Anti-inflammatory mechanisms of resveratrol in activated HMC-1 cells: Pivotal roles of NF-κB and MAPK

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Abstract

Resveratrol is a phytoalexin polyphenolic compound found in various plants, including grapes, berries, and peanuts. Recently, studies have documented various health benefits of resveratrol including cardiovascular and cancer-chemopreventive properties. The aim of the present study was to demonstrate the effects of resveratrol on the expression of pro-inflammatory cytokines, as well as to elucidate its mechanism of action in the human mast cell line (HMC-1). Cells were stimulated with phorbol 12-myristate 13-acetate (PMA) plus A23187 in the presence or absence of resveratrol. To study the possible effects of resveratrol, ELISA, RT-PCR, real-time RT-PCR, Western blot analysis, fluorescence, and luciferase activity assays were used in this study. Resveratrol significantly inhibited the PMA plus A23187-induction of inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8. Moreover, resveratrol attenuated cyclooxygenase (COX)-2 expression and intracellular Ca2+ levels. In activated HMC-1 cells, phosphorylation of extra-signal response kinase (ERK) 1/2 decreased after treatment with resveratrol. Resveratrol inhibited PMA plus A23187-induced nuclear factor (NF)-κB activation, IκB degradation, and luciferase activity. Resveratrol suppressed the expression of TNF-α, IL-6, IL-8 and COX-2 through a decrease in the intracellular levels of Ca2+and ERK 1/2, as well as activation of NF-κB. These results indicated that resveratrol exerted a regulatory effect on inflammatory reactions mediated by mast cells.

Introduction

Resveratrol (trans-3, 4′, 5-trihydroxystilbene) is a polyphenol found in grapes and other plants. Resveratrol has been known to protect plants against fungal infections and to exhibit anti-proliferative, anti-oxidative and anti-inflammatory properties [1], [2], [3]. Resveratrol suppressed the expression of pro-inflammatory markers including cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) in both macrophages and cancer cell lines [4]. Recently, resveratrol was found to exert a beneficial effect in the treatment of ischemia and neurodegenerative diseases [5], [6]. Also, the anti-inflammatory effects of resveratrol have been associated with inhibition of the transcription factor NF-κB [7], possibly mediated via inhibition of IκB kinase [8]. Some reports demonstrated the inhibitory effects of resveratrol on NF-κB activation and target gene expression induced by various pro-inflammatory stimuli [9], [10]. Although the activity of resveratrol in some biological events has been investigated, its direct molecular targets and the mechanism of action on mast cell-mediated inflammation such as allergic disease are not known.

Mast cells are one of the major effecter cells in the immune response system. Activated mast cells release pro-inflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, IL-13 and inflammatory mediators including histamine, leukotrienes, serotonin, prostaglandin (PG)E2 as well as PGD2 [11], [12], [13]. Cytokines, such as TNF-α, IL-6 and IL-8 are released in a coordinate network and play an important role in chronic inflammation. As such, the pattern of cytokine expression largely determines the nature and persistence of the inflammatory response [14]. TNF-α is either preformed and stored in granules of mast cells or is newly synthesized following mast cell activation; it is a multifunctional cytokine and an important mediator of the immune and inflammatory response. TNF-α is an autocrine stimulator as well as a potent inducer of other inflammatory cytokines, including IL-1β, IL-6, IL-8, and GM-CSF [15], [16]. Moreover, IL-6 is a potent mediator of inflammatory processes, and a pleiotropic inflammatory cytokine produced by T cells, macrophages, monocytes, and synovial fibroblasts. On the other hand, IL-8 from mast cells acts on surrounding cells such as neutrophils, T-lymphocytes and eosinophils, and plays a role in the activation of inflammatory effecter cells [17]. In recent years, important roles for COX-2 in various tumours and inflammatory diseases have been demonstrated [18]. COX-2 is strongly induced in activated monocytes and macrophages, one of the major mediators of inflammatory reactions. Recent studies demonstrated that a COX-2 metabolite released from activated mast cells was also essential for the pathogenesis of eosinophilic airway inflammations [19].

Calcium (Ca2+) acts as a second messenger during cell activation, and an increase in the intracellular Ca2+ level has been proposed as an essential trigger for mast cell activation [20], [21]. Moreover, it has been reported that the release of intracellular Ca2+ from internal stores is required for MAPK activation [22]. Recently studies demonstrated the involvement of Ca2+ in MAPK and NF-κB activation and that increased Ca2+ levels were capable of inducing the release of biological mediators including TNF-α, IL-6 and IL-8 [23], [24]. Moreover, NF-κB activation is required for the expression of many inflammatory proteins such as TNF-α, IL-6, COX-2, as well as iNOS [25]. Therefore, inhibition of NF-κB could reduce the expression of inflammatory genes and is a mechanism by which anti-inflammatory agents might elicit their anti-inflammatory effects [26].

The aim of this study was to investigate the mechanism by which resveratrol affected the production of inflammatory cytokines. This study was conducted using the human mast cell line (HMC-1). The effects of resveratrol were evaluated on PMA plus A23187-induced expression of pro-inflammatory mediators by inhibiting MAPK and IκBα/NF-κB signal pathways.

Section snippets

Reagents

Resveratrol (trans-3, 4′, 5-trihydroxystilbene) was purchased from Sigma–Aldrich and dissolved in dimethyl sulfoxide (DMSO); the final DMSO concentration was adjusted to below 0.01% (v/v) in the culture media. PMA, calcium ionophore A23187 (Calcymycin; C29H37N3O6), PD98059, SP600125, SB203580 and 3-[4,5-dimetylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Iscove's modified Dulbecco's medium (IMDM) was obtained from Gibco

Effects of resveratrol on pro-inflammatory cytokine production

Initially we examined the cytotoxicity of resveratrol on HMC-1 cells using the MTT assay. Resveratrol did not show any cytotoxic effects up to 100 μM (data not shown). To evaluate the effect of resveratrol on the production of pro-inflammatory cytokines, we pretreated cells with resveratrol (10 and 50 μM) before stimulation with PMA (50 nM) and A23187 (1 μM) for 8 h, and analysis using ELISA. As shown in Fig. 1, the levels of TNF-α, IL-8 were considerably increased after stimulation with PMA + A23187,

Discussion

Many recent studies on plant-derived anti-inflammatory compounds have investigated the potential inhibitory effects of natural products using in vivo and in vitro systems. Resveratrol, found in the powdered root of Polygonum cuspidatum (Polygonaceae), is an active ingredient of oriental medicine, and has been used since ancient times to cure diseases such as inflammation, allergy and hyperlipemia [32]. Peanuts, grapes and related products such as red wines are probably the most common

Acknowledgements

This work was supported by grant no. RTI 05-03-02 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE) in the Republic of Korea.

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