A total of 110 6-week SKH-1 female hairless mice were obtained from Orient Bio, Ltd., (Seungnam, Korea; body weight ranged in 20~23 g upon receipt) and 6 groups (each, n=8) were selected 5 weeks following DNCB sensitization based on body weight (intact control: 27.51±1.07 g; DNCB treated mice: 27.37±0.87 g), clinical skin severity scores (intact control: 1.00±0.76 scores; DNCB treated mice: 12.45±1.01 scores) and scratching behavior (intact control: 11.88±5.46 frequencies/30 min; DNCB treated mice: 478.93±94.64 frequencies/30 min). Animals were acclimatized for 10 days and placed in polycarbonate cages (4 mice/cage) in a temperature (20–25°C) and humidity (50–55%) controlled room. Mice were housed under a 12 h light/dark cycle and standard rodent chow (Samyang Corp., Seoul, Korea) and water were available ad libitum. All laboratory animals were treated according to the national regulations of the usage and welfare of laboratory animals, and approved by the Institutional Animal Care and Use Committee of Daegu Haany University (Gyeongsan, Korea; approval no. DHU2015-068). The mice were subdivided into six groups, comprising three control groups (intact, DNCB treatment for negative control and DEXA treatment for reference control) and three treatment groups (ESGWc 100-, 200- and 400-fold treatment).

Colorless clear solutions of ESGWc were prepared by AriBio Inc. (Seoul, Korea). Briefly, ESGW was collected around Hoojeong-ri (Gyeongsang, Korea) and a 50-fold concentrated solution was created using a rotary evaporator (N-1200B; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). In addition, appropriate amounts of Ca-lactate and NaCl were separately prepared to supplement for the reduction in minerals during the concentration process. Salinity and mineral compositions of ESGWc compared with ESGW and general underground water are listed in Table I. White powders of water-soluble DEXA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) were obtained and used in the present study as a potent reference agent. All test materials used in the present experiment were stored at 4°C in a refrigerator to protect from light and humidity until usage.

AD-like dermatitis was induced in mice by sensitization with 10 mg/ml DNCB (Sigma-Aldrich; Merck KGaA; dissolved in a 3:1 mixture of acetone and olive oil) once a day for 1 week and boosted by 5 mg/ml DNCB three times a week for 28 days, according to established previous methods (3,18). DNCB solutions were topically applied on the dorsal back skins of mice in a volume of 200 µl/mouse. In control mice, vehicle (3:1 mixtures of acetone and olive oil) was topically applied, instead of DNCB solution.

ESGWc 100-fold dilutions (2-fold dilutions of original ESGW) were selected as the highest concentrations based on a previous study investigating the effects of ESGW on DNCB hairless mice, in which favorable anti-AD effects were demonstrated at this concentration (18). In addition, 200- and 400-fold dilutions were selected as the middle and lowest concentrations, respectively, using common ratio 2. ESGWc were 100-, 200- and 400-fold diluted with distilled water, using 10, 5 and 2.5 ml ESGWc in 990, 995 and 997.5 ml of distilled water, respectively. Appropriate amounts of Ca-lactate and NaCl were added to supplement for the reduction in minerals during the concentration process with a Ca:Mg balance, salinity and conductance adjustment of 2.11 and 7.40, 1.06 and 3.70, 0.53 and 1.85 g in 100-, 200- and 400-fold dilutions of ESGWc, respectively. All mice in the ESGWc groups were allowed to swim freely in the ESGWc in mouse polycarbonate cages (200×260×130 mm; DJ-101; Daejong Instrument Industry Co., Seoul, Korea), which contained ~1,900 ml of warm water at ~37°C and bottom plates 4 cm deep, for 20 min/day.

DEXA treatment was performed as follows: Water-soluble DEXA was dissolved in distilled water to form a 1% solution and topically applied to the dorsal back skins (200 µl/mouse) once daily for 6 weeks, 5 weeks after initial DNCB sensitization. Intact and DNCB control mice bathed in distilled water instead of test waters in the present experiment to induce the same swimming stresses. Test waters and 1% DEXA solutions were warmed to ~37°C at least 30 min prior to bathing to avoid cooling irritations. A total bathing period of 6 weeks was selected for the present study, based on previous findings of the bathing effects of mineral-rich water (20) and the results of a previous study into the bathing effects of various seawaters collected around Korea (18).

Changes in body weight were measured once a week, from 1 day prior to initial DNCB sensitization to the end of the 6-week bathing period in the three different concentrations of ESGWc, distilled water or topical application of 1% DEXA, using an automatic electronic balance (Precisa Gravimetrics AG, Dietikon, Switzerland). To reduce individual differences, the total body weight gains during the 11-week whole experimental period and the total body weight gains during the 6-week bathing or topical application of 1% DEXA period were calculated.

Five signs of skin lesions, including pruritus/itching, erythema/hemorrhage, edema, excoriation/erosion and scaling/dryness were graded as follows: 0, (no symptoms); 1, (mild); 2, (moderate); and 3, (severe). Total scores (maximum=15) were regarded as the clinical skin severity score, in accordance with previous reports (3,18), with some modifications. Scoring was conducted once a week for 6 weeks, starting 5 weeks after DNCB sensitization and ending 24 h after the final bathing (11 weeks after DNCB sensitization). An additional scoring was conducted 3 days after initial bathing (38 days after initial DNCB sensitization).

Each mouse from each group was placed individually in a routine polycarbonate mouse cage and their behavior was monitored for 30 min once a week, from 5 weeks after initial DNCB sensitization to 24 h after final bathing (11 weeks after first DNCB sensitization) including 3 days after initial bathing (38 days after first DNCB sensitization), respectively. Scratching of the rostral back and biting of the caudal back were observed; scratching movements by the hind paw were defined as a scratching bout that ended when the mouse either licked its hind paw or placed its hind paw back on the floor. A series of one or more biting movements was counted as one episode that ended when the mouse returned to the straight-forward position (3).

At 6 weeks after the initial bathing, ~1 ml venous blood was collected from the vena cava under anesthesia with 2 to 3% isoflurane (Hana Pharm Co., Ltd., Hwasung, Korea) and maintained with 1 to 1.5% isoflurane in a mixture of 70% N₂O and 28.5% O₂, and serum was separated by centrifugation at 21,000 × g for 10 min at 4°C using a clotting-activated serum tube (BD Biosciences, San Jose, CA, USA). Total IgE levels in sera were determined by sandwich ELISA using the mouse IgE ELISA kit (cat. no. 555248, BD Biosciences), according to previous methods (3,6,18). Briefly, plates were coated with capture antibody in ELISA coating buffer and incubated overnight at 4°C. Subsequently, plates were washed with phosphate-buffered saline (PBS)-Tween-20 (0.05%) and blocked with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) 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 plates were incubated for 2 h at 20°C. Following three washes with PBS-Tween-20 (0.05%), biotin-conjugated anti-mouse IgE and streptavidin-horseradish peroxidase conjugate were added to the plates, according to the ELISA kit protocols, and plates were incubated for 1 h at 20°C. Finally, tetramethylbenzidine substrate solution was added to the plates and following 15-min incubation in the dark, a 2 N H₂SO₄ solution was added to stop the reaction. Optical densities were measured at 450 nm using an automated ELISA plate reader (Tecan Group, Ltd., Männedorf, Switzerland).

At 24 h after the end of the forty second bath, the mice were sacrified by exsanguination through vena cava under anesthesia with 3% isoflurane and dissected. The spleen and left submandibular lymph node (LN) in each mouse were collected following elimination of the surrounding connective tissues, muscles and debris. Individual weights of lymphatic organs were measured in grams to record absolute wet-weight. To reduce the individual body weight differences, the relative weight (% of body weight) of lymphatic organs was calculated using body weight at sacrifice and absolute organ weight according to previously established methods (18).

Splenic concentrations of TNF-α, IL-1β and IL-10 were measured by ELISA using commercially available kits, including a mouse TNF-α ELISA kit (cat. no. 558534, BD Biosciences), a mouse IL-1β ELISA kit (cat. no. MBS824757) and a mouse IL-10 ELISA kit (cat. no. MBS8244590; both MyBioSource, Inc., San Diego, CA, USA), respectively, as previously described (18). Tissue samples (10–15 mg) were homogenized in a tissue grinder containing 1 ml lysis buffer (PBS containing 2 mM phenylmethylsulfonyl fluoride and 1 mg/ml aprotinin, leupeptin, and pepstatin A, all obtained from Sigma-Aldrich; Merck KGaA), as described by Clark et al (21). Analysis was performed with 100 ml standard (diluted in lysis buffer) or 10, 50 or 100 ml tissue homogenate. Each sample was run in duplicate and a portion of the sample was analyzed for protein. Data were expressed as pg/mg of protein. For each assay, a standard curve was generated and based on replicates of the measured absorbance, demonstrated an average coefficient of variance of <10%.

Total RNA was extracted from skin tissue samples using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to a previously described method (18). cDNA was synthesized from 2 µg total RNA in a final reaction volume of 20 µl. RNA was reverse transcribed using a High-Capacity cDNA Reverse Transcription kit (cat. no. 43-749-67; Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Briefly, a mixture of 2 µl RT buffer, 0.8 µl dNTP mix, 2 µl RT primers, 1 µl MultiScribe^™ reverse transcriptase, 1 µl RNase inhibitor and 3.2 µl nuclease-free distilled water was prepared and mixed with 10 µl RNA sample. Reverse transcription was performed as follows: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min. RT-qPCR analysis was carried out with a CFX96™ Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using iTaq™ SYBR-Green (Bio-Rad Laboratories, Inc.). Nuclear free water, forward primer (10 µM), reverse primer (10 µM) and SYBR Green were mixed with cDNA in a 10 µl reaction volume. The PCR cycling conditions included an initial pre-denaturation of 95°C for 1 min, denaturation for 15 sec, annealing of 55–65°C for 20 sec, and extension of 72°C for 30 sec. A total of 50 cycles were performed. All reactions were done in triplicate. The expression of GAPDH mRNA was used as a control for tissue integrity in all samples. The sequences of the PCR oligonucleotide primers for TNF-α, IL-4, IL-5, IL-13 and GAPDH are listed in Table II. For quantitative analysis, the intact control skin tissue was used as the control, and the relative expression of TNF-α, IL-4, IL-5 and IL-13 was calculated using the 2^−ΔΔCq method (22).

Cutaneous GSH levels were determined using a fluorescence assay, as previously described (23). Firstly, skin (1:3, w/w dilution) was homogenized in 100 mM NaH₂PO₄ (pH 8.0; Sigma-Aldrich; Merck KGaA) containing 5 mM EDTA (buffer 1). Homogenates were then treated with 30% trichloroacetic acid (Sigma-Aldrich; Merck KGaA) and centrifuged twice, at 1,940 × g for 6 min at 4°C and at 485 × g for 10 min at 4°C and the fluorescence of the resulting supernatant was measured using a fluorescence spectrophotometer (RF-5301PC; Shimadzu Corp., Kyoto, Japan). Briefly, 100 µl supernatant was mixed with 1 ml buffer 1 and 100 µl o-phthalaldehyde (1 mg/ml in methanol; Sigma-Aldrich; Merck KGaA). Fluorescence was determined after 15 min (k_exc=350 nm; k_em=420 nm). The standard curve was prepared with various concentrations of GSH (0.0–75.0 µM). Protein levels in the skin homogenates were measured using the method described in a study by Lowry et al (24). Results were presented as µM GSH/mg of protein.

Firstly, the protein content of homogenate (10 mg/ml in 1.15% KCl) was measured using the method described by Lowry et al (24). The thiobarbituric acid reactive substances (TBARS) measurement was used to evaluate lipid peroxidation, as previously described (25). For this assay, 10% trichloroacetic acid (Sigma-Aldrich; Merck KGaA) was added to the homogenate to precipitate proteins. This mixture was then centrifuged for 3 min at 1,000 × g at 4°C. The protein-free sample was extracted and 0.67% thiobarbituric acid (Sigma-Aldrich; Merck KGaA) was added. The mixture was kept in a water bath at 100°C for 15 min. Levels of malondialdehyde (MDA), an intermediate product of lipoperoxidation, were determined by difference between absorbances at 535 and 572 nm on a microplate spectrophotometer reader (Tecan Group, Ltd., Männedorf, Switzerland) and the results were reported as nM/mg of protein (18).

The quantification of superoxide anion production in skin tissue homogenates (10 mg/ml in 1.15% KCl; tissue was obtained after sacrifice) was performed using the nitroblue tetrazolium (NBT) assay, as previously described (26). Briefly, 50 µl homogenate was incubated with 100 µl NBT (1 mg/ml; Sigma-Aldrich; Merck KGaA) in 96-well plates at 37°C for 1 h. Subsequently, the supernatant was carefully removed and the reduced formazan solubilized by adding 120 µl 2 M KOH and 140 µl dimethyl sulfoxide. NBT reduction was measured at 600 nm using a microplate spectrophotometer reader (Tecan Group, Ltd.). Protein content was used for data normalization.

Samples from dorsal back skins, spleen and left submandibular LN were separated and fixed in 10% neutral buffered formalin at room temperature for 24 h, embedded in paraffin, sectioned into 3–4-µm sections and stained with hematoxylin and eosin (H&E) for general histopathology, Masson's trichrome (MT) for collagen fiber or toluidine blue for mast cells, according to previously described methods (18). Following this, the histopathological profiles of each sample were observed under a light microscope (Eclipse 80i; Nikon Corporation, Tokyo, Japan). Mean epithelial thicknesses of the epidermis (µm), mean numbers of inflammatory and mast cells infiltrated in the dermis (cells/mm² of dermis), total splenic thicknesses (mm/central regions), mean numbers of white pulp (white pulps/mm² of splenic parenchyma) and red pulp cells (x10³ cells/mm² of splenic parenchyma), total submandibular LN thicknesses (mm/central regions), cortex lymphoid follicle numbers (follicles/mm² of cortex) and mean thicknesses (µm/LN) of submandibular LN were calculated for general histomorphometrical analysis using a computer-assisted image analysis program (iSolution FL v. 9.1; IMT i-solution Inc., Irvine, CA, USA) under H&E stain and collagen fiber occupied regions in the dermis (%/mm² of dermis) under an MT stain, according to previously established methods (18,27). The histopathologist was blinded to the groups when this analysis was performed.

Following deparaffinization of prepared skin using xylene, submandibular LN or spleen histological paraffin sections, citrate buffer antigen (epitope) retrieval pretreatment was conducted, as previously described (18). Briefly, a water bath with a staining dish containing 10 mM citrate buffer (pH 6.0) was pre-heated until the temperature reached 95–100°C. Slides were immersed in the staining dish and the lid was placed loosely on the staining dish. Slides were incubated in the water bath for 20 min and then the water bath was turned off. Subsequently, the staining dish was placed at room temperature for 20°C to allow the slides to cool. Following epitope retrieval, sections were immunostained using avidin-biotin complex (ABC) methods for caspase-3, poly ADP ribose polymerase (PARP), nitrotyrosine (NT), 4-hydroxynonenal (4-HNE), MMP-9, interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), IL-1β, IL-2 and TNF-α, following protocols recorded in previous studies (18,27). Briefly, endogenous peroxidase activity was blocked by incubation in methanol and 0.3% H₂O₂ at room temperature for 30 min, and non-specific binding of immunoglobulin was blocked with 1% normal horse serum blocking solution (1:100; Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature for 1 h in a humidity chamber. Primary antiserum (listed in Table III) was treated overnight at 4°C in a humidity chamber and then incubated with biotinylated universal secondary antibody (Vectastain Elite ABC kit; 1:50; Vector Laboratories, Inc.) and ABC reagents (Vectastain Elite ABC kit; 1:50; Vector Laboratories, Inc.) for 1 h at room temperature in a humidity chamber. Finally, a DAB peroxidase substrate kit (cat. no. SK-4100; Vector Laboratories, Inc.) was applied for 3 min at room temperature. All sections were rinsed in 0.01 M PBS three times between steps. The cells or fibers occupied by >30% of immunoreactivities, the density of each antiserum (for caspase-3, PARP, NT, 4-HNE, MMP-9, IFN-γ, iNOS, IL-1β, IL-2 and TNF-α) as compared with intact dermal keratinocytes or dermal fibers, were regarded as positive, and the mean numbers of caspase-3, PARP, NT and 4-HNE immunoreactive cells in the epidermis (cells/100 epithelial cells), and mean IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cell numbers in the dermis (cells/mm² of dermis), spleen (cells/mm² of spleen) and submandibular LN (cells/mm² of LN) were also counted using an automated image analysis process (iSolution FL v. 9.1; IMT i-Solution Inc., Irvine, CA, USA), as previously described (18,27) with some modifications. In addition, the occupied percentages by MMP-9 immunoreactive fibers were also calculated in the dermis (%/mm² of dermis) as MMP-9 immunoreactivities in the present experiment. The histopathologist was blinded to the group distribution when performing the analysis.

All data were expressed as the mean ± standard deviation of eight hairless mice. Multiple comparison tests for different dose groups were conducted. Variance homogeneity was examined using Levene's tests. If the Levene's test indicated no significant deviations from variance homogeneity, the obtained data were analyzed by one way analysis of variance, followed by least-significant differences multi-comparison test to determine which pairs of group comparison were significantly different. In the case of significant deviations from variance homogeneity being observed in the Levene's test, a non-parametric comparison test, Kruskal-Wallis H test, was conducted. When a significant difference was observed with the Kruskal-Wallis H test, the Mann-Whitney U test was conducted to determine the specific pairs of group comparisons that were significantly different. Statistical analyses were conducted using SPSS v. 14.0 for Windows (SPSS, Inc., Chicago, IL, USA). In addition, the percentage changes between intact vehicle and DNCB control were calculated to observe the severities of AD-like lesions induced by DNCB in the present study, and the percentage changes as compared with DNCB control and hairless mice bathing in seawaters or receiving topical application of 1% DEXA were also calculated to aid the understanding of the efficacy of seawater treatment, as described previously (28).

No significant differences in body weight gains were observed among any of the groups during the total 11-week experimental period. During the 6-week bathing period, DNCB control mice demonstrated similar body weights to the intact vehicle control mice, apart from significant decreases in body weight identified in the DNCB-sensitized (DEXA-treated and ESGWc 100-, 200-, or 400-fold) mice on days 6 and 7 days after initial DNCB sensitization compared with the intact vehicle control mice (P<0.05). Topical application of 1% DEXA and bathing with all three concentrations of ESGWc did not significantly alter body weight compared with mice in the DNCB control group throughout the entire experimental period of the present experiment (Fig. 1).

DNCB control mice exhibited significant increases in clinical skin severity scores from 24 h before initial bathing up until the end of the experimental period compared with intact vehicle control mice (P<0.05; Table IV). However, significant decreases in clinical skin severity scores were observed in mice receiving topical application of 1% DEXA, from 1 week after initial topical application up until the end of the experiment, as compared with DNCB control mice (P<0.05). Significant decreases in clinical skin severity scores were detected from 2 weeks after initial bathing in ESGWc 100-, 200- and 400-fold dilutions, as compared with DNCB control mice throughout the experimental period (P<0.05; Table IV).

DNCB control mice exhibited significant increases in scratching behavior from 24 h before initial bathing up until the end of the experimental period, as compared with intact vehicle control mice (P<0.05; Table V). However, these increases in scratching behavior were significantly inhibited by topical application of 1% DEXA from 1 week after initial topical application until the end of the experimental period, compared with DNCB control mice (P<0.05). In addition, significant decreases in scratching behaviors were detected from 2 weeks after initial bathing in all three concentrations of ESGW in a concentration-dependent manner, compared with DNCB control mice throughout the experimental period (P<0.05; Table V).

Significant increases in total serum IgE levels were detected in DNCB control mice compared with intact vehicle control mice (P<0.05; Fig. 2). However, significant decreases of total serum IgE levels were detected by topical treatment of 1% DEXA and also by bathing in ESGWc at 100-, 200- and 400-fold dilutions, in a concentration-dependent manner, compared with DNCB control mice (P<0.05; Fig. 2).

Significant increases of submandibular LN and spleen absolute and relative weights were detected in DNCB control mice compared with intact vehicle control mice (P<0.05; Table VI). However, significant decreases in both absolute and relative submandibular LN and spleen weights were detected following topical application of 1% DEXA, and also following bathing in all three ESGWc dilutions, concentration-dependently, compared with DNCB control mice (P<0.05; Table VI).

Significant increases in splenic tissue TNF-α, IL-1β and IL-10 contents were detected in DNCB control mice compared with intact control vehicle mice (P<0.05). However, significant decreases in splenic tissue TNF-α, IL-1β and IL-10 contents were detected following topical treatment of 1% DEXA and also concentration-dependent decreases were observed following bathing in ESGWc 100-, 200- and 400-fold dilutions, compared with DNCB control mice (P<0.05; Table VII).

Significant increases in the expression of TNF-α, IL-4, IL-5 and IL-13 mRNA in the dorsal back skin tissue were detected in DNCB control mice compared with intact vehicle control mice according to RT-qPCR analysis (P<0.05; Table VIII). However, significant decreases in expression of TNF-α, IL-4, IL-5 and IL-13 mRNA were observed in skin tissues following topical treatment with 1% DEXA. Concentration-dependent decreases were also observed following bathing in the three dilutions of ESGWc, compared with DNCB control mice (P<0.05; Table VIII).

Significant decreases in dorsal back skin tissue GSH content and significant increases in skin lipid peroxidation and superoxide anion production were observed in DNCB control mice compared with intact vehicle control mice (P<0.05; Table IX). However, compared with DNCB control mice, there were significant increases in GSH content and significant decreases in lipid peroxidation and superoxide anion production in the skin tissues of mice bathing in ESGWc 100-, 200- and 400-fold dilutions, in a concentration-dependent manner (P<0.05). Topical application of 1% DEXA did not significantly influence skin tissue antioxidant defense systems compared with DNCB control mice (Table IX).

A significant increase in mean epithelial thickness due to hyperplasia/hypertrophy of epidermal keratinocytes was detected in the dorsal back skin tissues of DNCB control mice, with significant increases in the numbers of mast and inflammatory cells infiltrating into dermis, compared with mice in the intact vehicle control group (P<0.05; Table X; Fig. 3). Abnormal collagen depositions, elevations of caspase-3, PARP, NT and 4-HNE immunoreactive epidermal cells, dermal MMP-9 immunoreactivities, infiltrations of dermal IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells were also observed in the DNCB control group compared with the intact vehicle control group (Figs. 3–5). These results were confirmed by histomorphometrical analysis, which demonstrated significant increases in mean epithelial thickness, numbers of dermal infiltrated mast and inflammatory cells, percentages of collagen fiber-occupied dermal regions, caspase-3, PARP, NT and 4-HNE immunoreactive epidermal cells, dermal IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells in DNCB control mice compared with intact vehicle control mice (all P<0.05; Table XI). However, these histopathological hypersensitivity-related AD signs were significantly inhibited by bathing in the ESGWc 100-, 200- and 400-fold dilutions, in a concentration-dependent manner, compared with DNCB control mice (P<0.05; Tables X and XI). Topical application of 1% DEXA also significantly reduced the increases of mean epithelial thicknes, numbers of dermal infiltrated mast and inflammatory cells, caspase-3, PARP, NT and 4-HNE immunoreactive epidermal cells, dermal MMP-9 immunoreactivities, dermal IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells induced by NDCB treatment (P<0.05; Tables X and XI); however, 1% DEXA did not signifcantly influence the percentage of collagen fiber-occupied dermal regions, as compared with DNCB control mice (Table X).

Marked hypertrophic changes due to hyperplasia of red pulp lymphoid cells were detected in the splenic tissues of DNCB control mice, with marked increases of the numbers of IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells (Figs. 6 and 7). These results were confirmed by histomorphometrical analysis, which demonstrated significant increases in total splenic thicknesses, numbers of red pulp lymphoid cells, numbers of IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells in DNCB control mice compared with intact vehicle control mice (P<0.05; Table XII). However, these hypersensitivity-related splenic hypertrophic signs were significantly inhibited following topical treatment with 1% DEXA, and also by bathing in the three dilutions of ESGWc, concentration-dependently, as compared with DNCB control mice (P<0.05). No significant changes in the number of white pulp cells were demonstrated following treatment with DNCB compared with the intact vehicle control mice, nor by topical application of 1% DEXA or bathing in ESGWc 100-, 200- and 400-fold dilutions, as compared with DNCB control mice (Table XII).

Marked hypertrophic changes due to hyperplasia of cortex lymphoid cells were detected in submandibular LN tissues in DNCB control mice, with marked increases of the numbers of IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells (Figs. 8 and 9). These observations were confirmed by histomorphometrical analysis, which demonstrated significant increases in total submandibular LN thicknesses, numbers of cortex lymphoid follicles, cortex thicknesses, numbers of IFN-γ, iNOS, IL-1β, IL-2 and TNF-α immunolabeled cells in submandibular LN tissues of DNCB control mice compared with intact vehicle control mice (P<0.05; Table XIII). However, these submandibular LN hypersensitivity-related hypertrophic signs were significantly inhibited by topical treatment of 1% DEXA, and also concentration-dependently by bathing in all three dilutions of ESGWc, compared with DNCB control mice (P<0.05; Table XIII).