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Biomol Ther 22(1), 27-34 (2014) PGD2 generation was dose-dependently inhibited by curcum- in, with a concomitant suppression of COX-2 protein expres- sion. Yu et al., reported that the Syk downstream molecules PI3K/Akt pathway affects transcription factor NF-κB activation in gastric cancer cells and mast cells (Yu et al., 2010; Lu et al., 2011) and NF-κB has been identified as an essential transcrip- tion factor for the induction of several inflammatory mediators including, TNF-α, COX-2, and inducible NO synthase (Reddy et al., 2000; Tak and Firestein, 2000; Lu et al., 2011). Thus, we examined the effect of curcumin on Akt/NF-κB axis activation. When IgE-sensitized BMMCs were pretreated with curcumin for 1 h and then stimulated with Ag for 15 min, phosphory- lation of the Akt, IKK complex (p-IKKα/β) and IκBα (p-IκBα) was increased, with a concomitant decrease of total IκBα and nuclear translocation of NF-κB (C-NF-κB). As shown in Fig. 4B, both curcumin and Bay 61-3606 inhibited the phosphory- lations of Akt, IKKα/β, IκBα, IκBα degradation and the translo- cation of cytosolic p65 to nuclear (N-NF-κB), suggesting that Syk mediated Akt/NF-κB pathway regulate the reduction of COX-2 dependent PGD2 by curcumin. Curcumin inhibited the Syk pathway in IgE/Ag-induced BMMCs Previously, we and others reported that Syk plays an es- sential role in the initiation of FceRI-induced mast cells acti- vation and mediates LAT, resulting in the activation of down- stream signaling, including MAPKs, phosphatidylinositide 3-kinase (PI3K), and PLCγ (Lin et al., 1993; Lu et al., 2011). Furthermore, it has been reported that curcumin inhibited the releases of TNF-α and IL-4 from mast cells via a Syk depen- dent pathway, and that the inhibitions of these secretions by curcumin is dependent on its direct inhibition of Syk kinase activity rather than Syk phosphorylation (Lee et al., 2008). To determine whether curcumin affects Syk phosphorylation in IgE/Ag-induced BMMCs, we examined the effects of curcumin on the phosphorylation of Syk and its downstream signal mol- ecules LAT and PLCγ1. As shown in Fig. 5A, curcumin did not affect Syk phosphorylation, but significantly and dose-depend- ently inhibited the phosphorylations of LAT and PLCγ1 (Fig. 5B, C), as previously reported (Lee et al., 2008). Bay 61-3606 (the Syk inhibitor used as a positive control) completely inhibit- ed the phosphorylations of Syk, LAT and PLCγ1. These results suggest that curcumin inhibits PGD2, LTC4, and degranulation by regulating the Syk signal pathway. DISCUSSION Curcumin has been shown to have diverse biological ac- tivities, such as, antioxidant, anti-allergic, anti-inflammatory, antiviral, antibacterial, antifungal, and anticancer activities (Ammon and Wahl, 1991). Several groups have reported that curcumin shows anti-allergic activity through the inhibition of histamine release, TNF-α and IL-4 from activated mast cells and in vivo type I hypersensitivity animal model (Yano et al., 2000; Ram et al., 2003; Lee et al., 2008). In addition, curcumin also suppresses arachidonic acid metabolizing enzymes such as cPLA2 phsophorylation, COX-2 expression and recombi- nant 5-LO activity in LPS-stimulated RAW 264.7 cells and A23187-stimulated HT human colon cancer cells (Hong et al., 2004) and inhibits in vitro LOX and COX activities in TPA- and arachidonic acid-induced inflammation in mouse epider- mis (Huang et al., 1991). Furthermore, Curcumin modulates the inflammatory response by down-regulating the activity of COX-2 and inducible nitric oxide synthase (iNOS) enzymes through suppression of NF-κB activation (Surh et al., 2001). Even though they reported that curcumin inhibited generation of COX-dependent PGs and LOX-dependent hydroxyeicosa- tetraenoic acids (HETEs), previous reports were mainly exam- ined as a part of anticancer activity of curcumin. However, the effect of curcumin on generation of COX-2 dependent PGD2 and 5-LO dependent LTC4 generation in IgE/Ag-induced mast cells and IgE-mediated PSA reaction have not been studied to date. Therefore, we investigated the effects of curcumin on the generation of eicosanoid (PGD2 and LTC4) in BMMCs and on PSA reaction in mice. It has been well established that lipid mediators, like LTC4 and PGD2, are closely associated with various allergic and inflammatory diseases (Werz and Stein- hilber, 2006; Ricciotti and FitzGerald, 2011). Thus, the inhibi- tion of LTC4 and PGD2 generation by mast cells is an important therapeutic strategy in the context of allergic-inflammatory dis- ease. The aim of this study was to investigate the effect of curcumin on IgE/Ag-induced COX-dependent PGD2 and 5-LO dependent LTC4 generation in BMMCs and on IgE-induced passive systemic anaphylaxis (PSA) in mice. IgE binds to FceRI on mast cells, the Syk-LAT axis activates PLCγ, which increases intracellular Ca2+ influx and leads to degranulation and eicosanoid production (Siraganian, 2003). As described above, IgE binds to FceRI on BMMCs, it promptly elicits 5-LO dependent LTC4 generation, which is inhibited dose-depend- ently by curcumin (Fig. 2A). It has been reported that the syn- thesis of LTC4 in mast cells is regulated by two steps, namely, AA release from phospholipid by cPLA2α, MAPKs-mediated phosphorylation of cPLA2α and the conversion of free AA to LTC4 by 5-LO (Fischer et al., 2005). Translocation of both 5-LO and phospho-cPLA2α (C-p-cPLA2α) to the nuclear enve- lope are depend on the increase of cytosolic Ca2+ level (Werz, 2002; Lu et al., 2011; Lu et al., 2012). The present study showed that curcumin inhibited the translocations of both en- zymes, which concurred with its observed inhibitory effect on intracellular Ca2+ influx (Fig. 2B). Next, to elucidate the effect of curcumin on COX-2 dependent delayed PGD2 generation, BMMCs were pre-treated with aspirin to abolish preexisting COX-1 activity, and then stimulated with Ag for 7 h with or without curcumin. We found that curcumin also suppressed COX-2 expression and attendant PGD2 generation (Fig. 4A). It has been previously reported that curcumin inhibited the TPA- induced up-regulation of COX-2 and MMP-9 by suppressing ERK1/2 phosphorylation and NF-κB transactivation in epithe- lial cells (Lee et al., 2005), and that it exerts anti-inflammatory effects by inhibiting the NF-κB and MAPKs pathways (Cho et al., 2007). Recently, we also reported that COX-2-dependent PGD2 generation and COX-2 expression in BMMCs occurs via the activation of the NF-κB and MAPKs pathways (Lu et al., 2011; Lu et al., 2012), thus we investigated the effect of curcumin on the MAPKs and NF-κB pathways in IgE/Ag-in- duced BMMCs. As shown in Fig. 3B, curcumin suppressed the phosphorylations of ERK1/2, JNK, and p38 MAP kinase in a dose dependent manner, which implied that it affects the phosphorylation and translocation of cPLA2. Since curcumin or Bay 61-3606 suppressed intracellular Ca2+ influx (Fig. 2B), MAPKs phosphorylation (Fig. 3B) and Akt/ NF-κB axis activa- tion, these results suggest that Syk plays an important role in the generation of PGD2 and LTC4 in IgE/Ag-induced BMMCs. Therefore, we examined whether curcumin affects the phos- http://dx.doi.org/10.4062/biomolther.2013.092 32PDF Image | Curcumin Inhibits the Activation of Immunoglobulin
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