Preparation of DSE
The dried fructus of D. superbus (300 g) were extracted three times by sonication for 1 h with 3 L of 70% ethanol. The extracted solution was filtered through filter paper and evaporated to dryness (56.89 g). The yield of ethanolic extract obtained was 18.96%.
Specific pathogen-free female BALB/c mice (7 weeks old) were purchased from the Orient Co. (Seoul, Korea) and used after 1 week of quarantine and acclimatization. The mice were allowed sterilized tap water and standard rodent chow. All experimental procedures were approved by Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee. This study was performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals and Korean national animal welfare law.
OVA-induced allergic asthma
OVA sensitization and airway challenge were performed as described previously . In brief, mice were sensitized on days 0 and 14 by intraperitoneal injection of 20 μg OVA emulsified in 2 mg aluminum hydroxide in 200 μL PBS buffer (pH 7.4). On days 21, 22 and 23 after initial sensitization, mice received an airway challenge with OVA (1%, w/v, in PBS) for 1 h using an ultrasonic nebulizer (NE-U12; Omron Corp., Tokyo, Japan). DSE was dissolved in PBS and was prepared fresh daily before each treatment. DSE was administered by gavage to mice at doses of 100 mg/kg or 200 mg/kg once daily from day 18 to 23. Negative and positive control mice were orally administered PBS or montelukast (30 mg/kg in PBS), respectively. Montelukast was developed as a cysteinyl leukotriene (cys-LT)-1 receptor antagonist  and was introduced into the market after successful clinical evaluation in patients with aspirin-sensitive asthma, nocturnal exacerbation of asthma, and allergic asthma .
Following OVA challenge, bronchoalveolar lavage fluid (BALF) samples were obtained from the mice and processed, and inflammatory cells were counted as described previously . In brief, mice were sacrificed by intraperitoneal injection of pentobarbital (50 mg/kg; Hanlim Pharm. Co., Seoul, Korea) 48 h after the last challenge, and a tracheostomy was performed. To obtain BALF, ice-cold PBS (0.5 mL) was infused into the lung and withdrawn via tracheal cannulation three times (total volume 1.5 mL). Total inflammatory cell numbers were assessed by counting cells in at least five squares of a hemocytometer after exclusion of dead cells by Trypan blue staining. To determine differential cell counts, 100 μL of BALF was centrifuged onto slides using a Cytospin (Hanil Science Industrial, Seoul, Korea) (200 g, 4°C, 10 min). The slides were dried, and the cells were fixed and stained using Diff-Quik® staining reagent (B4132-1A; IMEB Inc., Deerfield, IL), according to the manufacturer’s instructions. The supernatant obtained from BALF was stored at −70°C for biochemical analysis.
Measurement of the levels of cytokines, chemokine, and IgE
The levels of IL-4, IL-13, and eotaxin in BALF were measured using enzyme-linked immunosorbent assay (ELISA) kits (BioSource International, Camarillo, CA) according to the manufacturer’s protocols. The levels of total IgE in BALF and plasma were measured using an ELISA. Microtiter plates were coated with anti-IgE antibodies (anti-mouse IgE; 10 g/mL; Serotec, Oxford, UK) in PBS-Tween 20, and incubated with BALF or plasma sample. The plates were then washed four times, and 200 μL of o-phenylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO) was added to each well. The plates were incubated for 10 min in the dark and the absorbance was then measured at 450 nm.
Equal amounts of total lung protein (30 μg) were heated at 100°C for 5 min, loaded onto 8% SDS-PAGE gels, and electrophoresed. The proteins were then transferred to a nitrocellulose membrane (at 100 V for 2 h). The membrane was blocked for 1 h with Tris-buffered saline containing 0.05% Tween-20 (TBST) plus 5% skim milk. It was incubated with anti-iNOS (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-actin (1:1000 dilution; Cell Signaling Technology, Danvers, MA) overnight at 4°C. The membrane was washed three times with TBST and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000 dilution; Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The membrane was again washed three times with TBST and developed using an enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden). For quantitative analysis, densitometric band values were determined using Chemi-Doc (Bio-Rad, Hercules, CA, USA).
After BALF samples were obtained, lung tissue was fixed in 10% (v/v) neutral buffered formalin. Tissues were embedded in paraffin, sectioned at 4 μm thickness, and stained with H&E solution (hematoxylin, Sigma MHS-16, and eosin, Sigma HT110-1-32) and periodic acid−Schiff (PAS) (IMEB Inc., San Marcos, CA) to estimate inflammation and mucus production, respectively.
Measurement of NO and prostaglandin E2 (PGE2) production in RAW 264.7 cells
The murine macrophage RAW 264.7 cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco Inc., Grand Island, NY, USA) supplemented with 5.5% heat-inactivated fetal bovine serum (Gibco Inc.), penicillin (100 U/mL), and streptomycin (100 μg/mL) in a 5% CO2 incubator at 37°C. RAW 264.7 cells were plated at a density of 2.5 х 105 cells/well in 48-well plates and incubated overnight. Cells were treated with lipopolysaccharide (LPS, 1 μg/mL) in the presence or absence of various concentrations of DSE. After incubation for 18 h, supernatants were analyzed for the levels of NO (Griess Reagent System, Promega Corp., Madison, WI, USA) and PGE2 (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturers’ protocols.
Image capture and photomicrography
Photomicrographs were obtained using a Photometric Quantix digital camera running a Windows program, and montages were assembled in Adobe Photoshop 7.0. Images were croppedand corrected for brightness and contrast, but were not otherwise manipulated.
Data are expressed as means ± standard error of the mean (SEM). Statistical significance was determined using analysis of variance (ANOVA) followed by a multiple comparison test with Bonferroni adjustment. P values < 0.05 or <0.01 were considered significant.