Gigantol Isolated from the Whole Plants of Cymbidium goeringii Inhibits the LPS-Induced iNOS and COX-2 Expression via NF-κB Inactivation in RAW 264.7 Macrophages Cells

• Abstract
• Introduction
• Materials and Methods
• Isolation of gigantol from Cymbidium goeringii
• Chemicals and antibodies
• Cell culture and sample treatment
• Nitrite, PGE₂, TNF-α, IL-1β and IL-6 assays
• Western blot analysis
• RNA preparation and RT-PCR
• Transient transfection and luciferase assay (reporter gene assay)
• Nuclear extraction and electrophoretic mobility shift assay (EMSA)
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

During our efforts to find bioactive natural products with anti-inflammatory activity, we isolated gigantol from the whole plants of Cymbidium goeringii (Orchidaceae) by activity-guided chromatographic fractionation. Gigantol was found to have potent inhibitory effects on LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 cells. Consistent with these findings, gigantol suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in RAW 264.7 cells in a concentration-dependent manner. Our data also indicate that gigantol is a potent inhibitor of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) release and influenced the mRNA expression levels of these cytokines in a dose-dependent manner. Furthermore, a reporter gene assay for nuclear factor kappa B (NF-κB) and an electromobility shift assay (EMSA) demonstrated that gigantol effectively inhibited the activation of NF-κB, which is necessary for the expression of iNOS, COX-2, TNF-α, IL-1β and IL-6. Thus, our studies suggest that gigantol inhibits LPS-induced iNOS and COX-2 expression by blocking NF-κB activation.

Introduction

During inflammatory processes, large amounts of the pro-inflammatory mediators nitric oxide (NO) and prostaglandin E₂ (PGE₂) are generated by the inducible isoforms of NO synthase (iNOS) and by cyclooxygenase-2 (COX-2) [1]. In mammalian cells, NO is synthesized by three different isoforms of nitric oxide synthase (NOS), namely, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Although nNOS and eNOS are constitutively expressed, iNOS is expressed in response to interferon-γ, lipopolysaccharide (LPS), and a variety of pro-inflammatory cytokines [1]. Cyclooxygenase (COX) converts arachidonic acid to PGs, and has two isoforms, i. e., COX-1 and COX-2 [2]. COX-1 is expressed constitutively in most tissues and appears to be responsible for maintaining normal physiological functions, whereas COX-2 is only detectable in certain types of tissues and is induced transiently by growth factors, pro-inflammatory cytokines, tumor promoters, and bacterial toxins [3].

LPS is a major inflammatory molecule, and triggers the productions of pro-inflammatory toxins and cytokines, like TNF-α, in various cell types [4]. Moreover, TNF-α plays key roles in the induction and perpetuation of inflammation caused by autoimmune reactions by activating T cells and macrophages and by up-regulating other pro-inflammatory cytokines and endothelial adhesion molecules, such as, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, which enhance the recruitment of leukocytes to sites of inflammation [5]. Likewise, IL-1β is one of the most important inflammatory cytokines secreted by macrophages and is induced by LPS in macrophages. During inflammation, increased IL-1β release leads to cell or tissue damage [6], and thus, reduction of IL-1β release from macrophages may retard the inflammatory response to LPS stimulation. Moreover, in terms of response to noxious stimuli, the signal transduction pathway of IκB/NF-κB activation contributes to the regulation of IL-1β [7]. Additionally, the production of IL-6 is induced by several other factors, such as TNF-α and IL-1β as well as the bacterial endotoxin, LPS. IL-6 is a pro-inflammatory cytokine that acts as an endogenous pyrogen and has multiple effects on the immune system, particularly on hematopoiesis [7]. It is also regulated by the signal transduction pathway of IκB/NF-κB activation [8].

Nuclear transcription factor kappa-B (NF-κB) is one of the most ubiquitous transcription factors and regulates genes involved in cellular proliferation, inflammatory responses, and cell adhesion. The activation of NF-κB has been reported to induce the transcriptions of many pro-inflammatory mediators, e. g., iNOS, COX-2, TNF-α and IL-1β, -6 and -8 [9]. Functionally active NF-κB exists mainly as a heterodimer and is composed of subunits of the Rel family, i. e., p50 and p65, and is normally sequestered in the cytosol as an inactive complex due to its binding with inhibitory κB (IκBs) in unstimulated cells [10]. The activation of NF-κB involves the phosphorylation of IκBs at two critical serine residues (Ser32, Ser36) via the IκB kinase (IKK) signalosome complex [10].

Although Cymbidium goeringii (Orchidaceae) has been used as a traditional treatment for burns, frostbite, and bleeding [11], no phytochemical or pharmacological studies have been undertaken to date, with the exception of a single study on the isolation of sterols from this plant. [12]. However, no report has been issued on its anti-inflammatory activity or on its mode of action. Thus, as a part of our on-going screening program to evaluate the anti-inflammatory potential of natural compounds, we investigated the in vitro inhibition of NO production by the methanol extract of whole C. goeringii using activity-guided fractionation, which led to the isolation of gigantol (Fig. [1]). We then evaluated the effects of gigantol on NO induction by LPS and on the LPS-induced productions of PGE₂, TNF-α, IL-1β, IL-6, and of other pro-inflammatory proteins and on their mRNA expressions in vitro.

Materials and Methods

Isolation of gigantol from Cymbidium goeringii

Cymbidium goeringii was collected on Mt. Baekyang, Jeongeup, Chonbuk province, Korea during April 2003. The plant was identified by Dr. Sung Hoon Bang at the Department of Horticulture, Kyung Hee University and a voucher specimen (03 057) has been deposited at the Laboratory of Natural Product Chemistry, Kyung Hee University. Fresh whole plants (10 kg) were cut and extracted three times with 80 % MeOH (3 × 18 L). Extract solutions were filtered and dried using a rotatory evaporator under reduced pressure to give the MeOH extract (344 g), which (300 g) was suspended in H₂O (2 L) and partitioned with EtOAc (2 L × 2). The EtOAc soluble portion obtained was concentrated under vacuum to yield the EtOAc extract (21 g). The aqueous layer was then successively partitioned with n-BuOH (2 L × 2), and these n-BuOH extracts were also evaporated under vacuum to give the n-BuOH extract (23 g) and the aqueous extract (256 g), respectively. The EtOAc extract (20 g) was found to be active by activity-guided fractionation and was chromatographed on a silica gel column (600 g, 7.0 × 60.0 cm; Merck; Darmstadt, Germany) using n-hexane-EtOAc (7 : 1 → 5 : 1 → 3 : 1 → 1 : 1) and CHCl₃-MeOH (17 : 1 → 10 : 1), to produce twenty-eight fractions (SOE-1 - SOE-28). SOE-8 (1.6 g) was subjected to a silica gel column chromatography (100 g, 5 × 17 cm) using CHCl₃-MeOH (5 : 1 → 3 : 1) as eluent to afford eleven fractions (SOE-8-1 - SOE-8-11). SOE-8-6 (152 mg) was purified by ODS column chromatography (70 g, 3 × 20 cm; Merck) and eluted with MeOH-H₂O (3 : 1) to give gigantol (72 mg). The structure of this compound was confirmed by comparing its spectral data (IR, ¹H-NMR, ¹³C-NMR, EI-MS) with those reported in the literature [13], [14]. Gigantol used for this study was checked by HPLC and was >98 % pure.

Gigantol: Colorless needles, m. p. 95 - 96 °C, IR (KBr window): ν_max = 3510, 3010, 2960, 1610, 1520, 1470 cm^-1; ¹H-NMR (CDCl₃, 400 MHz): δ = 6.82 (1H, d, J = 8.2 Hz, H-5′′), 6.77 (1H, dd, J = 8.2, 1.9 Hz, H-6′′), 6.65 (1H, d, J = 1.9 Hz, H-2′′), 6.28 - 6.29 (3H, br. s, H-2′,4′,6′), 3.85 (3H, s, H-OMe), 3.77 (3H, s, H-OMe), 2.83 - 2.85 (4H, m, H-1,2); ¹³C-NMR (CDCl₃, 100 MHz): δ = 160.8 (C-5′), 156.5 (C-3′), 146.0 (C-3′′), 144.4 (C-1′), 143.6 (C-4′′), 133.6 (C-1′′), 121.0 (C-6′′), 114.1 (C-2′′), 111.2 (C-5′′), 108.0 (C-2′), 106.7 (C-6′), 99.1 (C-4′), 55.8 (C-OMe), 55.1 (C-OMe), 38.0 (C-2), 37.0 (C-1); EI/MS: m/z = 274 [M]⁺, 137.

Chemicals and antibodies

Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). iNOS, COX-1, COX-2, and β-actin monoclonal antibodies and peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Enzyme immunoassay (EIA) kits for PGE_{2, TNF-α interleukin-1β (IL-1β) and IL-6 were obtained from R&D Systems (Minneapolis, MN, USA), and luciferase assay kits from Promega (Madison, WI, USA). pNF-κB-Luc reporter plasmid was purchased from BD Biosciences (San Jose, CA, USA). Superfect transfection reagent from Qiagen (Qiagen GmbH; Hilden, Germany), and RNA extraction kits from Intron Biotechnology (Seoul, Korea). iNOS, COX-2, TNF-α, IL-1β, IL-6 and β-actin oligonucleotide primers were from Bioneer (Seoul, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltertazolium bromide (MTT), aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol, Escherichia coli lipopolysaccharide (LPS), acetylsalicylic acid (aspirin), carrageenan, serotonin, indomethacin and all other chemicals were from Sigma (St. Louis, MO, USA).}

Cell culture and sample treatment

The RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were grown at 37 °C in DMEM supplemented with 10 % FBS, penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL) in a humidified 5 % CO₂ atmosphere. Cells were incubated with test compounds at different concentrations and stimulated with LPS 1 μg/mL for the indicated times. Gigantol dissolved in DMSO was added to the medium in serial dilution (the final DMSO concentration in all assays did not exceed 0.1 %).

Nitrite, PGE₂, TNF-α, IL-1β and IL-6 assays

Nitrite accumulation, an indicator of NO synthesis, was measured in culture media using the Griess reaction [15]. PGE₂, TNF-α, IL-1β and IL-6 levels in macrophage culture media were quantified using EIA kits, according to the manufacturer’s instructions (R&D Systems).

Western blot analysis

Cellular proteins were extracted from control and gigantol treated RAW 264.7 cells, and washed cell pellets were resuspended in extraction lysis buffer (50 mM HEPES (pH 7.0), 250 mM NaCl, 5 mM EDTA, 0.1 % Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 5 mM sodium fluoride (NaF), 0.5 mM sodium orthovanadate) containing 5 μg/mL of each of leupeptin and aprotinin, and then incubated for 30 min at 4 °C. Cell debris was removed by microcentrifugation, and supernatants were quick frozen. Protein concentrations were determined using Bio-Rad protein assay reagent according to the manufacturer’s instruction. Forty micrograms of cellular proteins from treated and untreated cell extracts were electroblotted onto nitrocellulose membranes after 10 % SDS-polyacrylamide gel electrophoresis. Immunoblots were incubated overnight with blocking solution (5 % skim milk) at 4 °C, and then incubated for 4 h with a 1 : 1000 dilution of monoclonal anti-iNOS, a 1 : 1000 dilution of anti-COX-2 antibody, and a β-actin antibody (Santa Cruz Biotechnology Inc.). Blots were washed twice with Tween 20/Tris-buffered saline (TTBS) and then incubated with a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc.) for 1 h at room temperature. Blots were again washed three times with TTBS and then developed using enhanced chemiluminescence kits (Amersham Life Science; Arlington Heights, IL, USA).

RNA preparation and RT-PCR

Total cellular RNA was isolated using Easy Blue® kits (Intron Biotechnology). RNA (1 μg) was reverse-transcribed (RT) from each sample using MuLV reverse transcriptase, 1 mM dNTP, and oligo (dT_{12 - 18}) 0.5 μg/μL. PCR analyses were performed on aliquots of the cDNA preparations to detect iNOS, COX-2, TNF-α, IL-1β and IL-6 (using β-actin as the internal standard) gene expression using a thermal cycler (Perkin Elmer Cetus; Foster City, CA, USA). Reactions were carried out in 25μL containing (final concentrations) 1 unit of Taq DNA polymerase, 0.2 mM dNTP, × 10 reaction buffer, and 100 pmol of 5′ and 3′ primers. After an initial denaturation for 2 min at 95 °C, 30 amplification cycles were performed for iNOS (1 min at 95 °C, annealing 1 min at 60 °C, and extension 1.5 min at 72 °C), COX-2 (denaturation 1 min at 94 °C, annealing 1 min at 60 °C, and extension 1 min at 72 °C), TNF-α (denaturation1 min at 95 °C, annealing 1 min at 55 °C, extension and 1 min at 72 °C), IL-1β (denaturation 1 min at 94 °C, annealing 1 min at 60 °C, and extension 1 min at 72 °C) and IL-6 (denaturation 1 min at 94 °C, annealing 1 min at 57 °C, and extension 1 min at 72 °C). The PCR primers used in this study are listed below and were purchased from Bioneer (Seoul, Korea): sense strand iNOS, 5′-AATGGCAACATCAGGTCGGCCATCACT-3′, anti-sense strand iNOS, 5′-GCTGTGTGTCACAGAAGTCTCGAACTC-3′; sense strand COX-2, 5′-GGAGAGACTATCAAGATAGT-3′, anti-sense strand COX-2, 5′-ATGGTCAGTAGACTTTTACA-3′; sense strand TNF-α, 5′-ATGAGCACAGAAAGCATGATC-3′, anti-sense strand TNF-α, 5′-TACAGGCTTGTCACTCGAATT-3′; sense strand IL-1β, 5′-TGCAGAGTTCCCCAACTGGTACATC-3′; anti-sense strand IL-1β, 5′-GTGCTGCCTAATGTCCCCTTGAATC-3′; sense strand IL-6, 5′- GAGGATACCACTCCCAACAGACC-3′; anti-sense strand IL-6, 5′- AAGTGCATCATCGTTGTTCATACA-3′; sense strand β-actin, 5′-TCATGAAGTGTGACGTTGACATCCGT-3′, anti-sense strand β-actin, 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′. After amplification, PCR reaction products were electrophoresed on 2 % agarose gels and visualized using ethidium bromide and UV [15].

Transient transfection and luciferase assay (reporter gene assay)

RAW 264.7 cells were transfected using Superfect reagent (Qiagen GmbH) and pNF-κB-Luc reporter plasmid (BD Biosciences; San Jose, CA, USA), as instructed by the manufacturer. Cells were incubated for 2 h before adding 5 mL of DMEM/10 % FBS. Forty-eight hours after the start of transfection, cells were pretreated with gigantol for 1 h and stimulated with LPS (1 μg/mL). Following 3 h of stimulation, cells were lysed and luciferase activities were determined using the Promega luciferase assay system (Promega) and a luminometer (Perkin Elmer Cetus). Luciferase activities were normalized versus sample protein concentrations.

Nuclear extraction and electrophoretic mobility shift assay (EMSA)

RAW 264.7 macrophages in 100-mm dishes (1 × 10⁶ cells/mL) were preincubated with various concentrations of gigantol (25, 50, 100 μg/mL) and then stimulated with LPS (1 μg/mL) for 1 h. The cells were washed once with PBS, scraped into 1 mL of cold PBS, and pelleted by centrifugation. Nuclear extracts were prepared as described previously with slight modification [15]. Briefly, cell pellets were resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, 10 μg/mL aprotinin) and incubated on ice for 15 min. They were then lysed by adding 0.1 % Nonidet P-40 and vortexing vigorously for 10 s. Nuclei were pelleted by centrifugation at 12,000 × g for 10 min at 4 °C and resuspended in high salt buffer (20 mM HEPES, pH 7.9, 25 % glycerol, 400 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate). Nuclear extract (10 μg) was mixed with double-stranded NF-κB oligonucleotide. 5′-AGTTGAGGGGACTTTCCCAGGC-3′ end-labeled by [γ-³²P] dATP (the underline indicates a κB consensus sequence or a binding site for the NF-κB/cRel homodimeric or heterodimeric complex). Binding reactions were performed at 37 °C for 30 min in 30 μL of reaction buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 4 % glycerol, 1 μg of poly(dI-dC), and 1 mM DTT. DNA-protein complexes were separated from the unbound DNA probe on native 5 % polyacrylamide gels at 100 V in 0.5 × TBE buffer. Gels were vacuum dried for 1 h at 80 °C and exposed to X-ray film at -70 °C for 24 h.

Statistical analysis

Results are expressed as mean ± S.D. of at least three experiments performed using different cell preparations in vitro. Statistically significant values were compared using a non-parametric multiple comparisons test (Kruskal-Wallis test) followed by Dunn’s test. Statistical significance was set at P < 0.05.

Results

Since it was recently suggested that inflammatory diseases are primarily associated with excessive NO formation [16], we undertook to identify the active principle of the MeOH extract of C. goeringii by focusing on its inhibitory effect on NO formation. The MeOH extract of C. goeringii was fractionated and aliquots were subjected to nitrite assays using RAW 264.7 macrophage cells. The MeOH extract has an inhibitory effect on NO formation in LPS-induced macrophage cells (data not shown), and this extract was further fractionated into EtOAc and BuOH extracts. The EtOAc fraction showed higher activity than the BuOH extract, and silica gel column chromatography and successive ODS column chromatography of this extract afforded gigantol (Fig. [1]). Our physical and spectroscopic data were in accord with those published for gigantol [13], [14].

To assess the effect of gigantol on LPS-induced NO production in RAW 264.7 cells, cells pretreated with/without gigantol for 1 h were treated with LPS (1 μg/mL) for 24 h; controls contained neither LPS nor sample. Cell culture media were harvested and NO concentrations were measured using the Griess reaction. Gigantol was found to inhibit LPS-induced NO production in a concentration-dependent (25, 50, 100 μg/mL) manner with an average IC₅₀ of 42.1 μg/mL for three separate experiments (Fig. [2] A). l-N ⁶-(1-Iminoethyl)lysine (NIL) was used as a positive control. To examine whether gigantol inhibits PGE₂ production, cells were pre-incubated with gigantol for 1 h, and then activated with 1 μg/mL LPS for 24 h. As shown in Fig. [2] B, PGE₂ production was significantly inhibited by gigantol in a concentration-dependent manner. The cytotoxicity of gigantol was evaluated in the presence or absence of LPS by MTT assays, and it was found that gigantol had no affect on RAW 264.7 cell viability even at 150 μg/mL for 24 h. However, gigantol in the presence of LPS reduced cell viability by 27.5 % at the highest concentration used (150 μg/mL) (see Fig. 1S in Supporting Information).

Since gigantol was found to inhibit both NO and PGE₂ production, we investigated whether these inhibitory effects are related to iNOS and COX-2 modulation using Western blot and RT-PCR. In unstimulated RAW 264.7 cells, iNOS and COX-2 at the protein and mRNA levels were undetectable. However, both iNOS and COX-2 were markedly induced by LPS treatment, and gigantol significantly inhibited these expressions in a concentration-dependent manner (Fig. [3] A). RT-PCR also showed that iNOS and COX-2 mRNA expressions were correlated with their protein levels. However, gigantol did not affect the expression of β-actin, the housekeeping gene. These results are consistent with the inhibitory effects of gigantol on NO and PGE₂ production (Fig. [2]).

We also tested the effect of gigantol on LPS-induced TNF-α, IL-1β and IL-6 release using enzyme immunoassays (EIAs) and RT-PCR. We found that gigantol pretreatment reduced TNF-α, IL-1β and IL-6 expression at the protein (Fig. [4] A, B and C) and mRNA (Fig. [4] D) levels in RAW 264.7 macrophage cells in a concentration-dependent manner.

Since the activation of NF-κB is critically required for the activation of iNOS, COX-2, TNF-α, IL-1β and IL-6 by LPS [9], [17], we examined the effect of gigantol on LPS-stimulated NF-κB-dependent reporter gene expression. In this study, we used a pNF-κB-luc plasmid, which was generated by inserting four spaced NF-κB binding sites into pLuc-promoter vector. RAW 264.7 cells were transiently transfected with pNF-κB-luc plasmid and then stimulated with 1 μg/mL LPS in the presence or absence of gigantol. It was found that gigantol treatment significantly reduced the LPS-induced increase in NF-κB-dependent luciferase enzyme expression (Fig. [5] A). In addition, LPS-induced NF-κB-DNA binding and transcriptional activity in RAW 264.7 macrophage cells was significantly inhibited by gigantol in a concentration-dependent manner (Fig. [5] B). Taken together, the above findings demonstrate that gigantol suppresses iNOS, COX-2, TNF-α, IL-1β and IL-6 expressions at least in part via an NF-κB-dependent mechanism.

Discussion

During our continued searches for novel anti-inflammatory agents from natural products, we found that the ethyl acetate fraction of C. goeringii inhibited LPS-induced NO production in RAW 264.7 cells. Activity-guided fractionation led to the isolation of the bibenzyl derivative, gigantol. Although it has been previously reported that gigantol has various pharmacological effects, i. e., anti-mutagenic, spasmolytic, antifungal and antibacterial effects [18], [19], [20], [21], its anti-inflammatory effect and the mechanism involved have not been addressed.

In murine macrophage RAW 264.7 cells, LPS induces iNOS transcription and transduction, and this is followed by NO production. Furthermore, LPS stimulation is known to induce IκB proteolysis and NF-κB nuclear translocation [10]. Therefore, RAW 264.7 cells provide us with an excellent model for drug screening and for subsequently evaluating potential inhibitors of the pathway leading to the induction of iNOS and the production of NO. The reactive free radical NO is a major macrophage-derived inflammatory mediator and has also been reported to be involved in the pathogenesis of inflammatory diseases [22]. In addition, a large amount of evidence suggests that prostaglandins (PGs) are involved in various pathophysiological processes, including those of inflammation and carcinogenesis, and that the inducible isoform of cyclooxygenase (COX-2) is mainly responsible for the production of large amounts of these mediators [23]. Based on these findings, the anti-inflammatory activities of gigantol on LPS-induced NO, PGE₂ and TNF-α production in murine macrophage RAW 264.7 cells were studied. To further investigate the inhibitory effect of gigantol on these pro-inflammatory molecules, we investigated the expression of iNOS and COX-2 proteins and of iNOS, COX-2 and TNF-α mRNA. Inhibition of the expression of iNOS and COX-2 at the gene level was evidenced by reductions in their mRNA levels in a concentration-dependent manner. Thus, the inhibition of NO and PGE₂ release may be attributed to the suppression of iNOS and COX-2 mRNA transcriptions. Importantly, the inhibition of the LPS-induced expression of these molecules in RAW 264.7 cells by gigantol was not due to gigantol’s cytotoxicity, as assessed by MTT assay and expression of the housekeeping gene β-actin.

It has been reported that cytokines such as TNF-α, IL-1β and IL-6 are pro-inflammatory in vitro and in vivo [6]. Moreover, the production of TNF-α is crucial for the synergistic induction of NO synthesis in IFN-γ and/or LPS-stimulated macrophages [24]. TNF-α elicits a number of physiological effects, such as septic shock, inflammation, cachexia, and cytotoxicity [25]. In particular, IL-1 is a major pro-inflammatory cytokine, and mainly released by macrophages and is believed to play a considerable role in the pathophysiology of endometriosis [26]. IL-6 is also a pivotal pro-inflammatory cytokine, and is regarded as an endogenous mediator of LPS-induced fever. In the present study, we found that gigantol also significantly inhibits the releases of TNF-α and IL-1β, -6 and their mRNA expression.

NF-κB is known to play a critical role in the regulation of cell survival genes and to coordinate the expressions of pro-inflammatory enzymes and cytokines, such as, iNOS, COX-2, TNF-α, and IL-1β, IL-6 [9]. Since the expressions of these pro-inflammatory mediators are known to be modulated by NF-κB, we examined the possibility that gigantol inhibits NF-κB activity. Thus, we examined NF-κB activation and the DNA binding activity of p65 to confirm the inhibitions of the expressions of iNOS, COX-2, TNF-α, IL-1β and IL-6 because we believed that the inhibitory effects of gigantol on NO, PGE₂, TNF-α, IL-1β and IL-6 production were regulated by the NF-κB signaling pathway. Our results suggest that NF-κB activation and the DNA binding activity of p65 are in fact inhibited in a concentration-dependent manner by gigantol.

In conclusion, we found that gigantol is a potent inhibitor of LPS-induced NO, PGE₂, TNF-α, IL-1β and IL-6 production, and that it acts at the transcription level. These inhibitions were found to be caused by the prevention of NF-κB activation in RAW 264.7 macrophages. Therefore, we conclude that gigantol appears to have potential to prevent inflammatory disease.

Acknowledgements

This research was supported by a grant from the ‘Investigation of biological activity components and evaluation of pharmacological efficacy in Ganghwa indigeous Crops’.

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• 23 Simon L S. Role of regulation of cyclooxygenase-2 during inflammation.  Am J Med. 1999;  106 S37-42
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• 26 Bergqvist A, Bruse C, Carlberg M, Carlstrom K. Interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in endometriotic tissue and in endometrium.  Fertil Steril. 2001;  75 489-95
  CrossrefPubMedSearch in Google Scholar

Kyung-Tae Lee, Ph. D.

Department of Pharmaceutical Biochemistry

College of Pharmacy

Kyung-Hee University

Dongdaemun-Ku

Hoegi-Dong 130-701

Seoul

Korea

Phone: +82-2-961-0860

Fax: +82-2-966-3885

Email: ktlee@khu.ac.kr

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  PubMedSearch in Google Scholar
• 26 Bergqvist A, Bruse C, Carlberg M, Carlstrom K. Interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in endometriotic tissue and in endometrium.  Fertil Steril. 2001;  75 489-95
  CrossrefPubMedSearch in Google Scholar

Kyung-Tae Lee, Ph. D.

Department of Pharmaceutical Biochemistry

College of Pharmacy

Kyung-Hee University

Dongdaemun-Ku

Hoegi-Dong 130-701

Seoul

Korea

Phone: +82-2-961-0860

Fax: +82-2-966-3885

Email: ktlee@khu.ac.kr

• Supplementary Material