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Curcumin Inhibits the Activation of Immunoglobulin

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Curcumin Inhibits the Activation of Immunoglobulin ( curcumin-inhibits-activation-immunoglobulin )

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Biomol Ther 22(1), 27-34 (2014) Curcumin (diferuloyl methane) is a major constituent of the rhizome of Curcuma longa, and is used traditionally to treat in- flammation, gastrointestinal disorders, hepatic disorders, dia- betic wounds, skin wounds, rheumatism, sinusitis, and other disorders (Ammon and Wahl, 1991). Furthermore, scientific studies have shown that curcumin inhibits histamine release and the secretions of tumor necrosis factor-a (TNF-a) and interleukin-4 (IL-4) from mast cells triggered by IgE, calcium ionophore A23187, or compound 48/80 (Suzuki et al., 2005; Lee et al., 2008; Choi et al., 2010). Curcumin has also been reported to inhibit IgE-induced type I hypersensitivity and ovalbumin-induced airway hyperreactivity (Yano et al., 2000; Ram et al., 2003; Lee et al., 2008), and to inhibit COX-2 gene expression in phorbol ester-treated human gastrointestinal epithelial cells and mouse skin (Chun et al., 2003; Ricciotti and FitzGerald, 2011) and in vitro lipoxygenase and cyclooxy- genase activities in mouse epidermis (Huang et al., 1991). However, the effect of curcumin on IgE/Ag-induced COX-2 de- pendent PGD2 and 5-LO dependent LTC4 generation in mast cells and IgE-mediated systemic anaphylactic response have not been well investigated. In this study, we evaluated the effects of curcumin on the generation of eicosanoid (PGD2 and LTC4) in FcεRI-induced mast cells and on passive systemic anaphylaxis (PSA) re- sponse in mice. MATERIALS AND METHODS Plant material Curcumin was isolated from the ethyl acetate fraction of a methanol extract of the rhizome of Curcuma longa, as de- scribed previously (Kiuchi et al., 1993), and produced a sin- gle TLC spot and had a HPLC determined purity of >99.5%. Curcumin was prepared by dissolving it in dimethyl sulfoxide (DMSO). The final concentration of DMSO in culture media was adjusted to 0.1% (v/v). DMSO alone was run as a control in all cases. Chemicals and reagents Mouse anti-dinitrophenyl (DNP) IgE and DNP-human se- rum albumin (HSA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fexofenadine-HCl (Fexo), a histamine H1 receptor antagonist, was obtained from Korea Pharma (Seoul, Korea). The rabbit polyclonal antibodies specific for phospho-IκB, IKKa/b, ERK1/2, JNK, p38, Akt, b-actin, and to- tal form for IκB, ERK1/2, JNK, p38, and Akt were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal antibodies for phospho-cPLA2a (Ser505), cPLA2a, 5-LO, PLCγ1, IKKa/b lamin B and NF-κB p65 as well as secondary goat anti-rabbit IgG-HRP and rabbit anti-goat IgG-HRP antibodies, total Syk, total LAT, and Bay 61-3606 were purchased from Santa Cruz Biotechnology (Dallas, Tex- as, USA) and antibodies for phosphotyrosine was purchased from Millipore (Millipore, Billerica, MA, USA). The antibody- reactive bands were visualized with an enhanced chemilumi- nescence (ECL) system (Pierce Biotechnology, Rockford, IL, USA). The enzyme immnoassay (EIA) kits for PGD2, LTC4, histamine and the antibody for COX-2 were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Culture and activation of bone marrow derived mast cells (BMMCs) BMMCs were isolated from bone marrow of C57BL/6 mice and differentiated as described previously (Lu et al., 2011). Briefly, BMMCs were cultured in RPMI 1640 medium con- taining 10% fetal bovine serum, 100 U/ml penicillin, 10 mM HEPES, 100 mM MEM non-essential amino acid solution (Invit- rogen, Grand Island, NY, USA) and 20% pokeweed mitogen- spleen cell conditioned medium as a source of IL-3. For stimu- lation, 106 cells/ml were sensitized overnight with 500 ng/ml anti-DNP IgE, pretreated with indicated concentration of cur- cumin or Bay 61-3606, and stimulated for appropriate periods with 100 ng/ml DNP-HSA. The reactions were terminated by centrifugation of the cells at 3,000 rpm for 5 min at 4°C. Determination of LTC4 and PGD2 Concentration of LTC4 and PGD2 were determined as de- scribed previously (Lu et al., 2011). IgE sensitized BMMCs suspended in enriched medium at a cell density of 1×106 cells/ ml were pretreated with indicated concentration of curcumin or Bay 61-3606 for 1 h and stimulated with DNP-HSA for 15 min. Supernatants were isolated for further analysis by EIA kit. The concentration of LTC4 was determined using an EIA kit. To assess COX-2-dependent PGD2 synthesis, BMMCs were preincubated with 1 mg/ml of aspirin for 2 h to irreversibly in- activate preexisting COX-1. After washing, BMMCs were in- cubated with 100 ng/ml DNP-HSA at 37oC for 7 h in the pres- ence of curcumin or Bay 61-3606. PGD2 in the supernatants were quantified using PGD2 EIA kit and cells were used for immunoblots analysis. Under the conditions employed, LTC4 reached 4.75 ng/106 cells and PGD2 generation reached 2.12 ng/106 cells. All data were the arithmetic mean of triplicate de- terminations. Measurement of intracellular Ca2+ level Intracellular Ca2+ levels were determined using FluoForteTM Calcium Assay Kit (Enzo Life Sciences, Ann Arbor, MI, USA), as described previously (Hwang et al., 2013). Briefly, BMMCs (1×106 cells) were sensitized overnight with 500 ng/ml anti- DNP IgE. Sensitized BMMCs were preincubated with FluoFor- teTM Dye-Loading Solution for 1 h at room temperature. After washing the dye from cell surface with HBSS, cells (5×104) were seeded into 96-well microplates and pretreated with cur- cumin or Bay 61-3606 for 1 h before adding DNP-HSA. Fluo- rescence was measured using a fluorometric imaging plated reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm on a BMG Labtechnologies FLUOStar OPITIMA platereader (Offenburg, Germany). Preparation of nuclear and cytoplasmic extracts The nuclear and cytoplasmic extracts were prepared as de- scribed previously (Lu et al., 2011). BMMCs were sensitized to DNP-specific IgE (500 ng/ml, overnight) and pretreated with curcumin or Bay 61-3606 for 1 h, and then stimulated with DNP-HSA (100 ng/ml) for 30 min. Cultured BMMCs were collected by centrifugation, washed with PBS, and lysed in a buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF and 0.1% NP40 on ice for 10 min. Supernatants (cytosolic fractions) were obtained by centrifugation at 1,000 g for 4 min. Nuclear pellets were washed and lysed in a buffer containing 20 mM HEPES (pH 7.9), 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM http://dx.doi.org/10.4062/biomolther.2013.092 28

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