes, eosinophils and basophils are activated in the blood of patients with AD (17,30). In particular, eosinophils have been shown to be present in the majority of patients with AD, and they are correlated with disease activity (31). NGE significantly decreased the numbers of WBCs, basophils, monocytes, neutrophils, eosinophils and lymphocytes induced by DNCB (Fig. 4).

In the histological analysis, the repeated cutaneous application of DNCB increased dermal mast cell numbers and CD4⁺ T cell numbers, and this effect was suppressed by oral administration of NGE. Activated mast cells and CD4⁺ T cells secrete various chemokines and cytokines, including IL-4, IL-6 and TNF-α. To examine whether NGE decreases the inflammatory response, RT-qPCR analysis was performed to measure the mRNA expression of IL-4, IL-6 and TNF-α in the AD-like skin lesions (Fig. 5). The oral administration of NGE significantly suppressed the mRNA expression of IL-4, IL-6 and TNF-α in the AD-like skin lesions. These results indicated that NGE downregulated the DNCB-induced mRNA expression of inflammatory cytokines.

To obtain a better understanding of the mechanisms underlying the anti-inflammatory activity of NGE, the IgE and levels of inflammatory cytokines in serum were measured by ELISA. The hyperproduction of IgE is a major characteristic of AD, and patients with AD often exhibit elevated levels of total and allergen-specific IgE antibodies in their serum. As shown in Fig. 6, the total level of IgE was markedly elevated in the DNCB-treated group compared with that in the vehicle group. However, the increase in the serum level of IgE induced by DNCB was significantly decreased by NGE treatment. In addition, the repeated topical application of DNCB significantly increased the levels of IL-10 and IL-12 in mouse serum, whereas NGE significantly inhibited this increase. These results demonstrated that NGE suppressed the DNCB-induced elevation of IgE and inflammatory cytokine levels, thereby leading to the inhibition of skin inflammation.