SG-HQ2 inhibits mast cell-mediated allergic inflammation through suppression of histamine release and pro-inflammatory cytokines

Abstract

In this study, we investigated the effect of 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2), a synthetic analogue of gallic acid (3,4,5-trihydroxybenzoic acid), on the mast cell-mediated allergic inflammation and the possible mechanism of action. Mast cells play major roles in immunoglobulin E-mediated allergic responses by the release of histamine, lipid-derived mediators, and pro-inflammatory cytokines. We previously reported the potential effects of gallic acid using allergic inflammation models. For incremental research, we synthesized the SG-HQ2 by the modification of functional groups from gallic acid. SG-HQ2 attenuated histamine release by the reduction of intracellular calcium in human mast cells and primary peritoneal mast cells. The inhibitory efficacy of SG-HQ2 was similar with gallic acid. Enhanced expression of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, interleukin-4, and interleukin-6 in activated mast cells was significantly diminished by SG-HQ2 100 times lower concentration of gallic acid. This inhibitory effect was mediated by the reduction of nuclear factor-κB. In animal models, SG-HQ2 inhibited compound 48/80-induced serum histamine release and immunoglobulin E-mediated local allergic reaction, passive cutaneous anaphylaxis. Our results indicate that SG-HQ2, an analogue of gallic acid, might be a possible therapeutic candidate for mast cell-mediated allergic inflammatory diseases through suppression of histamine release and pro-inflammatory cytokines.

Keywords: Allergic inflammation; histamine; mast cells; pro-inflammatory cytokine.

Figure 1

Effects of SG-HQ2 on the histamine release from mast cells: (a) HMC-1 cells (1 × 10⁶/well) were pretreated with or without SG-HQ2 for 30 min and then stimulated with PMA (40 nmol/L) plus A23187 (1 μmol/L) and (b) RPMC (2 × 10⁴/well) were pretreated with or without SG-HQ2 for 30 min and then stimulated with compound 48/80 (5 μg/mL). Histamine level was detected by fluorescent plate reader. Each data represents the mean ± SE of three independent experiments. *Significant difference at P < 0.05. SG-HQ2: (3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide); HMC-1: human mast cells; PMA: phorbol 12-mystate 13-acetate; RPMC: rat peritoneal mast cells; SE: standard error

Figure 2

Effects of SG-HQ2 on the intracellular calcium in mast cells: (a) HMC-1 cells (1 × 10⁵/well) were pretreated with or without SG-HQ2 for 30 min and then stimulated with PMA (40 nmol/L) plus A23187 (1 μmol/L) and (b) RPMC (1 × 10⁴/well) were pretreated with or without SG-HQ2 for 30 min and then stimulated with compound 48/80 (5 μg/mL). EGTA, a calcium chelator, was used as a positive control. Intracellular calcium was detected using fluorescent plate reader. Each data represents the mean ± SE of three independent experiments. *Significant difference at P < 0.05. SG-HQ2: (3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide); HMC-1: human mast cells; PMA: phorbol 12-mystate 13-acetate; RPMC: rat peritoneal mast cells; EGTA: Ethylene glycol tetraacetic acid; SE: standard error

Figure 3

Effects of SG-HQ2 on the expression of pro-inflammatory cytokines. HMC-1 cells (1 × 10⁶/well) were pretreated with or without SG-HQ2 for 30 min and then stimulated with PMA (40 nmol/L) plus A23187 (1 μmol/L). Extraction and analysis of mRNA was performed as described in Materials and methods. The gene expression of pro-inflammatory cytokines was determined by real-time PCR. Each data represents the mean ± SE of three independent experiments. *Significant difference at P < 0.05. SG-HQ2: (3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide); HMC-1: human mast cells; PMA: phorbol 12-mystate 13-acetate; PCR: polymerase chain reaction; SE: standard error

Figure 4

Effects of SG-HQ2 on the activation of NF-κB. Effects of SG-HQ2 on the expression of pro-inflammatory cytokines. HMC-1 cells (2 × 10⁶/well) were pretreated with or without SG-HQ2 for 30 min and then stimulated with PMA (40 nmol/L) plus A23187 (1 μmol/L). Nuclear translocation of NF-κB was assayed by Western blot (N-NF-κB, nuclear NF-κB). The band is a representative of three independent experiments. SG-HQ2: (3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide); HMC-1: human mast cells; PMA: phorbol 12-mystate 13-acetate; NF-κB: nuclear factor-kappa B

Figure 5

Effects of SG-HQ2 on the compound 48/80-induced systemic anaphylaxis. Mice (n = 5/group) were given an intraperitoneal injection of 8 mg/kg BW of the mast cell degranulator compound 48/80. SG-HQ2 was administered intraperitoneally at doses of 0.1–10 mg/kg BW 1 h before the injection of compound 48/80. The blood was obtained from the abdominal artery of each mouse to measure serum histamine level. Serum histamine level was detected by fluorescent plate reader. Each data represents the mean ± SE of three independent experiments. *Significant difference at P < 0.05. SG-HQ2: (3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide); BW: body weight; SE: standard error

Figure 6

Effects of SG-HQ2 on the IgE-mediated PCA. Ear skins of mice (n = 5/group) were sensitized with an intradermal injection of anti-DNP IgE (0.5 μg/site) for 48 h. SG-HQ2 was administered intraperitoneally at doses of 0.1–10 mg/kg BW 1 h before the intravenous injection of DNP-HSA (1 μg/mouse) and 4% Evans blue (1:1) mixture. Thirty minutes after the challenge, both ear skins were collected for measurement of the pigment dye. Each amount of dye was extracted as described in Materials and methods and detected by spectrophotometry. Each data represents the mean ± SE of three independent experiments. *Significant difference at P < 0.05. (A color version of this figure is available in the online journal.) SG-HQ2: (3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide); IgE: immunoglobulin E; PCA: passive cutaneous anaphylaxis; anti-DNP: anti-dinitrophenyl; DNP: dinitrophenyl; HSA: human serum albumin; SE: standard error