The animal protocol used in this study was reviewed and approved based on the ethical and scientific care procedures of the Pusan National University-Institutional Animal Care and Use Committee (PNU-2013–0378). All the animals were handled in the Pusan National University-Laboratory Animal Resources Center accredited by AAALAC International in accordance with the USA NIH guidelines (accredited unit no. 001525) and the Korean Food and Drug Administration (FAD) in accordance with the Laboratory Animals Act (accredited unit no. 00231). The mice were housed under specific pathogen-free (SPF) conditions and a strict light cycle (lights on at 08:00 and off at 20:00) at a temperature of 22±2°C and 50±10% relative humidity and were provided with standard irradiated chow diet (Purina Mills Inc., Seongnam, Korea) ad libitum.

Nine-week-old IL-4/Luc/CNS-1 Tg mice (n=20) were randomly divided into four groups, with five mice per group. The first group of Tg mice [acetone-olive oil (AOO), n=5] had 100 ml of AOO repeatedly spread on the dorsum of their ears three times a week for 4 weeks. In the second group (PA, n=10), 100 ml of 15% PA solution in AOO (4:1, v/v) was repeatedly spread on the dorsum of the ears three times a week for 4 weeks. The second group was further divided into the PA + Vehicle and PA + CKJ treatment groups, which received a comparable volume of water daily via oral administration and 50 mg/kg body weight of CKJ for 4 weeks, respectively. Age-matched Tg mice (n=5) were used as a no treatment group.

IL-4/Luc/CNS-1 Tg mice used in this study were obtained from the National Institute of Food and Drug Safety Evaluation of the Korean FDA (Osong, Korea). Large numbers of IL-4/Luc/CNS-1 Tg mice and non-Tg littermates were produced by mating IL-4/Luc/CNS-1 Tg mice and HR1 mice. Founder mice, into which the IL-4/Luc/CNS-1 transgene was inserted, were identified by PCR analysis of tail-derived genomic DNA. For PCR, 10 pmol each of sense (5′-CTC GCA TGC CAG AGA TCC TA-3′) and antisense (5′-CCA CAA CCT TCG CTT CAA AA-3′) primers were added into a mixture containing genomic DNA template, and the reaction mixtures were subjected to 35 cycles of amplification (1 min at 94°C; 1 min at 56°C and 1 min at 72°C) using a Perkin-Elmer thermal cycler (PerkinElmer, Waltham, MA, USA). The amplified PCR products were separated by 1% agarose gel electrophoresis and band patterns were detected using a Kodak Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY, USA). IL-4/Luc/CNS-1 Tg mice were screened from founder mice through the detection of PCR products with 467 bp.

CKJ extract was prepared as previously described (24,25). The soybean (Daepung strain) used to manufacture CKJ was kindly supplied by the National Institute of Crop Science (Miryang, Korea) while B. subtilis MC31 was obtained from the Food Microbiology Laboratory at Pusan National University. To manufacture the CKJ extract, 15 g of soybeans were washed and then soaked in three volumes of tap water at room temperature for 24 h. The soybeans were then treated with hot steam at 121°C for 50 min, after which they were allowed to cool to 45°C. The steamed soybeans were inoculated with 2% (w/w) B. subtilis MC31 and fermented for 72 h at 40°C. The fermented soybeans were then powdered by freeze-drying, homogenization and sifting. The final sample of CKJ extract was stored at −75°C until use.

GABA concentration was measured in a spectrophotometric assay containing GABase enzyme (Sigma-Aldrich, St. Louis, MO, USA) using the method described by Zhang and Bown (26). Briefly, the supernatant of the CKJ extract was collected from the lyophilized powder of CKJ (0.3 g) that had been soaked in 99% ethanol (1.2 ml) for 5 h. This supernatant (0.1 ml) was then mixed with 0.4 ml of MeOH and completely dried at 70–80°C for 30 min. Then, 70 mM LaCl₃ (1 ml) was added and the mixture was agitated for 10 min, centrifuged at 9,800 × g for 5 min. The supernatant (0.8 ml) was then mixed with 0.1 M KOH solution (0.16 ml) for 3–5 min, after which it was purified by centrifugation and filtration. This solution (0.55 ml) of CKJ was then dispensed into individual cuvettes, each of which contained 0.2 ml of 0.5 mM K₄P₂O₇ buffer (pH 8.6), 0.15 ml of 4 mM NADP and 0.05 ml of GABase (2 U/ml). The initial absorbance was then read at 340 nm using a spectrophotometer (Optizen POP; Mecasys Co., Ltd., Daejeon, Korea), after which 0.05 ml of 20 mM α-ketoglutarate was added and the samples were incubated for 60 min at room temperature. The absorbance was read at the same wavelength. The final concentration of GABA was then calculated by comparing the differences of the two absorbances and by comparison with a standard curve.

To determine the concentration of diadzein and genistein in CKJ, the aqueous extract of CKJ was dissolved in 100 mg/ml of 50% MeOH and agitated at 200 rpm for 4 h. Following incubation for 12 h at room temperature, the sample was centrifuged at 3,000 rpm, after which the supernatant was harvested, diluted to 25 mg/ml in 50% MeOH and passed through a syringe filter (0.45 mm).

The CKJ was analyzed using an iLC 3000 HPLC system (Interface Engineering Co., Ltd., Seoul, Korea) equipped with a Corona^® CAD^® Detector (ESA Bioscience, Inc., Chelmsford, MA, USA). Chromatographic separation was performed using a YMC-triart C18 column (4.6×250 mm, particle size 5 μm; Shiseido Co., Ltd., Tokyo, Japan). The mobile phase consisted of solvent A (0.1% formic acid in deionized water) and solvent B (acetonitrile) using the following gradient elution program: 0–30 min, 20–40% of solvent B and 30–45 min, 40–70% of solvent B. A flow rate of 1.0 ml/min was used for sample analysis and the nebulizer gas was nitrogen. The gas flow rate and gas pressure were maintained at 1.53 l/min and 35±2 psi, respectively. The output signal of the detector was recorded using the Clarity™ Chromatography Software (DataApex, Prague, Czech Republic).

Alterations of body weight during the experimental procedure were measured using an electronic balance (Mettler Toledo-International, Inc., Greifensee, Switzerland) three times a week for 4 weeks. After final administration, the weights of the spleen, thymus, auricular lymph node (ALN) collected from the sacrificed mice were also measured by the same method. Ear thickness was measured using a thickness gauge (Digimatic Indicator; Matusutoyo Co., Tokyo, Japan) to determine the degree of allergic skin inflammation induced by PA treatment.

In vivo imaging was conducted using an IVIS imaging system (Xenogen Corp., Alameda, CA, USA) as previously described (11). Briefly, IL-4/Luc/CNS-1 Tg mice were anesthetized with Zoletil and injected i.p. with 150 mg/kg of D-luciferin (Sigma-Aldrich). Ten minutes after the D-luciferin injection, images of mice were captured for 3 min using an IVIS imaging system. Photons emitted from specific regions were then quantified using the Living Image software (Xenogen Corp.). In vivo luciferase activity was expressed in photons per second.

The serum IgE concentration was measured using an ELISA kit (Shibayagi, Co., Ltd., Gunma, Japan) according to the manufacturer’s instructions. Briefly, capture antibodies were plated in the Nunc C bottom immunoplate supplied in the kit. The wells were then washed with washing solution (50 mM Tris, 0.14 M NaCl, 0.05% Tween-20, pH 8.0) three times, after which serum samples and standards diluted with buffer solution were added to the wells, and the plate was incubated for 2 h. The wells were then washed again with washing solution, after which 50 μl of biotin-conjugated anti-IgE antibodies (1,000-fold dilution) were added to each well and the samples were incubated for another 2 h to bind with captured IgE. The wells were then washed again with washing solution, after which horseradish peroxidase-conjugated detection antibodies (2,000-fold dilution) were added to each well and samples were incubated for 1 h. An enzyme reaction was then initiated by adding tetramethylbenzidine (TMB) substrate solution (100 mM sodium acetate buffer pH 6.0, 0.006% H₂O₂) and the samples was incubated at room temperature in the dark for 20 min. The reaction was terminated by adding acidic solution (reaction stopper, 1 M H₂SO₄), and the absorbance of yellow product was measured spectrophotometrically at 450 nm. The final concentration of IgE was calculated using a standard curve.

Ear tissues were removed from mice, fixed with 10% formalin, embedded in paraffin wax, routinely processed, and sectioned into 4 μm slices. The ear tissue sections were then stained with hematoxylin and eosin, after which they were examined by light microscopy (Leica Microsystems, Heerbrugg, Switzerland) for the presence of edema and inflammatory cell accumulation. Thickness levels of the epidermis and dermis and the number of crypts were also measured using the Leica Application Suite (Leica Microsystems).

Mast cells were detected by staining with Toluidine blue (Sigma-Aldrich) according to previously described methods (27). Subsequent to deparaffinization and dehydration, ear tissue sections were stained with 0.25% Toluidine blue (Sigma-Aldrich) and examined by light microscopy for the presence of mast cells. The number of cells per specific area was measured with Leica Application Suite (Leica Microsystems).

Ear tissues from a subset of the groups (n=5 per group) were homogenized using a PRO-PREP™ Solution kit (Intron Biotechnology, Seongnam, Korea) supplemented with 1/2 of a protein inhibitor cocktail tablet (Roche Diagnostics GmbH, Penzberg, Germany), followed by centrifugation at 13,000 rpm for 5 min. The prepared proteins were then electrophoresed through a 10% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, Corston, UK) for 2 h at 40 V in transfer buffer (25 mM Trizma-base, 192 mM glycine and 20% methanol). The efficiency of the transfer and equal protein loading were determined by staining the gel with Coomassie Blue (Sigma-Aldrich). Appropriate dilutions of primary antibodies, anti-IL-6 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-VEGF antibody (Peprotech, Inc., Rocky Hill, NJ, USA), and anti-β-actin (Sigma-Aldrich) were added to the membranes and allowed to hybridize overnight at 4°C. After the antibodies were removed, the membranes were washed three times in a solution composed of 10 mM Trizma-base (pH 7.6), 150 mM NaCl, and 0.05% Tween-20 for 10 min. This was followed by incubation with horseradish peroxidase-conjugated anti-secondary antibody for 1 h at room temperature. The membrane was washed again as described above and developed using an enhanced chemiluminescence detection system (Amersham Bioscience). The results were then quantified using the Image Analyzer System (2000MM; Estman Kodak) and expressed as the fold-increase over control values. Results were confirmed by two independent investigators who performed the experiments at least twice.

One-way ANOVA was used to identify significant differences between the PA- and AOO-treated groups (SPSS for Windows, Release 10.10, Standard Version; SPSS, Inc., Chicago, IL, USA). Additionally, response differences between the Vehicle- and CKJ-treated group in the PA-treated group were evaluated by a post hoc test (SPSS for Windows, Release 10.10, Standard Version; SPSS, Inc.) of the variance and significance levels. All the values are expressed as the means ± standard deviation (SD). P<0.05 was considered to indicate statistical significance.

The distribution of three functional compounds in CKJ was analyzed by enzyme assay and HPLC analysis. GABA was found to be present at 200.00 μg/g, while the levels of the flavonoids daidzein and genistein were 85.68 and 132.51 μg/g, respectively (Fig. 1). Therefore, above results indicated that CKJ extract contained high concentrations of GABA although the concentration of flavonoids was slightly low.

To determine whether CKJ treatment suppressed changes in ear phenotype induced by PA treatment, ear morphology and thickness were observed in IL-4/Luc/CNS-1 Tg mice after CKJ treatment for 4 weeks. The outline of the ear vein became distinct or thickened in the PA + Vehicle-treated group compared to the AOO-treated group, and ear color changed from flesh tint to dark brown. These alterations were slightly reversed in the PA + CKJ co-treated group (Fig. 2B). In addition, ear thickness rapidly increased in PA + Vehicle-treated mice compared to non- or AOO-treated mice. However, these levels in PA + CKJ-treated mice were significantly lower than those in the PA + Vehicle-treated group (Fig. 2C). Therefore, these findings demonstrated that CKJ treatment successfully decreases ear and vein thickness induced by PA-treatment.

To quantify the therapeutic effects of CKJ on the allergenic response to PA treatment using the luciferase reporter system, luciferase signals from the whole body and eight organs were measured using the Living Image software in IL-4/Luc/CNS-1 Tg mice after individual PA + Vehicle or PA + CKJ treatment. In the whole body image, the luciferase signal was highly detected in the abdominal region of IL-4/Luc/CNS-1 Tg mice treated with PA + Vehicle, whereas the no treatment or AOO-treated group showed no luciferase signal. However, this signal was greatly reduced in groups treated with PA + CKJ (Fig. 3A). Following organ image analysis, a high luciferase signal was detected in the thymus of IL-4/Luc/CNS-1 Tg mice treated with PA + Vehicle, however, the signal was markedly reduced in the thymus of IL-4/Luc/CNS-1 Tg mice treated with PA + CKJ (Fig. 3B). Overall, these results indicated that the effects of CKJ on the allergic response induced by PA treatment could be quantified using IL-4/Luc/CNS-1 Tg mice without sacrificing the animals.

Alterations in body weight were measured during all experimental periods to examine the effects of CKJ treatment on whole body growth. However, no significant changes in body weight were in observed in any of the groups (Fig. 4A).

It is well known that the weight of some immune organs increase in response to the topical application of agents that have allergenic or sensitizing potential (28,29). As shown in Fig. 4B and C, PA treatment induced an increase in the weight of the spleen and lymph node in IL-4/Luc/CNS-1 Tg mice compared to the AOO-treated group. However, their weights were significantly reduced in the PA + CKJ-treated group, although not to the level of the AOO-treated group. The weight of the thymus was maintained in the PA + Vehicle and PA + CKJ-treated group (Fig. 4D). Taken together, these results suggested that CKJ treatment contributes to the reduction of spleen and lymph node weight observed in IL-4/Luc/CNS-1 Tg mice in response to PA treatment.

To verify the suppressive effects of CKJ treatment on ear histology, the histological analysis of ear tissue from IL-4/Luc/CNS-1 Tg mice was performed. The epidermis and dermis of the ear tissue were thicker in the PA + Vehicle-treated group than in the AOO-treated group. The thickness of the dermis greatly decreased in the PA + CKJ-treated group, but was not completely recovered to that of the AOO-treated group (Fig. 5A and Bb). The thickness of the epidermis increased in the PA + CKJ-treated group compared to the PA + Vehicle-treated group (Fig. 5A and Ba). Taken together, these results showed that CKJ may improve the dermis thickness among the AD responses induced by PA-treatment.

Mast cells play important roles in asthma, eczema, itch, allergic rhinitis, and allergic conjunctivitis (30). Therefore, ear tissue sections were stained with Toluidine blue and observed under a microscope to examine the effects of CKJ on the infiltration of mast cells. The number of mast cells stained blue was significantly greater in the PA + Vehicle-treated group than that in the AOO-treated group (Fig. 6). However, their number was significantly reduced following PA + CKJ-treatment, although not to that of the AOO-treated group (Fig. 6A and Ba). These data suggest that CKJ contributes to the suppression of mast cell infiltration in the dermis of ear skin.

The serum IgE concentration was measured in the four groups of mice to determine whether CKJ suppressed the allergic response induced by PA treatment. Repeated topical application of PA solution induced a significant increase in serum IgE concentration in IL-4/Luc/CNS-1 Tg mice. However, a significant decrease of IgE concentration was not observed in the PA + CKJ-treated group (Fig. 6Bb). Overall, these results suggested that CKJ treatment did not contribute to the reduction of IgE concentration in IL-4/Luc/CNS-1 Tg mice observed in response to PA treatment.

To determine whether CKJ induced the alteration of atopic response-related cytokine expression, the expression levels of VEGF and IL-6 were measured in ear tissues of IL-4/Luc/CNS-1 Tg mice. A high expression of VEGF protein was observed in the PA + Vehicle-treated group, whereas a low expression was detected in the AOO-treated group and a reduction of VEGF protein expression was observed in the PA + CKJ-treated group (Fig. 7A and Ba). Similar results were observed following analysis of IL-6 expression. The increased IL-6 expression after PA treatment was reversed by CKJ treatment, but not to levels of the AOO-treated group (Fig. 7A and Bb). The above results suggested that CKJ treatment may relieve the allergic response induced by PA treatment through the regulation of VEGF and IL-6 expression.