Chemicals and reagents
Dulbecco's Modified Eagle Medium (DMEM)-1640, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Hyclone (Logan, USA). Escherichia coli lipopolysaccharide (LPS) was purchased from sigma (St. Louis, USA). β-actin, i-NOS, COX-2, p65, p-IκBα, PE-B7-1, and FITC-B7-2 anti-bodies were purchased from BD Pharmingen™ (San Jose, USA). Enzyme immunoassay kits for the measurement of PGE2, IL-1β, IL-6, and TNF-α were purchased from R&D system (Minneapolis, USA).
Isolation of arctiin from Forsythiae Fructus
Forsythiae Fructus obtained from an herbal market in Seoul, Korea, was extracted three times with hot MeOH (3 hours) and then evaporated at 40°C under reduced pressure to dryness. The MeOH extract was then resuspended in distilled water and successively partitioned with CHCl3, EtOAc, and n-BuOH. The BuOH fraction was then loaded onto a silica gel column and eluted with MeOH-CHCl3 mixtures (1:5 to 1:1). The result was white amorphous powders, which were identified as authentic samples using spectrometric data of nuclear magnetic resonance (1H-NMR), mass spectrometry (MS). 1H-NMR (300 MHz), DMSO-d6 : δ 6.97(1H, d, J = 8.3, H-5), 6.82 (1H, d, J = 8.0, H-5'), 6.77 (1H, d, J = 1.7, H-2), 6.65 (1H, dd, J = 8.3, 1.7, H-6), 6.65 (1H, s, H-2'), 6.59 (1H, dd, J = 8.0, 1.8, H-6'), 4.83 (1H, d, J = 7.3, H-1''), 4.00 (2H, m, H-9'), 3.70 (3H, s, OCH3), 3.69 (6H, s, OCH3), 2.76 (2H, m, H-7'), 2.54 (4H, m, H-7, 8, 8'). Positive FABMS (m/z): 557 (M + Na+), 372 (M + gluco), 154, 136. The white amorphous powder compound analyzed by NMR and MS was identified to arctiin (Figure 1A).
ICR mice (6-8 weeks old, specific pathogen-free) were obtained from Orient-Bio Co. (Seongnam, Korea). Animals were fed with standard laboratory chow (PMI Lab Diet, Richmond, USA) and autoclaved distilled water (DW). They were acclimatized in an animal facility (Sahmyook University, Korea) and maintained at 22 ± 2°C in 50 ± 10% relative humidity and a light/dark (12 hrs/12 hrs) cycle for at least 7 days prior to the experiments.
Male ICR mice (6-8 weeks) were intraperitoneally injected with 1.5 ml thioglycollate broth for recruitment of macrophages. RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Rockville, USA). These cells were grown at 37°C in DMEM medium supplemented with 10% FBS and 1% (v/v) penicillin (10,000 U/ml)/streptomycin (10,000 U/ml) in a humidified 5% CO2-95% air incubator under standard conditions.
Cell viability assay
A commercially available cell viability assay was employed to evaluate the cytotoxic effect of arctiin using thiazolyl blue tetrazolium bromide (SIGMA, USA). RAW264.7 cells (2 × 105 cells/well) were plated with various concentrations of arctiin in 96-well microtiter plates (Nunc, Roskilde, Denmark) and were then cultured at 37°C in a 5% CO2 incubator. Subsequently, 50 μl of MTT solution was added to each well, and the cells were then cultured for 4 hrs at 37°C in the same incubator. Following this, 100 μl of solubilized solution were added to each well and the plate was allowed to stand overnight in the incubator. The optical density (OD) was then measured at 560 nm by a microplate reader (Molecular devices, USA).
RAW 264.7 cells were added to each well (200 μl; 1 × 106 cells/ml) of a flat-bottomed 96-well plate according to the following treatment condition: LPS (100 ng/ml), LPS/arctiin (12.5, 25, 50, 100 μg/ml), and media only (DMEM-10). Nitric oxide was measured in culture supernatants by reaction with the Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthy]-ethylenediamine dihydrochloride in 5% phosphoric acid; Roche) to 100 μl of culture supernatant for 15 min at room temperature in the dark. The absorbance at 540 nm was then determined using a microplate reader (Molecular devices, USA) and a standard curve was generated using NaNO2.
Determination of pro-inflammatory cytokines and PGE2
RAW 264.7 cells and primary macrophages were cultured in 12-well flat plates at a density of 5 × 106 cells/well. The cells were then treated with various concentrations of arctiin and subsequently stimulated with LPS (100 ng/ml) at 37°C for 48 hrs in humidified air with 5% CO2. The supernatants were then collected and measured for TNF-α, IL-1β, IL-6, and PGE2 by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol.
RT-PCR (reverse transcription polymerase chain reaction)
Total RNA was extracted from macrophages using the RNeasy Mini Kit (QIAGEN, USA) in an RNase-free environment. The reverse transcription of 1 μg RNA was carried out using M-MLV reverse transcriptase (Promega, USA), oligo (dT) 16 primer, dNTP (0.5 mM) and 1 U RNase inhibitor. After incubation at 65°C for 5 min and 37°C for 60 min, M-MLV reverse transcriptase was inactivated by heating at 70°C for 15 min. The polymerase chain reaction (PCR) was performed in 50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl2, and 2.5 mM dNTPs with 5 units of Taq DNA polymerase and 10 pM of each primer set for IL-1β, IL-6, TNF-α, iNOS, and COX-2. The cDNA was amplified by 35 cycles of denaturing at 94°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 1 min. Final extension was performed at 72°C for 5 min. The PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The primer sequences were as follows: 5'- AGC TCC TCC CAG GAC CAC AC-3' (forward), 5'-ACG CTG AGT ACC TCA TTG GC-3' (reverse) for i-NOS, 5'-AAG AAG AAA GTT CAT TCC TGA TCC C-3' (forward), 5'-TGA CTG TGG GAG GAT ACA TCT CTC-3' (reverse) for COX-2, and 5'-GTG GGC CGC CCT AGG ACC AG-3' (forward), 5'- GGA GGA AGA GGA TGC GGC AG T-3' (reverse) for β-actin as a control for PCR. The band intensity was quantified by densitometric analysis (Infinity 3026, Vilber Lourmat, France).
Preparation of cytosolic and nuclear extracts
The cells were collected after culture and washed twice with cold PBS, resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.2 mM PMSF, 0.5 mM DTT, 10 μg/ml aportinin). After 15 min incubation on ice, the cells were lysed by the addition of 0.1% NP-40 and vigorous vortexing for 1 min. The nuclei were pelleted by centrifugation at 12,000 × g for 1 min at 4°C and resuspended in high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 400 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate). The cytosolic and nuclear extracts were stored in aliquots at -70°C.
Western blot analysis
RAW264.7 cells were washed with phosphate-buffered saline (PBS) and lysed using lysis buffer (1% SDS, 1.0 mM sodium vanadate, 10 mM Tris-Cl buffer, pH 7.4) for 5 min. Further, 20 μg of protein from the cell lysates were applied to 8-12% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were blocked in 5% skim milk solution for 1 h. They were then incubated with anti-TNF-α, anti-IL-1β, anti-IL-6, anti-iNOS, anti-COX-2, anti-p-IκBα, or anti-p65 monoclonal antibodies for 2 h and subsequently washed 3 times with PBS. After incubation with an AP-labeled secondary antibody for 2 h, the bands were visualized using an alkaline phosphatase substrate (VECTOR, USA).
RAW 264.7 cells (1 X 106 cells/ ml) were cultured in Petri-dishes. The cells were treated with various concentrations (12.5, 25, 50, 100 μg/ ml) of arctiin in the presence of LPS (100 ng/ ml). The dishes were incubated at 37°C for overnight in humidified 5% CO2 incubator under standard conditions. The cells were then washed with PBS. The washed cells were blocked with staining buffer containing 10% normal mouse serum (NMS) for 20 min on ice. The blocked cells were incubated with co-stimulatory molecules such as B7-1 and B7-2 antibody (BD Biosciences, San Jose, USA) for 20 min on ice. The incubated cells were washed three times with staining buffer and then fixed by 1% paraformaldehyde in PBS. The fixed cells were measured by flow cytometry (Beckman coulter, Brea, USA).
All data are presented as mean ± SEM values. Significant differences (P < 0.05) between groups were evaluated using a one-way analysis of variance with SPSS (Chicago, IL, USA) for Windows and Duncan's Multiple Range Test where appropriate.