Hyun Mi Choi

Anti-inflammatory and antiarthritic effects of piperine in human interleukin 1β-stimulated fibroblast-like synoviocytes and in rat arthritis models

IntroductionThe objective of this study was to determine the anti-inflammatory, nociceptive, and antiarthritic effects of piperine, the active phenolic component in black pepper extract.MethodsThe in vitro anti-inflammatory activity of piperine was tested on interleukin 1β (IL1β)-stimulated fibroblast-like synoviocytes derived form patients with rheumatoid arthritis. The levels of IL6, matrix metalloproteinase (MMPs), cyclo-oxygenase 2 (COX-2), and prostaglandin E2 (PGE2) were investigated by ELISA and RT-PCR analysis. The analgesic and antiarthritic activities of piperine were investigated on rat models of carrageenan-induced acute paw pain and arthritis. The former were evaluated with a paw pressure test, and the latter by measuring the squeaking score, paw volume, and weight distribution ratio. Piperine was administrated orally to rats at 20 and 100 mg/kg/day for 8 days.ResultsPiperine inhibited the expression of IL6 and MMP13 and reduced the production of PGE2 in a dose dependant manner at concentrations of 10 to 100 μg/ml. In particular, the production of PGE2 was significantly inhibited even at 10 μg/ml of piperine. Piperine inhibited the migration of activator protein 1 (AP-1), but not nuclear factor (NF)κB, into the nucleus in IL1β-treated synoviocytes. In rats, piperine significantly reduced nociceptive and arthritic symptoms at days 8 and 4, respectively. Histological staining showed that piperine significantly reduced the inflammatory area in the ankle joints.ConclusionsThese results suggest that piperine has anti-inflammatory, antinociceptive, and antiarthritic effects in an arthritis animal model. Thus, piperine should be further studied with regard to use either as a pharmaceutical or as a dietary supplement for the treatment of arthritis.

Adiponectin may contribute to synovitis and joint destruction in rheumatoid arthritis by stimulating vascular endothelial growth factor, matrix metalloproteinase-1, and matrix metalloproteinase-13 expression in fibroblast-like synoviocytes more than proinflammatory mediators.

IntroductionThe role of adiponectin in the pathogenesis of arthritis is still controversial. This study was performed to examine whether adiponectin is involved in joint inflammation and destruction in rheumatoid arthritis (RA) in relation to the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs).MethodsSynovial cells from RA patients were treated with adiponectin or interleukin (IL)-1β for 24 hours. The culture supernatant was collected and analyzed for the levels of IL-6, IL-8, prostaglandin E2 (PGE2), VEGF, and MMPs by enzyme-linked immunosorbent assay. The levels of adiponectin, VEGF, MMP-1, and MMP-13 in the joint fluids from 30 RA or osteoarthritis (OA) patients were also measured.ResultsAdiponectin at the concentration of 10 μg/mL stimulated the production of IL-6, IL-8, and PGE2 in RA fibroblast-like synoviocytes (FLSs), although the level of these was much lower than with 1 ng/mL IL-1β. However, adiponectin stimulated the production of VEGF, MMP-1, and MMP-13 at the same level as IL-1β. In addition, the level of adiponectin and MMP-1 in the joint fluid of RA patients was significantly higher than in OA patients. Adiponectin was positively correlated with VEGF in RA patients but not in OA patients, while the level of MMPs in joint fluid was not correlated with adiponectin in either RA or OA patients.ConclusionsAdiponectin may play a significant role in the pathogenesis of RA by stimulating the production of VEGF and MMPs in FLSs, leading to joint inflammation and destruction, respectively.

Expression levels and association of gelatinases MMP-2 and MMP-9 and collagenases MMP-1 and MMP-13 with VEGF in synovial fluid of patients with arthritis.

This study was performed to provide evidence, albeit indirectly, as to which matrix metalloproteinases (MMPs), among the gelatinases MMP-2 and MMP-9 and the collagenases MMP-1 and MMP-13, play a more proactive role in the angiogenic process in arthritic joint. Joint fluid was collected from 33 patients with rhuematoid arthritis (RA) and osteoarthritis (OA), and protein (MMPs and vascular endothelial growth factor (VEGF)) levels were measured by ELISA, and the association of MMPs with VEGF was evaluated in joint fluid of patients with RA or OA. The levels of collagenases (total MMP-1 and total MMP-13) and gelatinases (total MMP-2 and total MMP-9) in RA joint fluid were significantly higher than those in OA fluid. Total MMP-9 levels were significantly associated with VEGF levels in RA fluids, but not in OA fluid, while total MMP-13 levels were strongly associated with VEGF levels in both RA and OA fluid. However, total MMP-2 and total MMP-1 levels were not associated with VEGF levels in either RA or OA joint fluid. Our results indirectly suggest that in RA and OA, MMP-9 and MMP-13 may play a more important role in angiogenesis than MMP-2 and MMP-1.

Hypoxia differentially affects IL-1β-stimulated MMP-1 and MMP-13 expression of fibroblast-like synoviocytes in an HIF-1α-dependent manner

OBJECTIVES To further understand the expression regulation of MMP-1 and MMP-13 under physiological and pathological conditions, we investigated the combined effects of hypoxia and pro-inflammatory stimuli on the expression of MMP-1 and MMP-13 in rheumatoid synovial fibroblasts. METHODS Synovial fibroblasts were cultured under either hypoxic or normoxic conditions in the presence of IL-1β stimulation. The culture supernatant was analysed for secreted levels of VEGF, MMP-1 and MMP-13. Their gene expression was quantified with real-time and semi-quantitative PCR. Another group of cells was transfected with small-interfering RNA (siRNA) specific for hypoxia-inducible factor-1 α (HIF-1α). The protein levels of HIF-1α were detected by western blot analysis. RESULTS In response to 10 ng/ml of IL-1β under normoxia, the levels of MMP-1 and MMP-13 increased compared with the levels observed under hypoxia. IL-1β stimulation under hypoxia induced a 2-fold increase in the level of MMP-1 and a 2-fold decrease in the level of MMP-13 compared with cells cultured under normoxia. A similar pattern of differential expression for MMP-1 and MMP-13 was observed with 1 and 5 ng/ml IL-1β, but not at 0.1 ng/ml. The differential expression of MMPs under the combined effect of IL-1β and hypoxia was significantly attenuated by silencing HIF-1α with siRNA. CONCLUSIONS Hypoxia in arthritic joints may differentially affect the IL-1β-stimulated expression of MMP-1 and MMP-13 in rheumatoid synovial fibroblasts. This effect is dependent on HIF-1α expression. This hypoxia-mediated differential effect should be taken into consideration when testing the efficiency of therapies that target HIF-1α.

Synergy between adiponectin and interleukin-1β on the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in fibroblast-like synoviocytes.

To determine whether adiponectin may have synergistic effects in combination with the proinflammatory cytokine interleukin (IL)-1β regarding the production of proinflammatory mediators during arthritic joint inflammation, synovial cells from rheumatoid arthritis (RA) patients were treated with adiponectin, IL-1β, and their combination for 24 h. Culture supernatant was collected and analyzed by enzyme-linked immunosorbent assay for levels of IL-6, IL-8, prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs). Adiponectin-mediated intracellular signaling pathways were investigated to elucidate the molecular mechanisms underlying their synergy. The association of proinflammatory mediators with adiponectin was investigated in the synovial fluid of arthritis patients. Adiponectin functioned synergistically with IL-1β to activate IL-6, IL-8, and PGE2 expression in RA fibroblast-like synoviocytes; Levels of VEGF, MMP-1, and MMP-13 were not synergistically stimulated. Adiponectin and IL-1β each increased the expression of both adiponectin receptor 1 and IL-1 receptor 1. However, adiponectin and IL-1β did not synergistically support the degradation of IκB-α or the nuclear translocation of NF-κB. Synergistically increased gene expression was significantly inhibited by MG132, an NF-κB inhibitor. Supporting the in vitro results, IL-6 and IL-8 levels were positively associated with adiponectin in synovial joint fluid from patients with RA, but not osteoarthritis (OA). In conclusion, adiponectin and IL-1β may synergistically stimulate the production of proinflammatory mediators through unknown signaling pathways during arthritic joint inflammation. Adiponectin may be more important to the pathogenesis of RA than previously thought.

Pyrrolidine dithiocarbamate, a NF-κB inhibitor, upregulates MMP-1 and MMP-13 in IL-1β-stimulated rheumatoid arthritis fibroblast-like synoviocytes

Activated NF-kappaB plays an important role in the expression of matrix metalloproteinase (MMP)-1 and MMP-13 in rheumatoid arthritis and osteoarthritis. The objective of this study was to determine the effects of the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) on the expression of MMPs in IL-1beta-stimulated fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis patients. FLSs were treated with IL-1beta (10 ng/ml) for 24 h in the presence or absence of PDTC. The level of MMP-1 and MMP-13 increased in response to PDTC in time- and dose-dependent manners in IL-1beta-stimulated FLSs; the expressions of IL-6 and vascular endothelial growth factor (VEGF) decreased in a PDTC concentration-dependent manner. However, PDTC-mediated repression of IL-6 and VEGF expression was not observed in TNF-alpha-stimulated rheumatoid arthritis FLSs. In contrast, other NF-kappaB inhibitors, such as fenofibrate, N-acetylcysteine and MG132, decreased MMP expression in IL-1beta-stimulated FLSs. The stimulatory effect of PDTC on MMP expression was not mimicked by specific inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway. Treatments with 100 muM PDTC did not inhibit the phosphorylation of p-ERK1/2, p-P38, and p-JNK, or the transnuclear migration of NF-kappaB through degradation of IkappaB-alpha in IL-1beta-stimulated FLSs. These results suggest that the increase of MMP expression may occur in a stimuli-specific manner or by an NF-kappaB independent mechanism. Therefore, therapeutic NF-kappaB inhibitors should be thoroughly studied before their clinical use in treating rheumatoid arthritis, as undesirable genes may be upregulated through unknown mechanisms, possibly resulting in worse symptoms.

Differential effect of IL-1β and TNF-α on the production of IL-6, IL-8 and PGE2 in fibroblast-like synoviocytes and THP-1 macrophages

Inflammation in the joint of rheumatoid arthritis is a complex immune reaction facilitated by various factors, such as cytokines, cells and hypoxia. Thus, we evaluated their relative capacity to produce proinflammatory mediators in response to IL-1β, TNF-α or IL-17 under hypoxia or normoxia in fibroblast-like synoviocytes (FLSs) and macrophages. The level of IL-6 expression was strongly increased in both FLSs and THP-1 macrophages in response to IL-1β and TNF-α, but the level by TNF-α was less than that by IL-1β. In contrast, the expression of IL-8 in both cell types was strongly stimulated by both IL-1β and TNF-α. In FLSs, PGE2 production increased only in response to IL-1β; and no effect was observed in THP-1 cells and TNF-α-stimulated FLSs. In addition, the production by IL-17 was extremely low when compared with those induced by IL-1β or TNF-α in FLSs and THP-1 cells. Hypoxia (2% O2) decreased IL-1β-stimulated production of PGE2, even though it increased the expression of mRNA and protein of COX-2. These results suggest that IL-1β and TNF-α differentially regulate gene expression in FLSs and macrophages under hypoxia or normoxia.

Increased levels of thymosin β4 in synovial fluid of patients with rheumatoid arthritis: association of thymosin β4 with other factors that are involved in inflammation and bone erosion in joints

Aim:  Thymosin (Tβ4) may have various biological effects that are relevant to the pathogenesis of rheumatoid arthritis (RA). This study was performed to gain insight into the relevance of Tβ4 in the pathogenesis of inflammatory arthritis.

Analgesic and anti-inflammatory effects in animal models of an ethanolic extract of Taheebo, the inner bark of Tabebuia avellanedae.

Taheebo, the purple inner bark of the Bignoniaceae tree Tabebuia avellanedae Lorentz ex Griseb, which is found in tropical rain forests in northeastern Brazil, has been used as a traditional medicine for various diseases for more than 1,500 years. In the current study, various animal models were used to demonstrate the analgesic and anti-inflammatory properties of its ethanolic extract, thereby investigating its potential as a therapeutic treatment for diseases with pain and inflammation. In the hot plate and writhing tests for the in vivo analgesic effect test of Taheebo, a 200 mg/kg dose of the extract induced a significant anti-nociceptive effect and increased the pain threshold by approximately 30% compared with the control. In vascular permeability and tetradecanoylphorbol acetate (TPA)‑, arachidonic acid- and carrageenan-induced paw edema tests for anti-inflammatory effects, treatment with 200 mg/kg Taheebo led to significant anti-inflammatory effects and inhibited inflammation by 30-50% compared with the control. At 100 mg/kg, the extract decreased the levels of pain and inflammation in all tested models, but the degree of inhibition was not statistically significant. The results suggest that the ethanolic extract of the inner bark of Tabebuia avellanedae has the potential to be developed as a therapeutic or supportive drug against diseases with accompanying pain and inflammation, including osteoarthritis.

Implication of MMP-9 and urokinase plasminogen activator (uPA) in the activation of pro-matrix metalloproteinase (MMP)-13

We examined whether the expression and activation of pro-matrix metalloproteinase (MMP)-1 varies from that of pro-MMP-13 in the joint fluid of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. To do this, joint fluid was collected from 34 RA and 34 OA patients. The collagenase (pro-MMP-1 and MMP-13, total MMP-1, and MMP-13), gelatinase (total MMP-2 and MMP-9), stromelysin (total MMP-3), matrilysin (total MMP-7), uPA, and tissue inhibitor of MMP (TIMP) levels were measured by ELISA. The level of total MMP-1 in RA joint fluids was similar to that of the OA joint fluid. In contrast, the level of total MMP-13 in the RA group was significantly higher than that of the OA group. Among various MMPs (MMP-2, MMP-3, MMP-7, and MMP-9), only MMP-9 was strongly associated with total MMP-13 in both RA and OA. The level of uPA was also strongly associated with MMP-13 in RA but not OA, while the level of TIMP-1 and TIMP-2 was not significantly different between RA and OA. In conclusion, MMP-9 and uPA might be involved in the activation of pro-MMP-13 through unknown mechanisms in arthritic diseases.