Imprinting control region (ICR) mice (n=150, male, aged 6 weeks) and Sprague-Dawley (SD) rats (n=10, male, aged 10 weeks) were purchased from Dae Han Biolink (Daejeon, Korea). The animals were housed 5 per cage in a laminar air flow room maintained at a temperature of 22±2°C and a relative humidity of 55±5% throughout the study. The care and treatment of the mice and rats 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 of Kyungpook National University (Daegu, Korea).

Compound 48/80, anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (HSA), pyrrolidine dithiocarbamate (PDTC), phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, and o-phthaldialdehyde (OPA) were all purchased from Sigma Aldrich (St. Louis, MO, USA). Forward and reverse primers for human TNF-α, IL-6 and IL-8 were purchased from Genotech (Daejeon, Korea) and ELISA kits were purchased from BD Biosciences (San Diego, CA, USA). Human mast cells (HMC-1; kind gift from Professor D.K. Kim, Department of Immunology, School of Medicine, Chonbuk National University, Jeonju, Korea) and rat peritoneal mast cells (RPMCs) were grown in Iscove's modified Dulbecco's medium (IMDM) and α-minimum essential medium (Gibco, Grand Island, NY, USA), respectively, supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), 100 U/ml penicillin G (HyClone), 100 µg/ml streptomycin (HyClone) and 250 ng/ml amphotericin B (HyClone) at 37°C in 5% CO₂. HMC-1 cells at passages 4–8 were used throughout the study.

The peritoneal cells were isolated from SD rats as previously described (18). In brief, the rats were anesthetized with CO₂ and injected with 20 ml of Tyrode's buffer A (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl₂, 1 mM MgCl₂, 5.6 mM glucose and 0.1% bovine serum albumin) into the peritoneal cavity, and the abdomen was gently massaged for approximately 90 sec. The peritoneal cavity was carefully opened, and the fluid containing peritoneal cells was aspirated using a Pasteur pipette. The peritoneal cells were sedimented at 150 g for 10 min at room temperature and resuspended in Tyrode's buffer A. Mast cells were separated from the major components of rat peritoneal cells, i.e., macrophages and small lymphocytes. Peritoneal cells were suspended in 1 ml of Tyrode buffer A, layered on 2 ml of Histodenz (Sigma-Aldrich) solution, and centrifuged at 400 × g for 10 min at 4°C. The cells remaining at the buffer-Histodenz interface were aspirated and discarded, and the cells in the pellet were washed and resuspended. Mast cell preparations were approximately 95% pure based on toluidine blue staining. More than 95% of the cells were viable based on trypan blue exclusion (data not shown).

P. cablin used in the present study was purchased from the oriental drug store, Bohwa Dang (Jeonju, Korea) and identified by Dr D.K. Kim at the College of Pharmacy, Woosuk University (Jeonju, Korea). A voucher specimen was deposited at the Herbarium of the College of Pharmacy, Woosuk University. The sample was extracted with purified water at 70°C for 5 h (2 times) in a water bath. The extract was then filtered, lyophilized and stored at 4°C. The yield of dried extract from the starting crude materials was approximately 15.3%. The dried extract of AEPC was dissolved in saline or Tyrode's buffer A prior to use.

The mice (n=10/group) were administered an intraperitoneal injection of 8 mg/kg body weight (BW) of the mast cell degranulation compound 48/80, as previously described (18). The mice were divided into the following groups: group A, compound 48/80 + saline; group B, compound 48/80 + AEPC 10 mg/kg; group C, compound 48/80 + AEPC 50 mg/kg; group D, compound 48/80 + AEPC 100 mg/kg; group E, compound 48/80 + AEPC 500 mg/kg; group F, compound 48/80 + AEPC 1,000 mg/kg; group G, AEPC 1,000 mg/kg only. AEPC was intraperitoneally administered at doses of 10–1,000 mg/kg BW 1 h prior to the injection of compound 48/80. For the time-dependent experiments, AEPC (1,000 mg/kg) was intra- peritoneally administered at 5, 10, 20 and 30 min after the injection of compound 48/80 (n=10/group). Mortality was monitored for 1 h after the induction of anaphylactic shock.

IgE-mediated PCA was induced as previously described (19). To induce the PCA reaction, the skin on the ears of the mice (n=5/group) was sensitized with an intradermal injection of anti-DNP IgE (0.5 µg/site) for 48 h. The mice were divided into the following groups: group A, saline only; group B, IgE/DNP-HSA; group C, IgE/DNP-HSA + AEPC 1 mg/kg, group D: IgE/DNP-HSA + AEPC 10 mg/kg, group E: IgE/DNP-HSA + AEPC 100 mg/kg, group F: IgE/DNP-HSA + AEPC 1,000 mg/kg AEPC was intraperitoneally administered at doses of 1–1,000 mg/kg BW 1 h prior to the intravenous injection of DNP-HSA (1 mg/mouse) and 4% Evans blue (1:1) mixture. Thirty minutes after the challenge, the mice were euthanized using carbon dioxide, and the skin of both ears was collected for measurements with pigment dye. The amount of dye was determined colorimetrically following extraction with a mixture of 1 ml of 1 M KOH and 9 ml of acetone and phosphoric acid (5:13). The absorbance intensity of the extract was detected using a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at 620 nm.

To assess mast cell degranulation, the release of histamine from mast cells was detected using the o-phthaldialdehyde spectrofluorometric procedure and a fluorescent plate reader (Molecular Devices) at an excitation wavelength of 360 nm and an emission wavelength of 440 nm, as previously described, and the level of β-hexosaminidase was read using the spectrophotometer at 405 nm, as previously described (20,21). The RPMCs (2×10⁴ cells/well in 24-well plates) were pre-treated with or without AEPC for 30 min and then stimulated with compound 48/80 (5 µg/ml) for 10 min. The cells were separated from the released histamine by centrifugation at 400 × g for 5 min at 4°C. β-hexosaminidase substrate buffer (100 mM sodium citrate, 1 mM 4-nitrophenyl N-acetyl-β-D-galactosaminide, pH 4.5) was added following by incubation for 1 h at 37°C. The reaction was terminated using stop solution (0.1 M Na₂CO₃ and NaHCO₃; Sigma Aldrich).

Intracellular calcium levels were measured with the use of the fluorescence indicator, Fluo-3/AM (Invitrogen, Carlsbad, CA, USA), as previously described (20). The RPMCs (1×10⁴ cells/well in 96-well plates) were pre-incubated with Fluo-3/AM for 1 h at 37°C. After washing the dye from the cell surface with Tyrode's buffer B (137 mM NaCl, 5.5 mM glucose, 12 mM NaHCO₃, 2.7 mM KCl, 0.2 mM NaH₂PO₄, 1 mM MgCl₂ and 1.8 mM CaCl₂), the cells were pre-treated with or without AEPC for 30 min and then stimulated with compound 48/80 (5 µg/ml). The fluorescence intensity was detected using a fluorescent plate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The intracellular calcium level was calculated using relative absorbance as the control value of 1.

Cell viability was assayed using an XTT assay kit (Welgene Inc., Seoul, Korea) according to the manufacturer's instructions and as previously described (22). The HMC-1 cells (1×10⁵ cells/well in 96-well plates) were pre-treated with various concentrations of AEPC (1–1,000 µg/ml) for 24 h and incubated with XTT plus phenazine methosulfate reagent for 2 h at 37°C. The absorbance intensity was detected using a spectrophotometer at 450 nm. Cell viability was calculated using relative absorbance as the control value of 100%.

Prior to the isolation of total cellular RNA, the HMC-1 cells (1×10⁶ cells/well in 24-well plates) were pre-treated with or without AEPC for 30 min and stimulated with PMA (40 nM) plus calcium ionophore A23187 (1 µM) (PMACI) for 2 h. RNAiso Plus reagent (Takara Bio Inc., Shiga, Japan) was used to extract the RNA, according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using the Maxime RT PreMix kit (iNtRON Biotechnology, Daejeon, Korea). To measure the expression levels of TNF-α, IL-6 and IL-8, RT-qPCR was carried out using the Maxime PCR PreMix kit (iNtRON Biotechnology) and the Thermal Cycler Dice TP850 (Takara Bio) according to the manufacturer's instructions. For RT-PCR, the total reaction mixture (20 µl) was composed of the following: 1 µl of cDNA (100 ng), 1 µl of each of forward and reverse primers (0.4 µM) and 17 µl of dH₂O. For quantitative (real-time) PCR (qPCR), the total reaction mixture (25 µl) was composed of the following: 1.5 µl of cDNA (200 ng), 1 µl of each of forward and reverse primers (0.4 µM), 12.5 µl of SYBR Premix Ex Taq (Takara Bio) and 9 µl of dH₂O. The primer sequences used were as follows: TNF-α forward, 5′-CCT ACC AGA CCA AGG TCA AC-3′ and reverse, 5′-AGG GGG TAA TAA AGG GAT TG-3′; IL-6 forward, 5′-AAA GAG GCA CTG GCA GAA AA-3′ and reverse, 5′-ATC TGA GGT GCC CAT GCT AC-3′; IL-8 forward, 5′-ACA GCA GAG CAC ACA AGC TT-3′ and reverse, 5′-CTG GCA ACC CTA CAA CAG AC-3′; β-actin forward, 5′-GGA CTT CGA GCA AGA GAT GG-3′ and reverse, 5′-AGC ACT GTG TTG GCG TAC AG-3′. The conditions for the qPCR steps were similar to those described in a previous study (22).

The secretion of pro-inflammatory cytokines was measured by ELISA, as previously described (8). The HMC-1 cells (1×10⁶ cells/well in 24-well plates) were pre-treated with or without AEPC for 30 min and stimulated with PMACI for 8 h. ELISA was performed using an ELISA kit (BD Biosciences) on a 96-well Nunc-Immuno plate (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. After terminating the reaction with a substrate, the absorbance intensity was detected using a spectrophotometer at 450 nm.

Nuclear and cytosolic proteins were both extracted as previously described (23). Prior to protein extraction, the HMC-1 cells (2×10⁶ cells/well in 6-well plates) were pre-treated with or without AEPC for 30 min and stimulated with PMACI for 2 h. Followikng suspension in 100 µl of cell lysis buffer A (0.5% Triton X-100, 150 mM NaCl, 10 mM HEPES, 1 mM EDTA/Na₃VO₄, 0.5 mM PMSF/DTT and 5 µg/ml leupeptin/aprotinin), the cells were vortexed, incubated for 5 min on ice and centrifuged at 400 × g for 5 min at 4°C, and the supernatant was then gathered as the cytosolic protein extract. The pellets were washed 3 times with 1 ml of PBS and suspended in 25 µl of cell lysis buffer B (25% glycerol, 420 mM NaCl, 20 mM HEPES, 1.2 mM MgCl₂, 0.2 mM EDTA, 1 mM Na₃VO₄, 0.5 mM PMSF/DTT and 5 µg/ml leupeptin/aprotinin), vortexed, sonicated for 30 sec, incubated for 20 min on ice and centrifuged at 15,000 × g for 15 min at 4°C, and the supernatant was then gathered as the nuclear protein extract. Samples of protein were electrophoresed using 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. NF-κB, IκBα, actin and p38 MAPK were assayed using the following antibodies purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-NF-κB (sc-109), anti-IκBα (sc-371), anti-actin (sc-8432; mouse monoclonal; 1:1,000), and Cell Signaling Technology Inc. (Danvers, MA, USA); anti-phospho-p38 (#9211) and anti-p38 (#9212). Immunodetection was carried out using a chemiluminescent substrate (Thermo Fisher Scientific).

The HMC-1 cells (2×10⁶ cells/well in 6-well plates) were seeded in serum/antibiotics-free IMDM 1 day prior to transient transfection. The expression vectors containing the NF-κB luciferase reporter construct (pNF-κB-LUC, plasmid containing the NF-κB binding site; Stantagen, Grand Island, NY, USA) or empty vectors were transfected using 8 µl of Lipofectamine 2000 reagent (Invitrogen). Following incubation for 5 h, the medium was replaced with IMDM containing 10% FBS and antibiotics. The cells were allowed to recover at 37°C for 20 h and subsequently were stimulated as indicated. The cell lysate was prepared and assayed for luciferase activity using a Luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Statistical analyses were performed using Prism5 (GraphPad Software, San Diego, CA, USA), and the effects of treatment were analyzed using one-way ANOVA followed by Dunnett's test. A P-value <0.05 was considered to indicate a statistically significant difference.

To determine the effectS of AEPC on allergic reaction in vivo, compound 48/80-induced systemic anaphylaxis and IgE-mediated PCA were induced in mice for immediate-type hypersensitivity. The intraperitoneal injection of compound 48/80 (8 mg/kg BW), a mast cell degranulator, induces lethal systemic anaphylaxis (18). The mice (n=10/group) were intraperitoneally administered 200 µl saline or various doses of AEPC (10–1,000 mg/kg BW) 1 h prior to the intraperitoneal injection of compound 48/80. Following the administration of compound 48/80, we monitored the mice for 1 h and the mortality rate was recorded. All mice injected with compound 48/80 and saline suffered fatal anaphylactic shock, whereas the mortality rate was reduced in the mice administered AEPC in a dose-dependent manner (Table I). In addition, the mortality rate of the mice administered AEPC (1,000 mg/kg BW) at 5, 10, 20 and 30 min after the injection of compound 48/80 was increased in a time-dependent manner (Table II).

Another animal model, of IgE-mediated PCA, was used to confirm the anti-allergic effects of AEPC (19). Both ears of each mouse were sensitized by an intradermal injection of anti-DNP IgE (0.5 µg/site) prior to the intravenous injection of DNP-HSA (1 mg/mouse) and a 4% Evans blue (1:1) mixture to induce the PCA reaction. After the antigen challenge, a blue spot was visible at the sensitized site due to the increased vascular permeability caused by the release of histamine from mast cells. AEPC was administered intraperitoneally 1 h prior to the challenge with antigen. The administration of AEPC reduced the size and blue color of the spot, thus inhibiting the PCA reaction in a dose-dependent manner (Fig. 1).

Histamine and β-hexosaminidase released from mast cells are important allergic mediators, and the inhibition of mast cell degranulation is the proper therapeutic target for allergic disorders (24). Thus, in this study, we evaluated the effects of AEPC on the release of histamine and β-hexosaminidase induced by compound 48/80. Treatment with compound 48/80 resulted in the increased release of histamine and β-hexsosaminidase from the RPMCs (data not shown). By contrast, AEPC suppressed the release of histamine and β-hexsosaminidase induced by compound 48/80 in a dose-dependent manner (Fig. 2A and B). These results support the hypothesis that AEPC inhibits compound 48/80-induced anaphylaxis by blocking degranulation of mast cells.

To investigate the mechanisms responsible for the inhibitory effects of AEPC on the release of histamine, we measured the intracellular calcium levels. Calcium influx in mast cells is critical to the release of histamine (25). Antigen cross-linking of IgE bound to FcεRI causes the calcium influx, which stimulates granule fusion-to-cell membranes consequentially through the binding of synaptotagmin to the soluble NSF attachment protein receptor (SNARE) complex (26). Compound 48/80 induced an increase in the intracellular calcium levels in the RPMCs; however, AEPC counteracted this increase (Fig. 2C). Therefore, our findings suggest that AEPC inhibits the release of histamine by blocking calcium movement into mast cells. The concentration of AEPC used in this study was not cytotoxic to the HMC-1 cells (Fig. 2D).

Pro-inflammatory cytokines are important to the progression of chronic allergic inflammation (27). Thus, we examined the effects of AEPC on the gene expression of pro-inflammatory cytokines in HMC-1 cells, such as TNF-α, IL-6 and IL-8. AEPC attenuated the PMACI-induced increase in the mRNA expression of TNF-α, IL-6 and IL-8 in a dose-dependent manner (Fig. 3A and B). To confirm the effects of AEPC on the mRNA expression of pro-inflammatory cytokines, cultured media were used in the ELISA for assaying the secretion of TNF-α, IL-6 and IL-8. Stimulation of the cells with PMACI for 8 h induced the secretion of cytokines. On the contrary, AEPC inhibited the secretion of TNF-α, IL-6 and IL-8 from the PMACI-stimulated HMC-1 cells (Fig. 3C).

To elucidate the mechanisms responsible for the inhibitory effects of AEPC on cytokine expression, we investigated the activation of the transcription factors, NF-κB and p38 MAPK, which have previously been reported to play important roles in immune and inflammatory responses (23). Stimulation of the HMC-1 cells with PMACI caused the degradation of IκBα and the translocation of p65 NF-κB into the nucleus. AEPC reduced the PMACI-induced activation of NF-κB by blocking the degradation of IκBα (Fig. 4A). To confirm the suppressive effects of AEPC on the activation of NF-κB, we performed an NF-κB-dependent gene reporter assay. PDTC, an inhibitor of NF-κB, was used as a positive control. The HMC-1 cells were transiently transfected with an NF-κB-luciferase reporter construct or an empty vector. Stimulation of the cells with PMACI increased the luciferase activity in the cells transfected with the NF-κB-luciferase reporter construct. AEPC significantly diminished the increased luciferase activity induced by PMACI (Fig. 4B). The MAPK pathway is also known to play a major role in the regulation of pro-inflammatory mediators (28,29). Thus, we evaluated the effects of AEPC on the activation of MAPKs. It is clear that AEPC attenuated PMACI-induced p38 MAPK phosphorylation (Fig. 4C).