To determine whether triptolide had an influence on the viability of RSF, we first evaluated the effects of various anti-rheumatic drugs on cell viability using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. Triptolide caused a marked decrease of cell viability in a concentration-dependent manner (Figure 1), while bucillamine, D-penicillamine, methotrexate, sulphasalazine, and sodium aurothiomalate did not decrease viability at a concentration of 10 μM. The mean (± S.D.) value of 50% inhibitory concentration (IC₅₀) of 6 independent experiments was 74.3 ± 9.5 nM.

Since triptolide reduced the viability of RSF, we also examined its effect on cell proliferation (DNA synthesis) by measuring the incorporation of 5-bromo-2'-deoxyuridine (BrdU) (Figure 2) using an enzyme-linked immunosorbent assay (ELISA). Triptolide caused marked suppression of cell proliferation in a concentration-dependent manner and it was almost completely inhibited at 100 nM. In contrast, the other drugs that were tested, such as bucillamine, D-penicillamine, and methotrexate, did not suppress the proliferation of RSF. The mean (± S.D.) value of IC₅₀ of 7 independent experiments was 20.4 ± 2.4 nM.

To determine whether the decrease of viability and suppression of proliferation when RSF were treated with triptolide was related to apoptosis, we examined the effect of triptolide on DNA fragmentation (a hallmark of apoptosis). An ELISA that specifically detected cytoplasmic histone-associated DNA fragments, mononucleosomes and oligonucleosomes, was used to quantitatively analyze DNA fragments. As shown in Figure 3, triptolide induced DNA fragmentation in cultured RSF, while bucillamine, D-penicillamine, and methotrexate did not affect cellular DNA. The mean (± S.D.) value of triptolide concentration at a half of maximum fold induction of 5 independent experiments was 35.9 ± 16.3 nM.

Cell morphology was observed with a light microscope after treatment of RSF with triptolide (Figure 4). When synovial cells were incubated with triptolide (100 nM) for 24 h, distinctive morphological changes occurred, such as cellular rounding, shrinkage, and membrane blebbing, and the cells became separated from neighbouring cells (panel b). The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay was used to confirm the apparent pro-apoptotic effect of triptolide. As shown in panel d, TUNEL-positive cells were observed after incubation with 100 nM triptolide for 24 h, accounting for 66 % of the cultured RSF. These results suggested that triptolide caused a decrease of RSF proliferation by the induction of apoptosis.

Caspases are responsible for many of the biochemical and morphological changes that occur during apoptosis, so we investigated whether triptolide induced the activation of caspase-3 in RSF. As shown in Figure 5, incubation of RSF with 100 nM triptolide for 24 h induced the activation of caspase-3, while activation was completely blocked by incubation with the pan-caspase inhibitor Z-VAD-FMK. We then examined effects of caspase inhibitors on triptolide induced-apoptosis in RSF. Triptolide-induced DNA fragmentation in RSF was suppressed by Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK, inhibitors of caspases-3, -8 and -9, respectively, in concentration dependent manner (Figure 6).

To explore whether proliferator-activated receptor (PPAR)γ activation was involved in the apoptotic effect of triptolide, we performed a luciferase reporter gene assay by co-transfection of RSF with a peroxisome proliferator response element (PPRE)-driven luciferase reporter plasmid and a PPARγ expression plasmid (Figure 7). Incubation with 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂), a PPARγ ligand, significantly induced PPRE-driven luciferase activity in this system. In contrast, triptolide had little influence on PPARγ activation despite causing the apoptosis of RSF.

Since triptolide was regarded as a major active compound of GTW, an extract of TWHF, we examined whether GTW induced apoptosis in RSF. As show in Figure 8, GTW induced DNA fragmentation in RSF at 10 to 30 μg/mL. With GTW at 50 μg/mL, the cells had undergone secondary (type 2) necrosis, which usually occurs after apoptosis during in vitro experiments.

RPMI-1640 medium, penicillin-streptomycin solution, fetal bovine serum (FBS), and trypsin-EDTA were obtained from Gibco BRL (Gaithersburg, MD, USA). Triptolide was purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA). 15dPGJ₂ was obtained from Cayman Chemical (Ann Arbor, MI, USA). Bucillamine, D-penicillamine, and salazosulfapyridine (sulphasalazine) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Sodium aurothiomalate was obtained from Shionogi & Co., Ltd. (Osaka, Japan). Z-DEVD-FMK (a caspase-3 inhibitor), Z-IETD-FMK (a caspase-8 inhibitor) and Z-LEHD-FMK (a caspase-9 inhibitor) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). GTW powder was kindly provided by Taizhou Pharmaceutical Co. (Jiang Su, China). All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

RSF were prepared from synovial tissues, as described previously 36. The synovial tissues were obtained during total knee replacement from RA patients who fulfilled the revised American Rheumatism Association criteria 37. Mean (± S.D.) age of 18 patients (14 female) with RA were 64.3 ± 5.8. The protocol for this study was approved by the ethics committee of St. Marianna University and all patients gave written consent to the use of their tissues for this research. Synovial tissues were digested for 2 h with 0.2 % (w/v) bacterial collagenase (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan) and then the synovial cells were suspended in RPMI-1640 medium with 10 % (v/v) FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were incubated at 37°C in 5 % CO₂ for several days, after which nonadherent cells were removed. The fibroblast-like adherent cells were used as RSF within 2 passages. These adherent cells contained no T cells (CD3⁺) or macrophage/monocytes (CD14⁺) on two-colour immunofluorescence and flow cytometry. All culture condition of RSF in RPMI-1640 medium containing 1 % (v/v) FBS under an atmosphere of 5 % CO₂.

Test drugs except GTW were dissolved in dimethyl sulfoxide (DMSO) as 1000 × stock solutions and then diluted with RPMI-1640 medium containing 1 % (v/v) FBS for cell culture experiments. GTW powder was dissolved in ethanol, filtrated, and then stored at -20°C. Test drug solutions were prepared freshly on the day of use. The final concentration of DMSO or ethanol (just for GTW study) for all treatments (including control cultures) was 0.1 % (v/v).

RSF (2 × 10⁴ cells/well) were incubated in 96-well plastic plates with test drugs in the condition described above. After 24 h, cell viability was assessed by measuring mitochondrial NADH-dependent dehydrogenase activity with a Cell Counting Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) using a sulfonated tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt, (WST-1) 38. Each measurement was done in triplicate and the results are presented as a percentage of the value for untreated control cultures.

The proliferation of RSF was assessed from the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Cells (1 × 10⁴ cells/well) were incubated in 96-well plastic plates with the test drugs in the condition described above. Then BrdU (10 μM) was added to the medium and the cells were incubated for another 18 h. Subsequently, the cells were fixed and BrdU incorporation was determined with a Cell Proliferation ELISA Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. Each measurement was done in triplicate and the results are presented as a percentage of the value for the untreated control culture.

DNA fragmentation was detected by ELISA and by TUNEL assay. RSF were plated in 96-well plastic plates (2 × 10⁴ cells/well) and cultured for 24 h. Then the cells were incubated for a further 24 h with test drugs in the condition described above. After incubation, DNA fragmentation was investigated using a Cell Death Detection ELISA^PLUS kit (Roche Diagnostics GmbH) 39. Each measurement was done in triplicate and the results are presented as the fold induction compared with untreated control cultures.

RSF (2.5 × 10⁴ cells/well) were incubated in 8-well chamber slides (Iwaki, Chiba, Japan) for 24 h with triptolide in the condition described above. Then morphological changes of the cells were observed under a microscope (BX51, Olympus Optical Co., Ltd., Nagano, Japan), after which the cells were fixed with 4 % (v/v) neutral buffered formalin for 10 min at room temperature. Subsequently, apoptotic synovial cells were identified by the TUNEL assay using an Apoptosis in situ Detection Kit (Wako Pure Chemical Industries) according to the manufacturer's instructions. Sections were counterstained with methylgreen (Wako Pure Chemical Industries) before observation.

RSF were incubated in tissue culture flask with or without triptolide and/or Z-VAD-FMK (a pan-caspase inhibitor) in the condition described above. After 24 h, caspase activity in the cells was measured using the CaspACE™ assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Cells were washed in ice-cold phosphate-buffered saline (PBS, 9.57 mM, pH 7.35–7.65, Takara Shuzo, Shiga, Japan), and resuspended in cell lysis buffer. After the cells were lysed by freezing and thawing, the lysates were centrifuged and the supernatant was used as the cell extract. The protein content of the extracts was determined by the BCA protein assay (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as the standard, and extracts were adjusted to a protein content of 1 mg/ml.

A luciferase reporter plasmid, containing 4 copies of the PPRE of the acyl-CoA oxidase gene promoter 40 at the Nhe I restriction site in the firefly luciferase expression vector PGV-P2 (Toyo Ink, Tokyo, Japan), was used to measure PPARγ activation 34. RSF (6 × 10⁴ cells/well) were seeded into 24-well culture plates in RPMI-1640 medium containing 10 % (v/v) FBS. After culture for 24 h at 37°C under an atmosphere of 5 % CO₂, the cells were co-transfected with the reporter plasmid (0.1 μg/well), with a PPARγ expression plasmid containing mouse PPARγ 2 cDNA 41 at the Hind III and Xba I restriction sites in the pRc/CMV expression vector (Invitrogen, Groningen, The Netherlands) (0.1 μg/well), and with an internal control plasmid (pRL-SV40; Promega) (0.01 μg/well) using Effectene Transfection Reagent (12 μg/mL, Qiagen, Hilden, Germany) according to the manufacturer's instructions. After incubation for a further 24 h at 37°C under an atmosphere of 5 % CO₂, the transfection mixture was replaced by RPMI-1640 medium containing 1% (v/v) FBS with or without one of the test drugs. After additional incubation for 18 h in the condition described above, luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA) according to the manufacturer's instructions. Each measurement was done in triplicate and firefly luciferase activity was normalized to Renilla luciferase activity.

Data are expressed as the mean ± S.D. Statistical analysis was done using multiple Student's t-tests with a Bonferroni's correction. A probability value of less than 0.05 was taken to indicate a significant difference.