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Imperatorin Suppresses Degranulation and Eicosanoid Generation in Activated Bone Marrow-Derived Mast Cells.

Jeong KT, Lee E, Park NY, Kim SG, Park HH, Lee J, Lee YJ, Lee E - Biomol Ther (Seoul) (2015)

Bottom Line: To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC.Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC.These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Research and Development Division, Korea Promotion Institute for Traditional Medicine Industry, Gyeongsan 712-210.

ABSTRACT
Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

No MeSH data available.


Related in: MedlinePlus

Effect of imperatorin on cell viability (A), degranulation (B), intracellular Ca2+ level (C), and PLCγ1 phosphorylation (D) in IgE/Ag-stimulated BMMC. Cell viability was measured using an MTS assay (A). BMMC sensitized overnight with IgE were pre-incubated with the indicated concentrations of imperatorin for 1 h and stimulated with DNP-HAS (Ag, 100 ng/ml) for 15 min. β-HEX release into the supernatant was determined (B) and BMMC were used for Western blotting (D). IgE-sensitized BMMC pretreated with FluoForteTM Dye-Loading Solution were seeded into 96-well microplates, pre-incubated with imperatorin, and stimulated with DNP-HAS. Relative Ca2+ levels were measured using Multilabel Plate Reader (C). The values are expressed as the means ± S.E.M. of three different samples. *p<0.05, **p<0.01, ***p<0.001, significantly different from the control as determined by one-way ANOVA.
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f1-bt-23-421: Effect of imperatorin on cell viability (A), degranulation (B), intracellular Ca2+ level (C), and PLCγ1 phosphorylation (D) in IgE/Ag-stimulated BMMC. Cell viability was measured using an MTS assay (A). BMMC sensitized overnight with IgE were pre-incubated with the indicated concentrations of imperatorin for 1 h and stimulated with DNP-HAS (Ag, 100 ng/ml) for 15 min. β-HEX release into the supernatant was determined (B) and BMMC were used for Western blotting (D). IgE-sensitized BMMC pretreated with FluoForteTM Dye-Loading Solution were seeded into 96-well microplates, pre-incubated with imperatorin, and stimulated with DNP-HAS. Relative Ca2+ levels were measured using Multilabel Plate Reader (C). The values are expressed as the means ± S.E.M. of three different samples. *p<0.05, **p<0.01, ***p<0.001, significantly different from the control as determined by one-way ANOVA.

Mentions: For assessing the cytotoxic activity, BMMC were treated with various concentrations of imperatorin for 7 h and cell viability was not affected upto 50 μM of imperatorin (Fig. 1A). When mast cells are activated, one of the primary released mediators is histamine, which is stored in preformed granules. Because the release of histamine by activated mast cells parallels β-HEX release, we first examined the inhibitory effect of imperatorin on β-HEX release. IgE-sensitized BMMC were pre-treated with different concentrations of imperatorin for 1 h followed by 15 min of stimulation with Ag. The supernatants were collected for determination of β-HEX. As shown in Fig. 1B, Ag treatment significantly increased β-HEX release and pre-treatment with imperatorin resulted in a dose-dependent suppression of β-HEX release, with an IC50 value of 15.9 μg/ml. Because elevated intracellular Ca2+ concentration is known to cause mast cell degranulation and eicosanoid generation (Fischer et al., 2005), we examined whether imperatorin affects intracellular Ca2+ levels in activated BMMC. The activation of BMMC induced an increase in the intracellular Ca2+ level and this increase was inhibited by imperatorin treatment (Fig. 1C). To further study the inhibitory effect of imperatorin on degranulation, we examined PLCγ1 activation in BMMC. The result showed that phosphorylation of PLCγ1 was upregulated by Ag treatment of IgE-sensitized BMMC. However, pre-treatment of BMMC with imperatorin suppressed PLCγ1 phosphorylation in a dose-dependent manner (Fig. 1D), indicating that the inhibitory effect of imperatorin on degranulation was mediated through inhibition of PLCγ1 phosphorylation.


Imperatorin Suppresses Degranulation and Eicosanoid Generation in Activated Bone Marrow-Derived Mast Cells.

Jeong KT, Lee E, Park NY, Kim SG, Park HH, Lee J, Lee YJ, Lee E - Biomol Ther (Seoul) (2015)

Effect of imperatorin on cell viability (A), degranulation (B), intracellular Ca2+ level (C), and PLCγ1 phosphorylation (D) in IgE/Ag-stimulated BMMC. Cell viability was measured using an MTS assay (A). BMMC sensitized overnight with IgE were pre-incubated with the indicated concentrations of imperatorin for 1 h and stimulated with DNP-HAS (Ag, 100 ng/ml) for 15 min. β-HEX release into the supernatant was determined (B) and BMMC were used for Western blotting (D). IgE-sensitized BMMC pretreated with FluoForteTM Dye-Loading Solution were seeded into 96-well microplates, pre-incubated with imperatorin, and stimulated with DNP-HAS. Relative Ca2+ levels were measured using Multilabel Plate Reader (C). The values are expressed as the means ± S.E.M. of three different samples. *p<0.05, **p<0.01, ***p<0.001, significantly different from the control as determined by one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556201&req=5

f1-bt-23-421: Effect of imperatorin on cell viability (A), degranulation (B), intracellular Ca2+ level (C), and PLCγ1 phosphorylation (D) in IgE/Ag-stimulated BMMC. Cell viability was measured using an MTS assay (A). BMMC sensitized overnight with IgE were pre-incubated with the indicated concentrations of imperatorin for 1 h and stimulated with DNP-HAS (Ag, 100 ng/ml) for 15 min. β-HEX release into the supernatant was determined (B) and BMMC were used for Western blotting (D). IgE-sensitized BMMC pretreated with FluoForteTM Dye-Loading Solution were seeded into 96-well microplates, pre-incubated with imperatorin, and stimulated with DNP-HAS. Relative Ca2+ levels were measured using Multilabel Plate Reader (C). The values are expressed as the means ± S.E.M. of three different samples. *p<0.05, **p<0.01, ***p<0.001, significantly different from the control as determined by one-way ANOVA.
Mentions: For assessing the cytotoxic activity, BMMC were treated with various concentrations of imperatorin for 7 h and cell viability was not affected upto 50 μM of imperatorin (Fig. 1A). When mast cells are activated, one of the primary released mediators is histamine, which is stored in preformed granules. Because the release of histamine by activated mast cells parallels β-HEX release, we first examined the inhibitory effect of imperatorin on β-HEX release. IgE-sensitized BMMC were pre-treated with different concentrations of imperatorin for 1 h followed by 15 min of stimulation with Ag. The supernatants were collected for determination of β-HEX. As shown in Fig. 1B, Ag treatment significantly increased β-HEX release and pre-treatment with imperatorin resulted in a dose-dependent suppression of β-HEX release, with an IC50 value of 15.9 μg/ml. Because elevated intracellular Ca2+ concentration is known to cause mast cell degranulation and eicosanoid generation (Fischer et al., 2005), we examined whether imperatorin affects intracellular Ca2+ levels in activated BMMC. The activation of BMMC induced an increase in the intracellular Ca2+ level and this increase was inhibited by imperatorin treatment (Fig. 1C). To further study the inhibitory effect of imperatorin on degranulation, we examined PLCγ1 activation in BMMC. The result showed that phosphorylation of PLCγ1 was upregulated by Ag treatment of IgE-sensitized BMMC. However, pre-treatment of BMMC with imperatorin suppressed PLCγ1 phosphorylation in a dose-dependent manner (Fig. 1D), indicating that the inhibitory effect of imperatorin on degranulation was mediated through inhibition of PLCγ1 phosphorylation.

Bottom Line: To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC.Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC.These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Research and Development Division, Korea Promotion Institute for Traditional Medicine Industry, Gyeongsan 712-210.

ABSTRACT
Imperatorin has been known to exert many biological functions including anti-inflammatory activity. In this study, we investigated the inhibitory effects of imperatorin on the production of inflammatory mediators in mouse bone marrow-derived mast cells (BMMC). Imperatorin inhibited degranulation and the generation of eicosanoids (leukotriene C4 (LTC4) and prostaglandin D2 (PGD2)) in IgE/antigen (Ag)-stimulated BMMC. To elucidate the molecular mechanism involved in this process, we investigated the effect of imperatorin on intracellular signaling in BMMC. Biochemical analyses of the IgE/Ag-mediated signaling pathway demonstrated that imperatorin dramatically attenuated degranulation and the production of 5-lipoxygenase-dependent LTC4 and cyclooxygenase-2-dependent PGD2 through the inhibition of intracellular calcium influx/phospholipase Cγ1, cytosolic phospholipase A2/mitogen-activated protein kinases and/or nuclear factor-κB pathways in BMMC. These results suggest that the effects of imperatorin on inhibition of degranulation and eicosanoid generation through the suppression of multiple steps of IgE/Ag-mediated signaling pathways would be beneficial for the prevention of allergic inflammation.

No MeSH data available.


Related in: MedlinePlus