The original stock of male imprinting control region (ICR) mice (6 weeks) and male Sprague-Dawley rats (8 weeks) were purchased from the Dae-Han Experimental Animal Center (Daejeon, Korea). The animals were housed 5 per cage in a laminar air flow room maintained under a temperature of 22±2°C and relative humidity of 55±5°C throughout the study. The care and treatment of the mice were in accordance with the guidelines established by the Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

Compound 48/80, anti-dinitrophenyl (DNP) IgE, DNP-human serum albumin (HSA), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The human mast cell line (HMC-1) was grown in Iscove’s media (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum at 37°C in 5% CO₂. Passages 4–8 of HMC-1 cells were used throughout the study.

The Sparassis crispa was purchased from the oriental drug store, Bohwa Dang (Jeonbuk, Korea) and identified by D.K. Kim, College of Pharmacy, Woosuk University. A voucher specimen was deposited at the Herbarium of the College of Pharmacy, Woosuk University. The S. crispa was ground (400 x g, 30 sec) at room temperature using Micro Hammer-Cutter Mill (Culatti Co., Zurich, Switzerland). The particle size was 0.5–2 mM after grinding. The plant sample (100 x g) was extracted twice with purified water (500 ml) at 70°C for 5 h in a water bath. The extract was passed through filter paper and the filtrate was lyophilized using a 0.45 μm syringe filter. The dried extract was dissolved in saline or Tyrode buffer A (HEPES 10 mM, NaCl 130 mM, KCl 5 mM, CaCl₂ 1.4 mM, MgCl₂ 1 mM, glucose 1.4 mM, 0.1% bovine serum albumin) before use.

Mice were given an intraperitoneal injection of 8 mg/kg body weight (BW) of the mast cell degranulator compound 48/80. WESC was administered intraperitoneally at doses of 10–1,000 mg/kg BW 1 h before the injection of compound 48/80 (n=10/group). Mortality was monitored for 1 h after induction of anaphylactic shock. After the mortality test, blood was obtained from the heart of each mouse to measure serum histamine contents.

An IgE-dependent cutaneous reaction was carried out as described previously (13). Briefly, mice were injected intradermally with 0.5 μg of anti-DNP IgE. After 48 h, each mouse (n=10/group) received an injection of 1 μg of DNP-HSA containing 4% Evans blue (1:4) via the tail vein. Thirty minutes after the challenge, the mice were sacrificed and the dorsal skin (diameter, 1 cm) was removed for measurement of the pigmented area.

Preparation of serum and determination of histamine contents were examined as previously described (14). Briefly, serum was withdrawn and the histamine contents were measured by the o-phthaldialdehyde spectrofluorometric procedure. The fluorescent intensity was measured at emission 438 nm and excitation 353 nm using a spectrofluorometer.

The intracellular calcium was measured with the use of the fluorescence indicator Fluo-3/AM (Molecular Probes, Eugene, OR, USA). HMC-1 cells were pre-incubated with Fluo-3/AM for 30 min at 37°C. After washing the dye from the cell surface, the cells were treated with WESC for 10 min before adding PMACI. It was excited at 488 nm, and the emission was filtered with 515 nm by flow cytometer (BD Biosciences Pharmingen, San Diego, CA, USA) and visualized by a fluorescence microscope Olympus BX51 (Olympus, Center Valley, PA, USA).

The total cellular RNA was isolated from the cells (1x10⁶/well in a 24-well plate) after stimulation with PMA (20 nM) and A23187 (1 μM) with or without WESC for 2 h using TRI reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s protocol. The first strand complementary DNA (cDNA) was synthesized using the Superscript II reverse-transcriptase (Invitrogen, Carlsbad, CA, USA). A reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of mRNA for TNF-α, IL-6, and β-actin (internal control). The conditions for the reverse transcription and PCR steps were similar to those described previously (13). The amplified products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide, documented using a Kodak DC 290 digital camera and digitized with UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT, USA). The band intensity was normalized to that of β-actin in the same sample.

The secretion of TNF-α and IL-6 was measured by the modification of an enzyme-linked immunosorbent assay (ELISA) as previously described (15). HMC-1 cells were cultured with media and resuspended in Tyrode buffer A. The cells were sensitized with PMACI for 8 h in the absence or presence of WESC. ELISA was performed by coating 96-well plates with 6.25 ng/well of monoclonal antibody with specificity for TNF-α and IL-6, respectively.

HMC-1 cells were washed 3 times with PBS and resuspended in lysis buffer. Samples were electrophoresed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described elsewhere (16), and then transferred to a nitrocellulose membrane. The nucleus and cytosolic p65 NF-κB and IκBα was assayed using anti-NF-κB (p65) and anti-IκBα antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The phosphorylation of ERK and p38 was determined using anti-phospho-p38 and anti-phospho-ERK antibodies (Cell Signaling Technology, Inc., Beverly, MA, USA). Immunodetection was performed the using Supersignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA).

For transient transfection, HMC-1 cells were seeded at 2x10⁶ in a 6-well plate 1 day before transient transfection. The expression vectors containing the NF-κB luciferase reporter construct (pNF-κB-LUC, plasmid containing NF-κB binding site; Stantagen, Grand Island, NY, USA) were transfected with serum- and antibiotics-free Iscove’s medium containing 8 μl Lipofectamine 2000 reagent (Invitrogen). After 5 h of incubation, the medium was replaced with Iscove’s medium containing 10% FBS and antibiotics. Cells were allowed to recover at 37°C for 30 h and subsequently were stimulated as indicated. Cell lysates were prepared and assayed for luciferase activity using the Luciferase Assay System (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

β-glucan contents in WESC were determined using a mushroom β-glucan assay kit (K-YBGL; Megazyme International, Wicklow, Ireland) according to the manufacturer’s protocol. The lyophilized extract of S. crispa (100 mg) and 1.5 ml of 37% hydrochloric acid (v/v, 10 N) were mixed vigorously, and incubated at 30°C for 45 min. The materials were mixed with 10 ml distilled water and incubated at 100°C for 2 h. After centrifugation at 1,500 x g for 10 min, 0.1 ml aliquots were combined with 0.1 ml of a mixture of exo-(1–3)-β-glucanase (20 U/ml) plus β-glucosidase (4 U/ml) in 200 mM sodium acetate buffer (pH 5.0) and incubated at 40°C for 60 min. To measure total glucan content, 3 ml of glucose oxidase/peroxidase mixture (GOPOD) was added and incubated at 40°C for 20 min. The absorbance of samples was analyzed spectrophotometrically at 510 nm against the reagent blank using a spectrophotometer (Shimadzu, UV-1201). To measure the α-glucan content, 2 ml of 2 M KOH was added, and the phytoglycogen and starch were dissolved by stirring for 20 min in an ice water bath. The suspension was added to 8 ml of 1.2 M sodium acetate buffer (pH 3.8), mixed with 0.2 ml of amyloglucosidase (16,300 U/ml) plus invertase (500 U/ml), and incubated in a water bath for 30 min at 40°C with intermittent mixing on a vortex stirrer. Tubes were centrifuged (10 min, 1,500 x g), and 0.1 ml aliquots were combined with 0.1 ml of sodium acetate buffer (200 mM, pH 5.0) plus 3 ml of GOPOD reagent and incubated for at 40°C for 30 min. The absorbance of samples was analyzed at 510 nm. The β-glucan content was determined by subtracting the α-glucan from the total glucan content.

Statistical analyses were performed using SAS statistical software (SAS Institute, Inc., Cary, NC, USA). Treatment effects were analyzed using analysis of variance, followed by Duncan’s multiple range tests. P<0.05 indicated significance.

To determine the effect of WESC in allergic reaction, an in vivo model of a systemic anaphylaxis was used. Compound 48/80 (8 mg/kg) was used as a model of induction for a systemic fatal allergic reaction. After the intraperitoneal injection of compound 48/80, the mice were monitored for 1 h, after which the mortality rate was determined. Injection of compound 48/80 into mice induced fatal shock in 100% of the animals. When WESC was intraperitoneally pretreated at doses ranging from 10 to 1,000 mg/kg for 1 h, the mortality was dose-dependently reduced. WESC completely inhibited compound 48/80-induced fatal shock at 1,000 mg/kg (Table I). In addition, the mortality of mice administered with WESC (1,000 mg/kg) 5, 10, 15 and 20 min after compound 48/80 injection time-dependently increased (Table II).

The effect of WESC on the compound 48/80-induced serum histamine release was investigated. The histamine level caused by compound 48/80 was decreased by WESC in a dose-dependent manner (Fig. 1A). To confirm the anti-allergic effects of WESC, we used a passive cutaneous anaphylaxis (PCA) model induced by anti-DNP-IgE and DNP-HSA. To compare to amount of dye with control, the left dorsal skin of these mice was injected with saline alone. WESC was intraperitoneally administered 1 h prior to the challenge with antigen. WESC dose-dependently inhibited PCA reaction (Fig. 1B).

We estimated the reducing effects of WESC on the histamine release from PMACI-induced HMC-1 cells. Mast cells released a high level of histamine when simulated with PMACI (Fig. 2A). When WESC was pretreated for 30 min, histamine was dose-dependently inhibited in PMACI-induced HMC-1. Up to 1 mg/ml of WESC did not show cytotoxicity (data not shown). To investigate the mechanism responsible for the reduction of histamine after WESC treatment, we assayed the intracellular calcium levels. Calcium movements across membranes of mast cells are critical to histamine release (17). When HMC-1 cells were stimulated with PMACI, intracellular calcium levels were significantly elevated (Fig. 2B). WESC (100 μg/ml) decreased the intracellular calcium level. The levels of intracellular calcium were also assessed by the relative fluorescent intensity.

We investigated the inhibitory effect of WESC on the expression of proinflammatory cytokines such as TNF-α and IL-6. The HMC-1 cell line is a useful tool for researching the cytokine activation pathway (18,19). Previously we reported that gene expression of TNF-α and IL-6 peaked 4 h after treatment of PMACI (20). Consequently, HMC-1 cells were stimulated by PMACI during 4 h, and the cells were preincubated with WESC for 30 min. Fig. 3A shows that the expression of proinflammatory cytokines was inhibited by WESC. To confirm the correlation of mRNA expression with protein production, we measured the secretion of TNF-α and IL-6. When HMC-1 cells were stimulated with PMACI for 8 h, the secretion of cytokines was remarkably induced. WESC dose-dependently inhibited the secretion of TNF-α and IL-6 in PMACI-stimulated HMC-1 cells (Fig. 3B).

To investigate the intracellular mechanism responsible for the inhibitory effect of WESC on the expression of proinflammatory cytokines, we examined the effect of WESC on the activation of transcription factors, NF-κB and MAPKs. NF-κB is an important transcriptional regulator of inflammatory cytokines and plays a crucial role in immune and inflammatory responses. Stimulation of HMC-1 with PMACI induced the nuclear translocation of p65 NF-κB and degradation of IκBα after 2 h of incubation. WESC inhibited the PMACI-induced nuclear translocation of NF-κB and degradation of IκBα (Fig. 4A). To confirm the inhibitory effect of WESC on the NF-κB activation, we examined the effect of WESC on the NF-κB-dependent gene reporter assay. HMC-1 cells were transiently transfected with a NF-κB-luciferase reporter construct or an empty vector. Exposure of cells to PMACI increased the luciferase activity in the cells transfected with the NF-κB-luciferase reporter construct (Fig. 4B). WESC significantly reduced PMACI-induced luciferase activity.

The MAPK signaling cascades also regulate important cellular processes including gene expression, cell proliferation, and cell survival and death (21). Previously we documented that PMACI activates all three types of MAPKs such as p38, JNK, and ERK at 15–30 min in HMC-1 (22). In the present results, stimulation of cells with PMACI induced phosphorylation of p38, JNK, and ERK, and WESC markedly attenuated PMACI-induced phosphorylation of ERK and p38 MAPK (Fig. 5). However WESC did not affect the phosphorylation of JNK (data not shown).