Ganghwaljetongyeum, an anti-arthritic remedy, attenuates synoviocyte proliferation and reduces the production of proinflammatory mediators in macrophages: the therapeutic effect of GHJTY on rheumatoid arthritis
© Jeoung et al.; licensee BioMed Central Ltd. 2013
Received: 15 December 2012
Accepted: 25 February 2013
Published: 26 February 2013
Ganghwaljetongyeum (GHJTY), a complex herbal decoction, is used to treat rheumatoid arthritis. However, the action mechanism of GHJTY is not still unclear on rheumatoid arthritis. In this study, we examined the beneficial effects and the action mechanisms of GHJTY on synoviocyte proliferation and inflammatory mediators.
To test the effect of GHJTY on synoviocyte proliferation, HIG-82 cells, rabbit knee synovial membrane cells, were treated with GHJTY under IL-1β. To evaluate the effects of GHJTY on proinflammatory mediators, we tested cytokine levels in RAW264.7 cells.
Proliferation of HIG-82 cells was significantly inhibited by GHJTY treatment. We found that GHJTY caused cytoskeleton damage to HIG-82 cells. In contrast, treatment of GHJTY did not show any cytotoxicity to other different origin cell lines, HeLa and RAW264.7 cells. GHJTY inhibited IL-1β-mediated NF-κB activation in HIG-82 cells and reduced the LPS-mediated production of proinflammatory cytokines, TNF-α, IL-12, and NO in RAW264.7 cells. In addition, the expression of cyclooxygenase in LPS-activated RAW264.7 cells was also decreased by GHJTY treatment.
These results suggest that GHJTY might effectively attenuate rheumatoid arthritis by inhibiting the production of proinflammatory mediators and the proliferation of synoviocytes.
KeywordsGanghwaljetongyeum Rheumatoid arthritis Synoviocytes Inflammation IL-1β NF-κB
Rheumatoid arthritis is a chronic inflammatory disorder accompanied by hyperplasia of the cartilage lining caused by infiltration of inflammatory cells, ultimately resulting in joint damage [1, 2]. One of the most striking features of inflammatory arthritis is hyperplasia of synovial fibroblasts. Synovitis was reported to play an essential role in the pathophysiology of rheumatoid arthritis [3, 4]. The progression of rheumatoid arthritis is accelerated by proinflammatory cytokines and chemokines, and tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are reported to be associated with the progression of rheumatoid arthritis . In response to these cytokines, synovial fibroblasts proliferate vigorously and form pannus tissues, which destroy the cartilage and bone of the joints [6–8]. Nuclear factor-κB (NF-κB) is a transcription factor which regulates gene expression associated with inflammation and is strongly activated by a range of stimuli including TNF-α, IL-1β, LPS, UV light, and oxidative stress . Cyclooxygenase (COX) is strongly induced by IL-1β and plays an important role in the pathophysiology of rheumatoid arthritis . Therefore, down-regulation of proinflammatory cytokines and NF-κB may be an appropriate therapeutic strategy for rheumatoid arthritis. Antiproliferative agents that modulate synoviocyte growth have been suggested to have potential as anti-rheumatic drugs.
Prescription of GHJTY in daily doses
Angelicae Koreanae Radix
Angelica Koreanum (Umbelliferae)
Atractylodes chinensis (Compositae)
Araliae Continentalis Radix
Aralia continentalis (Araliaceae)
Paeonia Radix Rubra
Paeonia obovata (Paeoniaceae)
Stephaniae Tetrandrae Radix
Sinomenium acutum (Menispermaceae)
Clematis mandshurica (Ranunculaceae)
Angelicae Gigantis Radix
Angelica gigas (Umbelliferae)
Poria cocos (Polyporaceae)
Alisma orientale (Alismataceae)
Akebia quinata (Lardizabalaceae)
Citrus unshiu (Rutaceae)
Chaenomeles sinensis (Rosaceae)
Phellodendron amurense (Rutaceae)
Glycyrrhiza uralensis (Leguminosae)
Juncus effuses (Juncaceae)
Gleditsia sinensis (Leguminosae)
Lonicera japonica (Caprifoliaceae)
Taraxacum platycarpum (Compositae)
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.
Results and discussion
GHJTY suppresses the proliferation of HIG-82 cells
GHJTY causes rearrangement of actin cytoskeleton of HIG-82 cells
GHJTY suppresses IL-1β-induced NF-κB activation in HIG-82 cells
GHJTY inhibits LPS-induced production of NO, TNF-α, and IL-12 in RAW264.7 cells
GHJTY suppresses the COX-1 expression in LPS-activated RAW264.7 cells
Rheumatoid arthritis is an autoimmune disease characterized by synovial proliferation, infiltration of lymphocytes and macrophage into the synovial lining, and a paucity of apoptosis . We evaluated the remedial value of GHJTY, a prescription for rheumatoid arthritis, by examining its effect on synoviocyte proliferation and inflammatory responses. First, GHJTY significantly inhibited the proliferation of HIG-82 cells, a rabbit knee synovial membrane cell line (Figure 1A) by causing cytoskeleton damage to the cells (Figure 2). In contrast, GHJTY did not show any cytotoxic effects on other origin cell lines, HeLa and RAW264.7 cells (Figure 1B and 1C). In addition to the inhibitory effect on synoviocyte growth, GHJTY reduced the IL-1β-mediated NF-κB activation in HIG-82 cells (Figure 3). Moreover, GHJTY decreased the LPS-mediated production of proinflammatory cytokines, TNF-α, IL-12, and NO in RAW264.7 cells (Figure 4A, 4B, and 4C). The expression of COX-1 in LPS-activated RAW264.7 cells was also reduced by GHJTY treatment (Figure 5). Consequently, GHJTY was shown to be effective in decreasing the hyperplasia of synovial fibroblasts and inflammatory responses, the most striking features of inflammatory arthritis. These results suggest that GHJTY might effectively attenuate rheumatoid arthritis by inhibiting the production of proinflammatory mediators from macrophage-like cells and the proliferation of synoviocytes.
Nuclear factor- κB
Tumor necrosis factor-α
Nonsteroidal anti-inflammatory drugs
This research was supported by the Grant from Graduate School of Dongshin University (Interdisciplinary of Oriental Medicine, Health & Welfare) in 2011.
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