Preparation of crude extract of Angelica sinensis
AS was collected from Jiri Mountain in South Korea and plant material was identified by an authority at the Rheumatology Laboratory and Research Center for Pulmonary Diseases, Chonbuk National University Medical School, Jeonju, Jeonbuk, South Korea. In total, 10 g of AS was extracted with 300 ml of 95% ethanol at 50°C for 3 hours twice. The total crude extract was evaporated under vacuum to yield a residue, and then the residue was suspended in 90% ethanol and successively partitioned with hexane, benzene, trichloromethane, ethyl acetate, n-butanol and water fractions, consecutively as described [21]. The ethyl acetate fraction was used in this study after well drying the fraction. And we used kaempferol 0.852 ug and decursin 2.116 ug in 1 mg/mL of extract by quantifying the content.
Reagents and antibodies
Recombinant human IL-1β was purchased from R&D system (Minneapolis, Minnesota, USA). Monoclonal antibody (mAb) against MMP-1, MMP-3, tissue inhibitor of metalloproteinase (TIMP), and COX-2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). mAb against extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), C-jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), p38, p-p38, nuclear factor κB (NF-κB) (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) and β-actin were purchased from Cell Signaling (Beverly, MD, USA). Fetal bovine serum (FBS) was obtained from Gibco BRL (Life Technologies, Grand Island, NY, USA).
Isolation and culture of RASFs
Synovial tissues obtained at the time of total knee arthroplasty in patients who fulfilled the American College of Rheumatology Criteria for RA [22], as previously described [23]. Synovial fibroblasts from passages 3–7 were used for each experiment and were morphologically homogeneous and had the appearance of RASFs with typical fibroblastoid configuration under inverse microscopy. The purity of the cells was tested by flow cytometry using phycoerythrin (PE)-conjugated anti-Thy-1 (CD90) or anti-CD14 and fluorescein isothiocyanate (FITC)-conjugated anti-CD3 mAb (BD Pharmingen, San Diego, CA). Informed consent was obtained from all patients, and the study protocol was approved by the Chonbuk National University Hospital Ethical Committee.
RNA Isolation and semiquantitative RT-PCR of COX, MMPs and TIMP
Total RNA was extracted from cultured cells using the TRIsol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. One microgram of RNA was reverse-transcribed using Maxime RT Premix Kit (iNtRON Biotechnology, Korea). cDNA was amplified using the following primer sets: COX-1 (sense) 5′-GCT ATT CCG GCC CCA ACT-3′ (antisense) 5′-GAT GAA GGT GGC ATT GAC AAA CT-3′, COX-2 (sense) 5′-TCC TTG CTG TTC CCA CCC ATG-3′ (antisense) 5′-CAT CAT CAG ACC AGG CAC CAG-3′, MMP-1 (sense) 5′-GAA GGA GAT GAA GCA GCC CAG ATG T-3′ (antisense) 5′-CAG TTG TGG CCA GAA AAC AGA AGT GAA A-3′, MMP-3 (sense) 5′GAC ACC AGC ATG AAC CTT GTT-3′ (antisense) 5′-GGA ACC GAG TCA GGA CTA TG-3′, TIMP-1 (sense) 5′-CCT TCT GCA ATT CCG ACC TCG TC-3′ (antisense) 5′-CGG GCA GGA TTC AGG CTA TCT GG-3′, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sense) 5′-ACC ACA GTC CAT GCC ATC AC-3′ (antisense) 5′-TCC ACC ACC CTG TTG CTG TA-3′. PCR products were electrophoresed by using 1% agarose gels and visualized by staining with ethidium bromide. Densitometric analysis was performed on the relative intensity of each band using the Multi Gauge program, version 3.0 (Fuji film, Tokyo, Japan).
Cell viability analysis
Cell viability was determined by a cell counting kit-8 (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions. Briefly, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitropenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (CCK-8) was reduced by dehydrogenases in cells to yield an orange-colored product (formazan) [24]. The amount of the formazan dye generated by dehydrogenases in cells was directly proportional to the number of living cells. RASFs (1 × 105 cells per well in complete RPMI-1640 media in a 96-well plate) were cultured in 200 mL medium per well without antigen stimulation in the presence or absence of 100 μg/mL EAAS for 2 days. CCK-8 (20 mL) was added to each well of the plate and the cells were incubated for 2–3 hours. The absorbance was measured at 450 nm using a microplate reader.
Assay of PGE2 production
RASFs were grown in 25 cm2 tissue-culture flasks for 48 hours before treatment. After washing with PBS (pH 7.4), cells were pretreated with IL-1β (1.0 ng/mL) or EAAS (100 μg.mL) at 37°C for 48 hours in DMEM containing 10% (v/v) FCS in an atmosphere of 5% CO2. The culture supernatant described above was collected at 2 days. The level of PGE2 in the medium was determined by ELISA kit (R&D Systems) in accordance with the instructions of the manufacturer.
Immunoblotting
RASFs (1 × 106 cells) were seeded on 100-mm culture dishes and harvested in phosphate buffered saline (PBS) after stimulation as described above. Cells were lysed in lysis buffer containing 50 mM Tris-CL, 150 mM NaCl, 5 mM EDTA 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitors. To determine the membrane COX-2 expression on RASFs, cell membranes were prepared from isolated RASFs, as described previously [25]. To analyze NF-kB (p65), nuclear extract was prepared using a previously described method [23]. To determine the cytoplasmic IkBa, cytoplasmic extracts were prepared from isolated RASFs as described previously [23]. The protein concentration was determined by the Bio-Rad protein assay regent (Bio-Rad Laboratories, USA). Samples (50 mg) were prepared with the four volume of 0.5 M Tris buffer (pH 6.8) containing 4% SDS, 20% glycerol and 0.05% bromophenol blue at 95°C for 5 min. SDS-PAGE was performed in 10% slab gel. Proteins were transferred to nitrocellulose paper. The membrane was washed in blocking buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 5% fat-free milk) for 60 min at room temperature with shaking and then washed with TBST (TBS, 0.01% Tween 20). Primary antibodies (10 mg/ml) against MMP-1, −3, TIMP-1, −2, COX-1, −2, ERK, p-ERK-1/2, p-38, p-p38 MAPK, JNK, p-JNK, NF-kB (p65), IkBa and β-actin was incubated at 4°C for 4 hr. The secondary HRP-conjugated antibody was goat anti-mouse IgG (Stressgen Bioreagents, Ann Arbor, MI, USA). Reactive proteins were detected using enhanced chemiluminescence (ECL, Amersham Life Sciences, Arlington, IL, USA) using Fuji film LAS-3000 (Tokyo, Japan).
Statistical analysis
All data were expressed as the mean ± SD of triplicates and all data were analyzed by the SPSS 18.0 program. Group mean values were compared by Student’s t test or ANOVA as appropriate. The significance of difference was defined as p values < 0.05, or p values < 0.01.
