Preparation of GHJTY extract
Herbal medicines used in this study were purchased from Hwalim Natural Drug Co. Ltd (Busan, Korea) and the origins were described in Table 1. The herbal formula of GHJTY (Table 1) was extracted with hot distilled water at 100°C for 2 hours at Dongshin University Oriental Hospital (Gwangju, Korea). The extract was centrifuged at 1,500 rpm for 20 min and the supernatant was concentrated by evaporation under vacuum with preferably low temperature (EYELA, Japan). The concentrated extract was lyophilized by a vacuum freeze drier at −80°C (Samwon Freezing Eengineering Co., Korea).
A rabbit knee synovial membrane cell line, HIG-82 (American Type Culture Collection, Manassas, VA, USA), was cultured in Ham’s F-12 nutrient mix medium (Ham’s F-12, GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with penicillin-streptomycin and 10% fatal bovine serum (FBS, GIBCO, Invitrogen) under an atmosphere of 5% CO2 in a humidified incubator. HeLa and RAW264.7 cells (Korea Cell LineBank, Korea) were maintained in DMEM (GIBCO, Invitrogen) and RPMI (GIBCO, Invitrogen), respectively under above cell culture conditions.
Effect of GHJTY on cell proliferation
Cells seeded in a 96-well microplate (Corning, NY, USA) at a density of 2 × 104 cells/well were cultured with or without IL-1β (5 ng/ml) (Sigma-Aldrich, Saint Louis, Missouri, USA). Celecoxib (Sigma-Aldrich) was used as a control drug of arthritis. GHJTY or celecoxib was added to the cells for 2 days. Cell proliferation was assayed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), according to manufacturer’s instructions (Promega, Madison, WI, USA). Absorbance was read by an ELISA microplate reader (Power Wavex340, NIO-TEK-INS TRUMENTS, INC) at 490 nm. Proliferation was calculated as the percentage (%) to not-treated cells.
Effect of GHJTY on actin cytoskeleton of HIG-82 cells
HIG-82 cells were seeded in an 8-well glass chamber plate (Nalge Nunc International) at a density of 3 × 104 cells/well and cultured with or without IL-1β (5 ng/ml) in Ham’s F-12 with 10% FBS for 2 days. After washing with HBSS (GIBCO, Invitrogen), the cells were fixed in 3.7% formaldehyde for 15 min, permeabilized with 0.1% Triton X-100 (Sigma), and incubated in a blocking solution of Image iT signal enhancer (Molecular Probes, Invitrogen) for 30 min. F actin was visualized by Alexa Fluor 594-conjugated Phalloidin (Molecular Probes) as described previously . Confocal images of specimens mounted with a ProLong gold antifade reagent with DAPI (Molecular probes) were acquired using a laser scanning confocal microscope (EZ-C1, Nikon).
Effect of GHJTY on IL-1β-induced NF-κB transcription activity in HIG-82 cells
HIG-82 cells seeded in a 96-well plate (Corning) at 1×104/well were transfected with a reporter plasmid, pNF-κB-luciferase  using Hily Max (Dojindo, Japan). One day after transfection, the cells were replaced with fresh Ham’s F-12 containing different concentrations of test agents and were incubated for 6 hours. The cells were treated with lysis buffer (Promega) and luciferase activities were assayed by a luminometer (MicroLumat Plus LB96V; Berthold, Wilbad, Germany).
Effect of GHJTY on the production of nitric oxide (NO) and proinflammatory cytokines in RAW264.7 cells
RAW264.7 cells seeded at 1×105/well in a 48-well plate (Corning) were preincubated with GHJTY or celecoxib for 2 hours. LPS (Sigma) was added to the cells for 20 hours. The levels of cytokines, TNF-α and IL-12 were measured in the cell supernatants using sandwich ELISA kits following the manufacturer’s experimental protocols (Biolegend, USA). Absorbance was read by an ELISA microplate reader (Power Wavex340, NIO-TEK-INS TRUMENTS, INC) at 450 nm. NO in the culture supernatant was measured using Griess Reagent (0.1% N-(1-naphthyl)-ethylendiamine dihydrochoride, 1% Sulfanilamide in 2.5% H3PO4).
Effect of GHJTY on the expression of COX in LPS-activated RAW264.7 cells
RAW264.7 cells were cultured in a 6-well plate (Corning, USA) at 1×106/well overnight. GHJTY or celecoxib was pretreated to the cells for 2 hours and LPS (1 μg/ml) was added for 18 hours. COX protein was detected by Western blot analysis using an antibody specific to COX-1 (Santa Cruz, USA) and a detection kit, Immobilon Western (Millipore, USA). The relative amount of COX-1 protein was analyzed by LAS-4000 mini (Fujifilm, Japan).
The results are expressed as mean ± SEM unless otherwise stated. Statistical differences were evaluated using Student's t-test, with a P value < 0.05 considered significant.