Preparation of extract
Roots of Arctium lappa L. (1 kg) were extracted with 30% ethanol under reflux (10 L, 24 h, twice). The extract solutions were filtered and then evaporated at 40°C under reduced pressure, yielding 88.8 g of dry powder. Approximately 50 g of the ethanol extract were resuspended in 1 L of water and then partitioned with equal volumes of n-hexane, AcOEt, and n-BuOH to give n-hexane, AcOEt, n-BuOH, and H2O fractions. The butanolic fraction weighed 22.0 g and the sample was named A. lappa butanolic extract (ALBE).
Cell culture and experimental animals
The RBL-2H3 rat mast cell line was obtained from the American Type Culture Collection (Rockville, MD, USA) and grown in minimum essential medium (MEM) with 15% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified incubator with a 5% CO2 /95% air atmosphere. Specific-pathogen-free 8-10-week-old male C57BL/6 mice were purchased from Orient Bio (Gyeonggi-do, Korea) and housed in an animal room at a temperature of 23 ± 1°C and a humidity of 55 ± 5%, with a 12/12-h light/dark cycle. The mice were fed a standard laboratory diet with tap water ad libitum.
Animal care and all experimental protocols were performed following the Institute for Laboratory Animal Research (ILAR) guidelines.
The anti-dinitrophenyl (DNP)-IgE and 4-nitrophenyl N-acetyl-β-D-glucosaminide were from Sigma-Aldrich, DNP-bovine serum albumin (BSA) was from Biosearch Technologies, minimum essential medium was from Invitrogen, fetal bovine serum (FBS) was from WelGENE, enzyme immunoassay reagents for cytokine assays, such as IL-4 and IL-5, were from BD Biosciences, the protein assay kit was from Bio-Rad Laboratories, anti-pERK, anti-ERK, anti-pJNK, anti-JNK, and anti-p-p38 were from Cell Signaling Technology, anti-p65 and anti-p38 were from Santa Cruz Biotechnology, anti-β-actin was from Sigma-Aldrich, anti-α-tubulin was from Abfrontier, the ECL chemiluminescence system was from GE Healthcare, and the polyvinylidene difluoride (PVDF) membrane was from Millipore. The polymerase chain reaction (PCR) oligonucleotide primers were custom synthesized by Bionics (Korea).
XTT assay for cell cytotoxicity and proliferation
Splenocyte cytotoxicity and proliferation were examined using the XTT assay kit, according to the manufacturer's instructions. The spleen was removed aseptically and dissociated into a single cell suspension in culture medium. Cells (5 × 105 cells/well) were incubated with various ALBE concentrations (1, 10, 100 μg/mL) in the presence or absence of ConA at 3 μg/mL for T cell activation. After incubating the cells for 72 h, a mixture of 25 μL of phenazine methosulfate (PMS; electron-coupling reagent) and 25 μL of XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] was added to each well. The cells were further incubated for 4 h to allow XTT formazan production. The absorbance was determined with a microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm.
β-Hexosaminidase release assay
Degranulation of RBL-2H3 cells was evaluated by measuring the activity of the granule-stored enzyme-β-hexosaminidase secreted in the extracellular medium. Cells were cultured in 24-well plates (2 × 105 cells/well) overnight. The cells were sensitized with anti-DNP-IgE (100 ng/mL) for 16 h at 37°C. After washing the cells with TGCM buffer (136 mM NaCl, 2.68 mM KCl, 0.36 mM NaH2PO2H2O, 1 mM CaCl2, 0.5 mM MgCl2, 11.9 mM NaHCO3, 5 mM dextrose, 1 g/L gelatin, pH 7.4), they were pretreated with ALBE (1, 10, 100 μg/mL) for 30 min and then treated with DNP-BSA (1 μg/mL) for 30 min at 37°C. Aliquots of the cellular supernatant (15 μL) were transferred to 96-well plates and incubated with 60 μL of substrate (1 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide in citrate 0.05 M, pH 4.5) for 60 min at 37°C. The cells were lysed with 0.1% Triton X-100 before removing the supernatant to measure the total β-hexosaminidase activity. The reaction was stopped by adding 150 μL of Na2CO3-NaHCO3 buffer 0.1 M, pH 10. The absorbance at 405 nm was measured with a microplate reader (Themo Labsystems). The results were presented as the percentage of total β-hexosaminidase content of the cells determined by cell lysis with 0.1% Triton X-100.
NA preparation and mRNA analysis by RT-PCR
Total splenocytes were plated at 3 × 107 cells/mL and treated with ALBE (100 μg/mL) and ConA (3 μg/mL) for 16 h. Total RNA from the treated cells was prepared with the TRIzol Reagent (Invitrogen), according to the manufacturer's protocol, and stored at -70°C until use. For detecting cytokines, including IL-4 and IL-5, total RNA was extracted after stimulation and treatment. The sequences of the primers used in this study were: IL-4 forward, 5'-ATG GGT CTC AAC CCC CAG CTA GT-3'; IL-4 reverse, 5'-GCT CTT TAG GCT TTC CAG GAA GTC-3'; IL-5 forward, 5'-AGC ACA GTG GTG AAA GAG ACC TT-3'; IL-5 reverse, 5'-TCC AAT GCA TAG CTG GTG ATT T-3'; GAPDH forward, 5'-GTG GCA AAG TGG AGA TTG TTG CC -3', and GAPDH reverse, 5'-GAT GAT GAC CCG TTT GGC TCC-3'. Each transcript was quantified as described in the instrument manual and normalized to the amount of GAPDH, a housekeeping gene.
Measurement of cytokine production (IL-4 and IL-5 secretion)
For cytokine immunoassays, total splenocytes were plated at 3 × 107 cells/mL and treated with ALBE (100 μg/mL) and ConA (3 μg/mL) for 16 h. Culture supernatants were collected and the amount of secreted IL-4 and IL-5 was measured using an enzyme-linked immunosorbent assay (ELISA) using the protocol supplied by BD Biosciences.
Cytosolic and nuclear extracts were prepared. In brief, splenocytes (5 × 107 cells/mL) were plated into 100-mm dishes and treated with ALBE (100 μg/mL) and ConA (3 μg/mL) for 4 h. The harvested cells were resuspended in 0.2 ml of buffer A (10 mM HEPES at pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 1 mM DDT, 0.1% NP-40, 0.2 mM PMSF). The cells were lysed on ice for 15 min, and centrifuged (5,000g, 5 min, 4°C). The supernatant was collected as cytosolic extracts. The nucleic pellet was washed with buffer A lacking NP-40, and resuspended in 0.025 ml of buffer C (20 mM HEPES, pH 7.5, 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DDT, 0.2 mM PMSF). After incubation on ice for 30 min, nuclear debris was spun down (13,000g, 10 min, 4°C). The supernatant was collected as nuclear extracts. The protein concentration was measured using a protein assay kit (Bio-Rad).
Total splenocytes were plated at 3 × 107 cells/mL and treated with ALBE (100 μg/mL) and ConA (3 μg/mL) for 15 min and then harvested and lysed in a lysis buffer containing 20 mM Tris, pH 7.6, 150 mM NaCl, and 1% Triton X-100 with a protease inhibitor cocktail. Protein contents were measured using a protein assay kit (Bio-Rad). Samples were diluted with 1 × lysis buffer containing 1% β-mercaptoethanol. Equal amounts of cellular protein (50 μg) were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking, membranes were incubated with the target antibody and then with horseradish peroxidase-conjugated secondary antibody to IgG. Immunoreactive proteins were visualized using the ECL Western blot detection system. The protein level was compared to a loading control, such as β-actin or non-phosphorylated protein.
Each experiment was repeated three or four times, and the results of a representative experiment are shown. The results are expressed as the means ± SEM and were compared using Student's t-test. A statistical probability of p < 0.05 was considered significant (# p < 0.05, ## p < 0.01, * p < 0.05, and ** p < 0.01).