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Planta Med 2007; 73(8): 769-773
DOI: 10.1055/s-2007-981553
Pharmacology
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

Allose Gallates Suppress Expression of Pro-Inflammatory Cytokines through Attenuation of NF-κB in Human Mast Cells

Seung Ho Lee1 [*] , Hyo-Hyun Park2 [*] , Jung-Eun Kim3 , Jeong-Ah Kim1 , Yeo Hyang Kim4 , Chang-Duk Jun5 , Sang-Hyun Kim2
  • 1College of Pharmacy, Yeungnam University, Kyongsan, Republic of Korea
  • 2Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
  • 3Department of Molecular Medicine, School of Medicine, Kyungpook National University, Daegu, Republic of Korea
  • 4Department of Pediatrics, Keimyung University School of Medicine, Daegu, Republic of Korea
  • 5Department of Life Science, Gwangju Institutive of Science and Technology, Gwangju, Republic of Korea
Further Information

Prof. Sang-Hyun Kim

Department of Pharmacology

School of Medicine

Kyungpook National University

Daegu 700-422

Republic of Korea

Phone: +82-53-420-4838

Fax: +82-53-423-4838

Email: shkim72@knu.ac.kr

Publication History

Received: January 16, 2007 Revised: May 14, 2007

Accepted: May 14, 2007

Publication Date:
28 June 2007 (online)

Also available at
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Table of Contents #

Abstract

Gallotannins are plant-derived, water-soluble polyphenols with wide-ranging biological activities. Mast cell-mediated allergic inflammation is known to cause many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 with immune regulatory properties. Expression of inflammatory cytokines is mainly regulated by a transcription factor, nuclear factor (NF)-κB. In the present study, the effect of eight gallotannins on the level of pro-inflammatory cytokines and NF-κB activation was investigated in human mast cell line (HMC-1). HMC-1 cells were sensitized by phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A23187). Among the eight gallotannins from Euphorbia species, three gallotannins such as 1,2,3,4,6-penta-O-galloyl-β-D-glucose, 1,2,6-tri-O-galloyl-β-D-allopyanose, and 1,2,3,6-tetra-O-galloyl-β-D-allopyranose suppressed the gene expression and secretion of pro-inflammatory cytokines in a dose-dependent manner. In addition, these three gallotannins blocked the activation of NF-κB as indicated by an NF-κB-dependent gene reporter assay. We conclude that these gallotannins may have potential for the treatment of inflammatory diseases through the down-regulation of NF-κB-mediated activation of mast cells.

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Abbreviations

HMC-1:human mast cell

IL:interleukin

NF-κB:nuclear factor-κB

PMA:phorbol 12-myristate 13-acetate

PMACI:PMA and calcium ionophore A23187

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Key words

Allose gallates - Euphorbia species - Euphorbiaceae - mast cells - pro-inflammatory cytokines - nuclear factor-κB

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Introduction

Mast cells are broadly distributed in the connective tissues of mammals and other vertebrates. Mast cells are central effector cells that cause hypersensitivity such as inflammatory diseases. Upon allergen provocation, cross-linkage of IgE bound on mast cells, via the high-affinity receptors triggers the release of an array of inflammatory mediators as well as various cytokines that are also involved in activation and recruitment of mast cells and other leukocytes [1], [2], [3].

Pro-inflammatory cytokines are important mediators of inflammation, proteolysis, cell proliferation and recruitment and play a key role in the pathogenesis of inflammatory diseases [2]. The human mast cell line (HMC-1) has previously been reported to release various cytokines/chemokines upon stimulation including interleukin (IL)-4, IL-5, IL-6, IL-8, IL-13 and tumour necrosis factor (TNF)-α [1]. HMC-1 cells can be activated by phorbol esters, calcium ionophore, SCF, and TNF-α [3], [4], [5]. HMC-1 cells are a useful model for the study of the expression of cytokines during inflammatory processes. Nuclear factor (NF)-κB is thought to play an important role in the modulation of pro-inflammatory cytokines that generate mediators of cellular responses [6]. NF-κB is a transcription factor that enhances the transcription of a variety of genes, such as cytokines, adhesion molecules, and growth factors. A number of approaches has been used to study NF-κB activation and its regulation of cytokine production in allergic inflammation [5], [7].

Tannins are water-soluble phenolic secondary metabolites of higher plants with the molar mass ranging from 300 Da to 3000 Da [8], [9]. Based on their structural characteristics, tannins can be separated into four major groups: gallotannins, ellagitannins, condensed tannins and complex tannins. Low molar mass monomeric, dimeric and trimeric oligomeric tannins are suggested to have noticeable biological and pharmacological activities [9], [10], [11]. The simplest hydrolyzable tannin, gallotannin, is a compound of polygalloyl esters of glucose. Gallotannins have been studied to exhibit diverse biological effects ranging from anti-inflammatory to anticancer and antioxidant effects [10], [12]. Most of these studies focused on the effects of gallotannins on immune cells with special ragard to mononuclear cells, macrophages, epithelial cells, and little is known regarding the fundamental effects in mast cells. The aim of this study is to evaluate the anti-inflammatory effect of gallotannins previously isolated from plants of the Euphorbia species (Euphorbiaceae) and to understand the mechanism of action in human mast cells.

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Materials and Methods

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Materials

Iscove's modified Dulbecco's medium (IMDM) was purchased from Gibco BRL (Grand Island, NY, USA). Phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187 (Calcymycin; C29H37N3O6), and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma (St. Louis, MO, USA).

Gallotannins were isolated from Euphorbia species. Gallotannins 1, 8, 15, 20, 21, 22, 23, 24 (Fig. [1]): 1-O-galloyl-β-D-glucose, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,3,4,6-penta-O-galloyl-β-D-glucose, 3-O-galloylquinic acid, 2-O-galloyl-D-galactose, 1,3,6-tri-O-galloyl-β-D-allose, 1,2,6-tri-O-galloyl-β-D-allopyanose, and 1,2,3,6-tetra-O-galloyl-β-D-allopyranose, respectively from E. jolkini and E. fisheriana [13], [14]. The purity of isolated gallotannins is more than 98 %.

Zoom Image

Fig. 1 Structures of gallotannins 15 (A), 23 (B) and 24 (C) from Euphorbia species.

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Cell culture and treatments

HMC-1 cells were grown in IMDM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 % heat-inactivated fetal bovine serum (FBS) at 37 °C in 5 % CO2 and 95 % humidity. HMC-1 cells (3 × 105 cells/mL) were treated with gallotannins (1 - 10 μg/mL) for 30 min prior to stimulation with PMACI (40 nM of PMA and 1 μM of A23187) and incubated at 37 °C for additional time.

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Reverse transcription-polymerase chain reaction (RT-PCR)

RT-PCR was used to analyze the expression of mRNA for TNF-α, IL-1β, IL-6, and β-actin (internal control) along with 1 - 10 μg/mL concentrations of gallotannins in PMACI-stimulated HMC-1. The conditions for RT-PCR were similar to ones previously described [7]. Amplified PCR products were separated by 2 % agarose gels and digitized using UN-SCAN-IT software (Silk Scientific; Orem, UT, USA). The relative expression level of each gene was determined with the Image J software by comparing the signal intensity of each gene in the array after normalization to the signal of a housekeeping gene.

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Enzyme-linked immunosorbent assay (ELISA)

HMC-1 cells were sensitized with PMACI for 8 h with or without gallotannins. The ELISA was performed by coating 96-well plates with 6.25 ng/well of monoclonal antibody with specificity for TNF-α, IL-1β and IL-6, respectively. Before use and between subsequent steps in the assay, the coated plates were washed twice with PBS containing 0.05 % Tween-20 and twice with PBS alone. For the standard curve, rTNF-α, rIL-1β and rIL-6 were added to the serum which was previously determined to be negative to endogenous TNF-α, IL-1β and IL-6. After exposure to the medium, the assay plates were exposed sequentially to biotinylated anti-human TNF-α, IL-1β or IL-6, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) tablet substrates. Optical density was read within 10 min of the addition of the substrate with a 405 nm filter.

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Transient transfection and luciferase activity assay

HMC-1 was seeded at 2 × 106 cells/mL in a 12-well plate 4 h before transient transfection. NF-κB luciferase reporter gene constructs (pNF-κB-LUC, plasmid containing NF-κB binding site; Stratagene; Grand Island, NY, USA) were transiently transfected into HMC-1 by using FBS- and antibiotics-free IMDM containing 8 μL of Lipofectamin 2000 reagent (Invitrogen; Carlsbad, CA, USA) [15]. Gallotannins (10 μg/mL) were pretreated to cells for 2 h prior to PMACI stimulation. Cells were harvested 20 h after stimulation and washed in cold PBS before lysis in 100 μL lysis buffer (Luciferase assay kit; Promega; Madison, WI, USA). Luciferase activity was measured with a MicroLumat Plus luminometer, according to the manufacturer's protocol. All transfection experiments were performed in at least three different experiments, with similar results. Total protein was assayed using bicinchoninic acid.

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Statistical analysis

The experiments shown are a summary of the data from at least three experiments. All data are presented as the mean ± SEM. Statistical analyses were performed using SAS statistical software (SAS Institute; Cary, NC, USA). The results were considered significant at a value of P < 0.05.

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Results

We examined whether isolated gallotannins could regulate pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 in HMC-1 cells. At first, we examined the cytotoxicity of gallotannins in HMC-1 cells using MTT assay. Gallotannins did not show a cytotoxic effect up to 100 μg/mL (data not shown). Next, we carried out RT-PCR to investigate the effects of gallotannins on the gene expression of pro-inflammatory cytokines in HMC-1 cells. Gene expression of TNF-α, IL-1β and IL-6 peaked at 4 h after treatment of PMACI (data not shown). Therefore, stimulation of HMC-1 with PMACI (40 nM of PMA and 1 μM of A23187) was induced during 4 h, and the cells were pretreated with gallotannins (10 μg/mL) for 30 min. Among the eight gallotannins, gallotannins 15, 23 and 24 (Fig. [1]) significantly reduced PMACI-induced gene expression of TNF-α, IL-1β and IL-6 (Fig. [2] A). In addition, gallotannins 15, 23 and 24 dose-dependently reduced PMACI-induced gene expression of TNF-α, IL-1β and IL-6 (Fig. [2] B). Culture supernatants were assayed for cytokine levels using ELISA. As shown in Fig. [3], gallotannins 15, 23 and 24 decreased the secretion of PMACI-induced TNF-α, IL-1β and IL-6. Gallic acid was used as a positive control [15]. Equal amounts of gallic acid showed similar or less effect compared with gallotannins 15, 23 and 24.

Zoom Image

Fig. 2 Inhibitory effect of gallotannins on the expression of cytokines in PMACI stimulated HMC-1 cells. A Cells (4 × 106 cells/mL) were separately pretreated with gallotannins (10 μg/mL) for 30 min and stimulated with PMA (40 nM) and A23187 (1 μM) for 4 h. B HMC-1 cell were separately pretreated with respective gallotannins 15, 23, 24 (0.1 - 10 μg/mL). mRNA expression of TNF-α, IL-1β and IL-6 was quantified by RT-PCR. β-Actin was used as a comparative control. The mRNA levels were quantitated by densitometry. Values are the mean ± SEM of duplicate determinations from three separate experiments. * P < 0.05: significantly different from the PMACI value. M = medium; GT = gallotannins; PMACI = PMA and A23187.

Zoom Image

Fig. 3 Inhibitory effect of gallotannins on the secretion of cytokines in PMACI-stimulated HMC-1 cells. A Cells (4 × 106 cells/mL) were pretreated with gallotannins (10 μg/mL) or gallic acid (10 μg/mL) for 30 min and stimulated with PMA (40 nM) and A23187 (1 μM) for 8 h. TNF-α, IL-1β and IL-6 level in supernatant was measured using ELISA and represented as the mean ± SEM of duplicate determinations from three separate experiments. * P < 0.05: significantly different from the PMACI value. GT = gallotannins; PMACI = PMA and A23187; GA = gallic acid.

Expression of TNF-α, IL-1β and IL-6 is known to be regulated by a transcription factor, NF-κB [16]. To evaluate the mechanism of action of gallotannins on the expression of pro-inflammatory cytokines, we examined the effect of gallotannins on the NF-κB-dependent gene reporter assay. We used PDTC, a potent inhibitor for the activation of NF-κB, as a positive control [17]. HMC-1 cells were transiently transfected with an NF-κB-luciferase reporter construct or the empty vector. Exposure of cells with PMACI increased the luciferase activity in the cells transfected with the NF-κB-luciferase reporter construct (Fig. [4]).

Zoom Image

Fig. 4 Effect of gallotannins on NF-κB-dependent transcriptional activity in PMACI stimulated HMC-1 cells. For NF-κB-dependent transcriptional luciferase activity, cells (2 × 106 cells/mL) were transfected for 24 h with the NF-κB luciferase reporter construct or empty vector, and transfected HMC-1 was stimulated with PMA (40 nM) and A23187 (1 μM) for 20 h. Respective gallotannins (10 μg/mL) and gallic acid (10 μg/mL) or PDTC (50 μM) were pretreated for 30 min prior to PMA and A23187 stimulation. Values are the mean ± SEM of duplicate determinations from three separate experiments. * P < 0.05: significantly different from the PMACI value. GT = gallotannins; PMACI = PMA and A23187; GA = gallic acid.

Gallotannins 15, 23 and 24 significantly reduced the PMACI-induced luciferase activity.

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Discussion

It is necessary to study inflammation using various cells and distinct pathways to provide useful therapeutic approaches to many common medical problems. Several laboratories have demonstrated that gallotannins exert potent anti-inflammatory effects. Most cellular studies aiming at exposing the mechanism of action of these anti-inflammatory effects used macrophages and epithelial cells. In macrophages, gallotannins would rather act as a pro-inflammatory stimulus [18]. As opposed to this, in epithelial cells, they act as an anti-inflammatory agent [8]. However, in mast cells, the effect of gallotannins was not characterized. In addition, the mechanism of action of gallotannins in inhibiting the gene expression of cytokines is not clear.

Mast cells play an important role in inflammatory processes that are relevant to the pathogenesis of human diseases [2], [3]. In response to diverse stimuli, mast cells release their mediators such as inflammatory cytokines to the extracellular space. TNF-α has an important amplifying effect in inflammation and it is also an autocrine stimulator as well as a potent inducer of other inflammatory cytokines, including IL-1β, IL-6, and IL-8, and granulocyte-monocyte-colony-stimulating factor [19], [20]. IL-1β is secreted mainly by macrophages and practically by every cell type in the body. It is produced in response to various stimulants and has a broad range of functions which includes activation of neutrophils, endothelial cells, monocytes and mast cells. IL-6 is a pleiotropic inflammatory cytokine produced by T cells, monocytes, macrophages, mast cells, and synovial fibroblasts. IL-6 is also produced from mast cells and its local accumulation is associated with a local allergic reaction. For this reason, the modulation of the secretion of these cytokines from mast cells can provide us with a beneficial therapeutic strategy for acute or chronic inflammatory disease.

In the present study, we demonstrated the inhibitory effect of gallotannins 15, 23 and 24 in human mast cells. They dose-dependently attenuated the PMACI-induced production of TNF-α, IL-1β and IL-6. Our data also showed that the inhibitory effect results from the inhibition of the NF-κB pathway. Many genes encode molecules important in inflammatory processes, such as cytokines and adhesion molecules [21], [22]. The role of NF-κB activation and its regulation of cytokine production in allergic inflammation have been characterized [23]. In PMACI-stimulated mast cells, gallotannins 15, 23 and 24 decreased the expression of NF-κB-dependent genes. These results demonstrate that gallotannins 15, 23 and 24 attenuate the activation of NF-κB and downstream pro-inflammatory cytokines.

It has been reported that gallotannin 15 attenuated TNF-α-induced NF-κB translocation in human umbilical vein endothelial cells [24]. Others investigated the effects of gallotannin 15 on gene expression of IL-8 and NF-κB activation. It inhibited IL-8 production and gene expression in human monocytic U937 cells stimulated with PMA. It also inhibited PMA-mediated NF-κB activation [25]. In activated macrophages, gallotannin 15 significantly inhibited LPS-induced NO production and COX-2 activity [26]. Our previous study reported that gallotannins 23 and 24 exhibited high inhibitory effects on Ca2+-activated hyaluronidase activity in the carrageenin-induced paw edema model. This enzyme is known to be related to vascular permeability and inflammatory process [27]. So far no other reports regarding anti-inflammatory effects of gallotannins 23 and 24 have been published. In the present study, we showed the effect of gallotannins 15, 23 and 24 on the mast cell-mediated inflammatory reaction. This is the first report of the inhibitory effect of gallotannins on cytokine release by HMC-1 cells. These results might imply a new pathway to explain the efficacy of gallotannins 15, 23 and 24 in the treatment of mast cell-mediated inflammatory responses. Our present study exhibited the accurate role that gallotannins play in a wide variety of conditions by different experimental techniques or different type of the cell line.

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Acknowledgements

This research was supported by the Yeungnam University research grants in 2004.

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References

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1 These authors contributed equally to this work

Prof. Sang-Hyun Kim

Department of Pharmacology

School of Medicine

Kyungpook National University

Daegu 700-422

Republic of Korea

Phone: +82-53-420-4838

Fax: +82-53-423-4838

Email: shkim72@knu.ac.kr

#

References

  • 1 Church M K, Levi-Schaffer F. The human mast cell.  J Allergy Clin Immunol. 1997;  99 155-60.
    CrossrefPubMedSearch in Google Scholar
  • 2 Metcalfe D D, Baram D, Mekori Y A. Mast cells.  Physiol Rev. 1997;  77 1033-79.
    PubMedSearch in Google Scholar
  • 3 Galli S J, Kalesnikoff J, Grimbaldeston M A, Piliponsky A M, Williams C M, Tsai M. Mast cells as ”tunable” effector and immunoregulatory cells: recent advances.  Annu Rev Immunol. 2005;  23 749-86.
    CrossrefPubMedSearch in Google Scholar
  • 4 Tsang C M, Wong C K, Ip W K, Lam C W. Synergistic effect of SCF and TNF-alpha on the up-regulation of cell-surface expression of ICAM-1 on human leukemic mast cell line (HMC)-1 cells.  J Leukoc Biol. 2005;  78 239-47.
    CrossrefPubMedSearch in Google Scholar
  • 5 Kim S H, Shin T Y. Effect of Dracocephalum argunense on mast-cell-mediated hypersensitivity.  Int Arch Allergy Immunol. 2006;  139 87-95.
    CrossrefPubMedSearch in Google Scholar
  • 6 Schoonbroodt S, Legrand-Poels S, Best-Belpomme M, Piette J. Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid.  Biochem J. 1997;  321 777-85.
    PubMedSearch in Google Scholar
  • 7 Kim S H, Choi C H, Kim S Y, Eun J S, Shin T Y. Anti-allergic effects of Artemisia iwayomogi on mast cell-mediated allergy model.  Exp Biol Med. 2005;  230 82-8.
    PubMedSearch in Google Scholar
  • 8 Erdelyi K, Kiss A, Bakondi E, Bai P, Szabo C, Gergely P. et al . Gallotannin inhibits the expression of chemokines and inflammatory cytokines in A549 cells.  Mol Pharmacol. 2005;  68 895-904.
    PubMedSearch in Google Scholar
  • 9 Li M, Kai Y, Qiang H, Dongying J. Biodegradation of gallotannins and ellagitannins.  J Basic Microbiol. 2006;  46 68-84.
    CrossrefPubMedSearch in Google Scholar
  • 10 Hagerman A E, Riedl K M, Rice R E. Tannins as biological antioxidants.  Basic Life Sci. 1999;  66 495-505.
    PubMedSearch in Google Scholar
  • 11 Rapizzi E, Fossati S, Moroni F, Chiarugi A. Inhibition of poly(ADP-ribose) glycohydrolase by gallotannin selectively up-regulates expression of proinflammatory genes.  Mol Pharmacol. 2004;  66 890-8.
    CrossrefPubMedSearch in Google Scholar
  • 12 Feldman K S, Sahasrabudhe K, Lawlor M D, Wilson S L, Lang C H, Scheuchenzuber W J. In vitro and in vivo inhibition of LPS-stimulated tumor necrosis factor-alpha secretion by the gallotannin beta-D-pentagalloylglucose.  Bioorg Med Chem Lett. 2001;  11 1813-5.
    CrossrefPubMedSearch in Google Scholar
  • 13 Lee S H, Tanaka T, Nonaka G I, Nishioka I. Tannins and related compounds CV. Monomeric and dimeric hydrolysable tannins having a dehydrohexahydroxydiphenoyl group, supinanin, euphorscopin, euphorhelin and jolkianin from Euphorbia species.  Chem Pharm Bull. 1991;  39 630-8.
    PubMedSearch in Google Scholar
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    CrossrefPubMedSearch in Google Scholar
  • 15 Choi C H, Kim S H. Role of gallic acid on the inflammatory allergic process.  Korean J Physiol Pharmacol. 2006;  10 101-8.
    PubMedSearch in Google Scholar
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1 These authors contributed equally to this work

Prof. Sang-Hyun Kim

Department of Pharmacology

School of Medicine

Kyungpook National University

Daegu 700-422

Republic of Korea

Phone: +82-53-420-4838

Fax: +82-53-423-4838

Email: shkim72@knu.ac.kr

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Fig. 1 Structures of gallotannins 15 (A), 23 (B) and 24 (C) from Euphorbia species.

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Fig. 2 Inhibitory effect of gallotannins on the expression of cytokines in PMACI stimulated HMC-1 cells. A Cells (4 × 106 cells/mL) were separately pretreated with gallotannins (10 μg/mL) for 30 min and stimulated with PMA (40 nM) and A23187 (1 μM) for 4 h. B HMC-1 cell were separately pretreated with respective gallotannins 15, 23, 24 (0.1 - 10 μg/mL). mRNA expression of TNF-α, IL-1β and IL-6 was quantified by RT-PCR. β-Actin was used as a comparative control. The mRNA levels were quantitated by densitometry. Values are the mean ± SEM of duplicate determinations from three separate experiments. * P < 0.05: significantly different from the PMACI value. M = medium; GT = gallotannins; PMACI = PMA and A23187.

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Fig. 3 Inhibitory effect of gallotannins on the secretion of cytokines in PMACI-stimulated HMC-1 cells. A Cells (4 × 106 cells/mL) were pretreated with gallotannins (10 μg/mL) or gallic acid (10 μg/mL) for 30 min and stimulated with PMA (40 nM) and A23187 (1 μM) for 8 h. TNF-α, IL-1β and IL-6 level in supernatant was measured using ELISA and represented as the mean ± SEM of duplicate determinations from three separate experiments. * P < 0.05: significantly different from the PMACI value. GT = gallotannins; PMACI = PMA and A23187; GA = gallic acid.

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Fig. 4 Effect of gallotannins on NF-κB-dependent transcriptional activity in PMACI stimulated HMC-1 cells. For NF-κB-dependent transcriptional luciferase activity, cells (2 × 106 cells/mL) were transfected for 24 h with the NF-κB luciferase reporter construct or empty vector, and transfected HMC-1 was stimulated with PMA (40 nM) and A23187 (1 μM) for 20 h. Respective gallotannins (10 μg/mL) and gallic acid (10 μg/mL) or PDTC (50 μM) were pretreated for 30 min prior to PMA and A23187 stimulation. Values are the mean ± SEM of duplicate determinations from three separate experiments. * P < 0.05: significantly different from the PMACI value. GT = gallotannins; PMACI = PMA and A23187; GA = gallic acid.