Recombinant human IL-1β was purchased from R&D Systems (Minneapolis, MN, USA) and kaempferol were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA) and dissolved in DMSO with a concentration of 100 mM stock solution. Monoclonal antibodies (mAbs) against COX-2, MMP-1, MMP-3 and TIMP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). mAbs against NF-κB (p65), IkBα, ERK, p-ERK, JNK, p-JNK, p38, p-p38 and β-actin were purchased from Cell Signaling Technology (Beverly, MD, USA). Fetal bovine serum (FBS) was obtained from Gibco BRL/Life Technologies (Grand Island, NY, USA).

Synovial tissues were obtained at the time of total knee arthroplasty from patients who fulfilled the American College of Rheumatology Criteria for RA (13), as previously described (14). Synovial tissue was digested for 2 h with 0.25% (w/v) collagenase and was then suspended in RPMI-1640 medium with 10% (v/v) FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin. The cells were incubated at 37°C in 5% CO₂ for several days, after which the non-adherent cells were removed. 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 an inverse microscope. The purity of the cells was determined 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, USA). Informed consent was obtained from all patients, and the study protocol was approved by the Chonbuk National University Hospital Ethics Committee.

Cell viability was determined by using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) according to the manufacturer’s instructions. CCK-8 allows convenient assays using Dojindo’s tetrazolium salt, 2-(2- methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt (WST-8), which produces a water-soluble formazan dye upon bioreduction in the presence of an electron carrier, 1-methoxy phenazinium methylsulfate (PMS) (15). CCK-8 solution is added directly to the cells; no pre-mixing of the components is required. CCK-8 is a sensitive non-radioactive colorimetric assay for determining the number of viable cells in cell proliferation and cytotoxicity assays. WST-8 is bioreduced by cellular dehydrogenases to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells. RASFs [1×10⁵ cells/well in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) FCS in a 96-well U-bottom plate] were cultured in 200 μl medium/ well in the presence or absence of 1.0 ng/ml IL-1β and/or kaempferol (100 μM) for 2 days according to the results of a dose-dependent examination and a previous report (15), while the control RASFs were incubated in DMEM with DMSO. CCK-8 (20 μl) was added to each well of the plate and the cells were incubated for 2–3 h. The absorbance was measured at 450 nm using a microplate reader to determine the cell viability in each well.

To evaluate the effects of kaempferol on the apotosis of RASFs, RASFs were incubated in DMEM for 24 h with kaempferol (100 μM), while the control RASFs were incubated in DMEM with DMSO. The cells were then trypsinized and collected for the detection of apoptosis with an Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s instructions. Briefly, the cells were washed twice with cold PBS and resuspended in 500 ml of binding buffer (10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a concentration of 0.5×10⁵ cells/ml. After the addition of 5 μl of Annexin V-FITC solution and propidium iodide (PI; 5 μl), the cells were incubated for 15 min at room temperature. The cells were analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA).

Total RNA was extracted from the cultured RASFs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA (1 μg) was reverse-transcribed using the Maxime RT Premix kit (iNtRON Biotechnology, Seoul, Korea). cDNA was amplified using the following primer sets: MMP-1 sense, 5′-GAA GGA GAT GAA GCA GCC CAG ATG T-3′ and 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′ and antisense, 5′-GGA ACC GAG TCA GGA CTA TG-3′; TIMP-1 sense, 5′-CCT TCT GCA ATT CCG ACC TCG TC-3′ and antisense, 5′-CGG GCA GGA TTC AGG CTA TCT GG-3′; COX-2 sense, 5′-TCC TTG CTG TTC CCA CCC ATG-3′ and antisense, 5′-CAT CAT CAG ACC AGG CAC CAG-3′; GAPDH sense, 5′-AAA TCA AGT GGG GCG ATG CT-3′ and antisense, 5′-AGC TTC CCG TTC AGC TCA GG-3′. PCR products were electrophoresed 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 software v3.0 (Fujifilm, Tokyo, Japan).

RASFs (1×10⁶ cells) were seeded on 100-mm culture dishes and harvested in phosphate-buffered saline (PBS). After washing with PBS, cell pellets were lysed with the lysis buffer [20 mM HEPES, pH 7.2, 1% Triton X-100, 150 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA and 1 μg/ml aprotinin]. Following incubation for 30 min at 4°C, cellular debris was removed by centrifugation at 100,000 × g for 30 min and the supernatants were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To determine membrane COX-2 expression in RASFs, cell membranes were prepared from isolated RASFs as previously described (16). To determine NF-κB (p65) expression, nuclear extracts were prepared using a previously described method (14). To determine cytoplasmic IkBα expression, cytoplasmic extracts were prepared as previously described (14).

Protein concentration was determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Samples (50 μg) were prepared with 4 vol 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 on a 10% slab gel. Proteins were transferred onto a nitrocellulose membrane. 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 antibodyies (10 μg/ml) against MMP-1, MMP-3, TIMP, COX-2, ERK, p-ERK-1/2, p-38, p-p38 MAPK, JNK, p-JNK, NF-κB (p65), IkBα and β-actin were then added followed by incubation at 4°C for 4 h. The secondary HRP-conjugated antibody was goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.). Reactive proteins were detected by enhanced chemiluminescence (ECL; Amersham Life Sciences, Arlington Heights, IL, USA) using Fujifilm LAS-3000 (Fujifilm).

RASFs (1×10⁴ cells) were grown in 25 cm² tissue-culture flasks for 48 h before treatment. After washing with PBS (pH 7.4), cells were pre-treated with IL-1β (1.0 ng/ml) or kaempferol (100 μM) at 37°C for 48 h in DMEM containing 10% (v/v) FCS in an atmosphere of 5% CO₂, while the control RASFs were incubated in DMEM with DMSO. The culture supernatant described above was collected at 2 days. The level of PGE2 in the medium was determined by ELISA (R&D Systems) in accordance with the instructions of the manufacturer.

All data are expressed as the means ± SD of the results of 3 experiments with different RASFs and all data were analyzed using SPSS 12.0 software. Group mean values were compared using the Student’s t-test or ANOVA where appropriate. P-values <0.05 were considered to indicate statistically significant differences.

The effect of kaempferol on the growth ability of RASFs was initially evaluated. Cell proliferation was measured following treatment with IL-1β for 3 days; IL-1β is a well known potent growth-promoting factor for RASFs (17). Cell proliferation was assayed as described in Materials and methods. IL-1β increased the proliferation of RASFs in a dose-dependent manner (from 0.1 to 10 ng/ml). To examine the effects of kaempferol on the IL-1β-induced proliferation of RASFs, kaempferol (100 μM) was added to the RASF cultures with/without IL-1β (1.0 ng/ml) for 2 days and the CCK-8 assay was performed. IL-1β significantly increased the proliferation of RASFs compared with the control cells cultured in DMSO without IL-1β and kaempferol (P<0.05) (Fig. 1A). Kaempferol significantly inhibited the proliferation of RASFs treated with or without IL-1β (P<0.05). Various doses of kaempferol (10, 50, 100, 200 μM) were added to the RASF cultures with IL-1β (1.0 ng/ml) for 2 days and CCK-8 assay was performed. The inhibitory effects of kaempferol were significantly enhanced in a dose-dependant manner (Fig. 1B).

To elucidate the underlying mechanisms by which kaempferol inhibits the IL-1β-induced proliferation of RASFs, the effects of kaempferol on the apoptosis of RASFs were examined by flow cytometry and staining with Annexin V and PI. The percentage of Annexin V-positive cells was significantly increased in the RASFs treated with kaempferol compared with the cells cultured in DMEM with DMSO without kaempferol (P<0.05) (Fig. 2).

RT-PCR was performed to determine the mRNA expression of MMP-1, MMP-3 and TIMP-1 in the monocultured RASFs. RASFs were stimulated with IL-1β (1.0 ng/ml) for 48 h in the presence or absence of kaempferol (100 μM). IL-1β enhanced the mRNA expression of MMP-1 and MMP-3 in RASFs (P<0.05), but not that of TIMP-1. Kaempferol inhibited the effects of IL-1β on the mRNA expression of MMP-1 and MMP-3 (P<0.05) (Fig. 3A). IL-1β also enhanced the mRNA expression of COX-2 in RASFs (P<0.01), but not that of COX-1 (data not shown). Kaempferol inhibited the IL-1β-induced COX-2 mRNA expression (P<0.05) (Fig. 3A). Kaempferol also significantly decreased the mRNA expression of MMP-1, MMP-3 and COX-2 compared with the control cells cultured in DMSO without IL-1β and kaempferol (P<0.05).

To determine the protein expression of MMPs, TIMP-1 and COX in the monocultured RASFs, we performed western blot analysis. RASFs were stimulated with IL-1β (1.0 ng/ml) for 48 h in the presence or absence of kaempferol (100 μM). IL-1β enhanced the protein expression of MMP-1 and MMP-3 in RASFs (P<0.05), but not that of TIMP-1; its effects on protein expression were similar to those on mRNA expression. Kaempferol inhibited the IL-1β-induced protein expression of MMP-1 and MMP-3 (P<0.05) (Fig. 3B). IL-1β also enhanced the protein expression of COX-2 in RASFs (p < 0.01), but not that of COX-1 (data not shown). Kaempferol inhibited the IL-1β-induced protein expression of COX-2 (P<0.05) (Fig. 3B). Kaempferol also significantly decreased the protein expression of MMP-1, MMP-3 and COX-2 compared with the control cells cultured in DMSO without IL-1β and kaempferol (P<0.05).

To confirm the effects of kaempferol on the IL-1β-induced production of PGE2 by RASFs, we examined the concentration of PGE2 in the culture supernatant. RASFs (1×10⁴ cells) were grown in 25 cm² tissue-culture flasks for 48 h before and after treatment with IL-1β (1.0 ng/ml) and/or kaempferol (100 μM). PGE2 production was increased following treatment with IL-1β (P<0.05) compared with the control cells cultured with DMSO without IL-1β, and it was significantly inhibited by treatment with kaempferol at 48 h (P<0.05) (Fig. 4); the effects of kaempferol on PGE2 were similar to those on COX-2 expression. Kaempferol also significantly decreased the production of PGE2 compared with the control cells cultured in DMSO without IL-1β and kaempferol (P<0.05) (Fig. 4).

To demonstrate the involvement of the signal transduction pathways and to elucidate the mechanisms behind the effects of kaempferol on IL-1β-induced RASF proliferation and the production of MMPs, COX-2 and PGE2, the activation of MAPKs and NF-κB was evaluated in the RASFs. IL-1β phosphorylated intracellular MAPKs, including ERK, p-38 and JNK and kaempferol significantly inhibited the IL-1β-induced intracellular MAPK activation (Fig. 5A). The activation of NF-κB, p65 and the degradation of cytoplasmic IkBα was observed in the RASFs treated with IL-1β. The effects of IL-1β on NF-κB activation were abrogated by kaempferol (Fig. 5B). Kaempferol also inhibited the activation of the above intracellular signaling pathways compared with the control cells cultured in DMSO without IL-1β and kaempferol. These results indicate that kaempferol inhibits the IL-1β-induced proliferation of RASFs, the expression of COX-2 and the production of PGE2 by inhibiting the activation of intracellular MAPK and NF-κB pathways.