Arazyme was purified from extracellular fractions of S. proteamaculan HY-3 (KCTC2390; Korean Collection for Type Culture, Daejeon, Korea) as previously described (8). In brief, extracellular fractions were collected by centrifugation of the Luria-Bertani (LB) culture medium (Sigma-Aldrich Korea, Seoul, Korea) at 5,000 × g for 10 min, or by filtration using a 0.2-μm membrane filter (Pall Life Sciences, Port Washington, NY, USA). Chromatography was subsequently performed on a DEAE-cellulose column (GE Healthcare Life Sciences, Little Chalfont, UK) equilibrated with 50 mM potassium phosphate buffer (pH 7.6; Sigma-Aldrich Korea). Bound proteins were then eluted with a 0.1–0.5 M sodium chloride (Sigma-Aldrich Korea) gradient at a flow rate of 400 ml/h and following this, each fraction was concentrated using a 10-kD cassette membrane (Pall Life Sciences). The protein solution was loaded onto a Sephadex G-75 column (GE Healthcare Life Sciences) equilibrated with 50 mM potassium phosphate buffer (pH 7.8) at a flow rate of 20 ml/h and fractions with proteolytic activity were concentrated with a 10-kD cassette membrane and stored at −20°C. Proteolytic activity was determined spectrophotometrically by measuring absorbance at 405 nm (SpectroQuest UV-2800, cat. no. S90424; Thermo Fisher Scientific, Inc., Waltham, MA USA), as previously described (8).

A total of 40 five-week-old female BALB/c mice (weight, 17–19 g) and 30 NC/Nga mice (weight, 19–21 g) were purchased from Japan SLC, Inc. (Hamamatsu, Japan) and acclimated for one week prior to the start of the experiments. Animals were housed in an air-conditioned animal unit at 23±2°C and a humidity of 50±10%. Mice were provided with solid feed (Rodfeed; Daehan Biolink Co., Ltd., Eumsung, Korea). A schematic diagram of the experimental procedure is provided in Fig. 1. Induction of AD was performed with 2,4-dinitrochlorobenzene (DNCB; Sigma-Aldrich, St. Louis, MO, USA) in BALB/c and NC/Nga mice. In brief, 1% DNCB was dissolved in an acetone-olive oil mixture (acetone/olive oil, 3:1; Sigma-Aldrich Korea). The dorsal hair of the mice was removed with an electric razor and no skin damage (e.g. chafed skin or hemorrhage) was observed. A total of 0.15 ml 1% DNCB solution was applied to the same area of dorsal skin. Following sensitization with 1% DNCB, the BALB/c mice were dorsally treated with 0.3% DNCB three times/week for 4 weeks, then once/week for 4 weeks. Nc/Nga mice were treated with 0.3% DNCB three times/week for 4 weeks and then twice per week for 6 weeks. The protocol for the care and treatment of the mice was approved by the Institutional Animal Care and Use Committee of Eulji University (Daejeon, Republic of Korea).

BALB/c mice were divided into the following eight groups (n=5 in each group): Untreated; DNCB; oral arazyme (10 mg/kg); 25 mg/kg dorsal arazyme; combined treatment with 5 mg/kg oral and 12.5 mg/kg dorsal arazyme; 5 mg/kg oral dexamethasone; 5 mg/kg dorsal dexamethasone; and combined treatment with 2.5 mg/kg oral and 2.5 mg/kg dorsal dexamethasone, which were all purchased from Sigma-Aldrich Korea. Nc/Nga mice were divided into the following six groups (n=5 in each group): Untreated; DNCB; 25 mg/kg arazyme; 50 mg/kg arazyme; 125 mg/kg arazyme; 5 mg/kg dexamethasone. The DNCB, arazyme and dexamethasone groups were dorsally administered with 1% DNCB and subsequently dorsally treated with 0.3% DNCB. The DNCB, arazyme and dexamethasone groups were treated with phosphate-buffered saline (PBS; Sigma-Aldrich Korea), arazyme and dexamethasone via gastric inoculation with a mouse-feeding needle (Cadence, Inc., Staunton, VA, USA) and/or application to the same area of the dorsal skin. The untreated group was treated with PBS without administration of DNCB, arazyme or dexamethasone.

Subsequent to sacrifice of the mice by CO₂ asphyxiation, the dorsal skin was removed, fixed in Carnoy’s solution (Sigma-Aldrich Korea), embedded in paraffin (Sigma-Aldrich Korea) and sectioned (5 μm-thick). The sections were then stained with hematoxylin-eosin solution (Sigma-Aldrich Korea) and subsequently examined by light microscopy (Leica Microsystems, Wetzlar, Germany) for histological evaluation. Specifically, the epidermis was evaluated for hypertrophy and infiltration by inflammatory cells, while the dermis was evaluated for infiltration by inflammatory cells.

Blood was collected from the tail of the mice every week. The serum was obtained by centrifugation and then stored at −70°C until required. Total IgE levels in the serum were measured using sandwich ELISA kits (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions.

The BALB/c mice were sacrificed and subsequently their spleens were removed under aseptic conditions. Splenocytes were then isolated from the spleens as previously described (11), after which the red blood cells were hemolyzed using red blood cell lysis solution (Sigma-Aldrich). Splenocytes were seeded in a 24-well plate at a concentration of 5×10⁶ cells/ml in RPMI-1640 medium with 1% penicillin-streptomycin and 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA).

Splenocytes were pretreated in the absence or presence of arazyme and then stimulated with 1 μg/ml concanavalin A (Sigma-Aldrich Korea) for 24 h. The cell supernatants were collected and the concentrations of interleukin (IL)-4, IL-5 and IL-13 were measured in the supernatant by a sandwich ELISA [OptEIA™ Human IL-4 and IL-5 sets; BD Biosciences; and Human IL-13 DuoSet kit, R&D Systems, Inc. (Minneapolis, MN, USA)] according to the manufacturer’s instructions. The concentration of each protein was calculated from the standard curves.

The severity of dermatitis was assessed macroscopically in a blinded experiment. The four indicators of skin lesions were: i) Erythema/hemorrhage, ii) edema/swelling, iii) excoriation/erosion and iv) dryness. Scoring was performed as follows: 0 (no symptoms), 1 (mild), 2 (moderate) and 3 (severe) (12).

The concentrations of ALT and AST were measured using the Reitman-Frankel method (13) in the serum of BALB/c mice using ALT and AST assay kits (Asan Pharm Co., Seoul, Korea) according to the manufacturer’s instructions.

Values are expressed as the mean ± standard deviation. Data were analyzed using Student’s t-test using SPSS software, version 10.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The therapeutic effects of arazyme were investigated in mice with DNCB-induced dermatitis by histological evaluation. Histological analysis of the skin of mice in the untreated group revealed that the tissue was normal, whereas mice in the DNCB group exhibited epidermal hypertrophy, hyperkeratosis of the epidermis and infiltration of inflammatory cells (Fig. 2A). Oral and oral/dorsal administration of arazyme markedly ameliorated the histopathological alterations as compared with those in the dexamethasone group, while dorsal administration of arazyme resulted in a small reduction in these alterations. Mice in the group administered oral/dorsal dexamethasone exhibited atrophy of the skin. Since IgE is known to act as an important pathogenic factor in AD (3,4), it was investigated whether the anti-inhibitory effects of arazyme were involved in the alteration of IgE production. Although the serum levels of IgE in the DNCB group were markedly upregulated compared with those in the untreated group, arazyme was not observed to effectively reduce these elevated IgE levels (Fig. 2B). The body weight of mice in the arazyme group was comparable to that of mice in the untreated and DNCB groups (Fig. 2C), while a reduction in body weight was observed in the dexamethasone group after 5 weeks.

Inflammatory cytokines, particularly Th2 cytokines, serve essential roles in allergic diseases such as AD (3,4); thus, the effects of arazyme on cytokine production were investigated. Splenocytes isolated from the spleens of BALB/c mice were treated with arazyme for 1 h and subsequently with concanavalin A for 24 h. The synthesis of IL-4, IL-5 and IL-13 was observed to increase in the supernatant of splenocytes following stimulation with concanavalin A for 24 and 48 h (Fig. 3). Pretreatment with arazyme inhibited the increased secretion of IL-4 and IL-13. IL-5 release remained unchanged in the arazyme-treated splenocytes.

As the oral treatment of arazyme proved effective at inhibiting histopathological features in AD-like BALB/c mice, the effects of oral administration of arazyme on Nc/Nga mice were investigated as an additional AD-like model. The DNCB group exhibited epidermal hyperplasia, hyperkeratosis and inflammation (Fig. 4A). Oral administration of arazyme resulted in suppression of the histological phenomena associated with AD at low concentrations (25 mg/kg), while treatment with high concentrations (50 and 100 mg/kg) of arazyme resulted in either a weak or absent effect. The severity of dermatitis was evaluated every week. Clinical signs and symptoms of AD developed subsequent to dorsal treatment with DNCB and these symptoms were observed to worsen with time following the initial treatment. As demonstrated in Fig. 4B, the DNCB group exhibited thick skin with severe erythema, hemorrhage, edema, erosion and excoriation. However, oral application of arazyme at low concentrations inhibited the development of these skin conditions. In addition, 25 mg/kg arazyme was observed to significantly reduce (^**P<0.01) the elevated IgE levels in the serum at weeks 9, 10 and 11 and the suppressive effect of arazyme was comparable to that produced by treatment with dexamethasone (Fig. 4C). However, high concentrations of arazyme had no inhibitory effect on the alterations in IgE levels. To confirm the cytotoxicity of arazyme, the alterations in body weight, ALT and AST levels in the serum were investigated. The levels of ALT and AST were unchanged by administration of arazyme (Fig. 5A). Body weight was only identified to be altered in the dexamethasone group (Fig. 5B).