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cumin using mast cells, major players in PSA. LTC4 is me- tabolites of arachidonic acid derived from action of 5-LO/LTC4 synthase in mast cells, and LTC4 have been implicated in in- flammation, proliferation and allergic conditions like asthma (Murphy and Gijon, 2007). Thus, we investigated the effects of curcumin on 5-LO dependent LTC4 generation in BMMCs. As shown in Fig. 2A, curcumin strongly inhibited LTC4 genera- tion in a dose-dependent manner. It is well known that Ca2+ is essential for arachidonic acid (AA) release from phospholipid and degranulation in IgE/Ag-induced mast cells (Kudo and Murakami, 1999; Yamaguchi et al., 1999), thus we examined the effect of curcumin on intracellular Ca2+ influx in IgE/Ag- induced BMMCs. As shown in Fig. 2B, intracellular Ca2+ level in activated BMMCs was about three fold higher than in rest- ing cells, and this increase was dose-dependently inhibited by curcumin. Bay 61-3606 (a Syk inhibitor) also strongly de- creased intracellular LTC4 generation as well as Ca2+ influx. Consistent with a previous report (Lee et al., 2008), the re- lease of b-Hex (a degranulation marker enzyme) was dose- dependently inhibited by curcumin (data not shown). Curcumin inhibited cPLA2 phosphorylation, translocation of phospho-cPLA2α and 5-LO and activation of MAPKs Recently, we and other group have reported that the re- lease of free AA from membrane phospholipid in activated mast cells requires the phosphorylation of cPLA2α (p-cPLA2α) by mitogen activated protein kinases (MAPKs) (Lin et al., 1993; Lu et al., 2011), and the translocation of p-cPLA2α is dependent on intracellular Ca2+ influx (Gijon and Leslie, 1999; Lu et al., 2011). To determine whether curcumin inhibits the phosphorylation and translocation of cPLA2α, we pretreated BMMCs with different concentrations of curcumin or Bay 61- 3606. As show in Fig. 3A, curcumin dose-dependently inhib- ited the phosphorylation (C-p-cPLA2α) and translocation of C-p-cPLA2α to the nuclear envelope (N-p-cPLA2α). The syn- thesis of 5-LO dependent LTC4 in IgE/Ag-induced mast cells is known to mediate the translocations of both p-cPLA2α and 5-LO to the nuclear envelope (Werz, 2002; Lu et al., 2011; Lu et al., 2012). Thus, we investigated the effect of curcumin on the translocation of 5-LO to the nuclear envelope. As was expected, curcumin or Bay 61-3606 inhibited the transloca- tion of cytosolic 5-LO (C-5-LO) to nuclear envelope (N-5-LO). Next, to confirm that the inhibition of cPLA2α phosphorylation by curcumin occurred via the inhibition of MAPKs phosphory- lation including extracellular regulated kinase1/2 (ERK1/2), c- jun N-terminal kinase (JNK), and p38 MAP kinase, therefore we examined the effect of curcumin or Bay 61-3606 on the phosphorylation of MAPKs. As shown in Fig. 3B, curcumin or Bay 61-3606 inhibited the phosphorylations of three MAPKs in a dose dependent manner. Curcumin inhibits COX-2 dependent PGD2 generation and NF-kB activation In mast cells, unlike 5-LO dependent LTC4 generation, PGD2 generation occurs in a biphasic manner. Immediate PGD2 gen- eration (occurring within 2 h), is associated with COX-1, and delayed PGD2 generation (during 2-10 h) is occurred by induc- ible COX-2 protein (Ashraf et al., 1996; Moon et al., 1998). To assess COX-2-dependent delayed PGD generation, BMMCs Fig. 5. Effect of curcumin on the Syk pathway. IgE-sensitized BMMCs were preincubated with curcumin or Bay 61-3606 for 1 h, and then stimulated with DNP-HSA for 5 min. Cell lysates were subjected to immunoprecipitation and immunoblot analysis for the phosphorylated forms of Syk, LAT and PLCγ1. Bay 61-3606 was used as a positive control with respect to the suppression of the Syk-mediated pathway. The relative ratios of p-Syk/Syk, p-LAT/LAT and p-PLCγ1/PLCγ1 protein levels were determined by measuring immunoblot band intensities by scanning densitometry (**p<0.01 and ***p<0.001). The results shown are representative of three in- dependent experiments. Li et al. Curcumin Inhibits Mast Cells Activation and Anaphylaxis 2 were pre-treated with aspirin to abolish preexisting COX-1 ac- tivity, followed by a brief wash, and then stimulated with Ag for 7 h with or without curcumin. As shown in Fig 4A, delayed 31 www.biomolther.orgPDF Image | Curcumin Inhibits the Activation of Immunoglobulin
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