• Journal of Pharmacopuncture
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• v.15 no.4
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• pp.32-41
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• 2012

Objectives: Rehmannia glutinosa pharmacopuncture solution (RGPS) was investigated to determine both its anti-allergic inflammatory effects on mast cells and its detailed mechanism of actions. Methods: We investigated whether RGPS suppress cytokines, enzymes, $Fc{\varepsilon}RI$ expression and $Fc{\varepsilon}RI$-mediated signaling in RBL-2H3 cells stimulated with anti-DNP IgE/DNP-HSA. The suppressive effects of RGPS on the levels of cytokines such as IL-$1{\beta}$, IL-6 and GM-CSF were measured using emzyme-linked immunospecific assay (ELISA). The mRNA expression levels of cytokines, enzymes (HDC2, COX-1, COX-2 and 5LO) and $Fc{\varepsilon}RI$ ${\alpha}{\beta}{\gamma}$ subunits were measured using reverse transcription polymerase chain reaction (RT-PCR) method. The activation of $Fc{\varepsilon}RI$-mediated signaling was examined using Western blot analyses. Results: RGPS suppressed production of proinflammatory cytokines (IL-$1{\beta}$, IL-6, and GM-CSF) in stimulated RBL-2H3 cells significantly (p < 0.05). RGPS also suppressed mRNA expression of inflammatory enzymes (HDC2, COX-1, COX-2, 5LO). In addition, mRNA expression levels of $Fc{\varepsilon}RI{\alpha}$, $Fc{\varepsilon}RI{\beta}$and $Fc{\varepsilon}RI{\gamma}$ were lowered by treatment with RGPS. Finally, RGPS prevented phosphrylation of Lyn, Syk, LAT, Gab2, PLC ${\gamma}1/2$, PI3K, Akt, cPLA2 and $I{\kappa}B{\alpha}$. Conclusions: RGPS effectively suppresses mast cell activations such as degranulation and inflammatory response via down-regulation of the $Fc{\varepsilon}RI$-mediated signaling pathways in IgE/Ag-stimulated mast cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.26 no.4
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• pp.469-474
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• 2012

Fagopyrum esculentum(FE) is an important food crop and medicinal plant that is used to improve diabetes, obesity, hypertension, hypercholesterolemia and constipation in Korea, but the underlying mechanisms involved in its anti-allergic activity are not fully understood. We investigated the effects on the release of inflammatory mediator and proximal signal events in $Fc{\varepsilon}RI$-mediated RBL-2H3 cell activation. FE reduced antigen (DNP-HSA)-induced release of histamine, prostaglandin D2 (PGD2) and cysteinyl Leukotriene (cysLT) in IgE-sensitized RBL-2H3 cells. In addition, it inhibited antigen-induced HDC2 and COX-2 and 5-LO mRNA expression in IgE-sensitized RBL-2H3 cells. FE also suppressed antigen-induced $Fc{\varepsilon}RI{\beta}$ and $Fc{\varepsilon}RI{\gamma}$ subunit mRNA expression in these cells. To identify the mechanisms underpinning the inhibition of release of inflammatory mediators such as histamine and PGD2 and cysLT by FE, we examined the proximal signal events of intracellular FceRI signaling molecules. FE suppressed antigen-induced phosphorylation of Lyn, Syk, LAT, $PLC{\gamma}1$, PI3K, Akt and cPLA2. Collectively, the anti-allergic effects of FE in vitro suggest its possible therapeutic application to inflammatory allergic diseases, in which its inhibition of inflammatory mediator and FceRI-dependent signaling events in mast cells may be hugely beneficial.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.6
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• pp.393-398
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• 2012

Mast cells are involved in allergic responses, protection against pathogens and autoimmune diseases. Dexamethasone (Dex) and other glucocorticoids suppress $Fc{\varepsilon}RI$-mediated release of inflammatory mediators from mast cells. The inhibition mechanisms were mainly investigated on the downstream signaling of Fc receptor activations. Here, we addressed the effects of Dex on Fc receptor expressions in rat mast cell line RBL-2H3. We measured mRNA levels of Fc receptors by real-time PCR. As expected, Dex decreased the mRNA levels of activating Fc receptor for IgE ($Fc{\varepsilon}R$) I and increased the mRNA levels of the inhibitory Fc receptor for IgG $Fc{\gamma}RIIb$. Interestingly, Dex stimulated transcriptions of other activating receptors such as Fc receptors for IgG ($Fc{\gamma}R$) I and $Fc{\gamma}RIII$. To investigate the mechanisms underlying transcriptional regulation, we employed a transcription inhibitor actinomycin D and a translation inhibitor cycloheximide. The inhibition of protein synthesis without Dex treatment enhanced $Fc{\gamma}RI$ and $Fc{\gamma}RIII$ mRNA levels potently, while $Fc{\varepsilon}RI$ and $Fc{\gamma}RIIb$ were minimally affected. Next, we examined expressions of the Fc receptors on cell surfaces by the flow cytometric method. Only $Fc{\gamma}RIIb$ protein expression was significantly enhanced by Dex treatment, while $Fc{\gamma}RI$, $Fc{\gamma}RIII$ and $Fc{\varepsilon}RI$ expression levels were marginally changed. Our data showed, for the first time, that Dex regulates Fc receptor expressions resulting in augmentation of the inhibitory receptor $Fc{\gamma}RIIb$.

• Korean Journal of Pharmacognosy
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• v.44 no.2
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• pp.118-124
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• 2013

Mast cells is the key effector cells for IgE-mediated allergic responses. In this study, we investigated whether Rhus trichocarpa extract (RT) inhibited IgE-mediated allergic responses in mast cells and an allergic animal model. We further tried to find its mechanism of action in mast cells. We found that RT suppressed antigen-stimulated degranulation and production of TNF-${\alpha}$ and IL-4 in rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMC), as well as IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. As the mechanism of action of RT, it inhibited the activation of spleen tyrosine kinase (Syk), a pivotal signaling molecule for activation of mast cells and that of LAT, a downstream adaptor molecule of Syk in $Fc{\varepsilon}RI$-mediated signal pathways. RT also suppressed the activation of mitogen-activated protein (MAP) kinases and Akt. The current results demonstrated for the first time that RT has the anti-allergic effect through inhibiting degranulation and secretion of cytokines by suppression of Syk in antigen-stimulated mast cells. Therefore, RT might be useful for allergic diseases.

• YAKHAK HOEJI
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• v.56 no.1
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• pp.26-34
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• 2012

Mast cells play an important role in early and late phase allergic reactions through allergen and IgE-dependent release of histamine, proteases, prostaglandins, and several multifunctional cytokines. In this study, we investigated whether Cudrania tricuspidata extract (CTE) suppresses IgE-mediated allergic responses in mast cells, an allergic animal model, and its mechanism of action in mast cells. We found that CTE inhibited IgE-mediated degranulation and cytokine production in rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMC), as well as passive cutaneous anaphylaxis (PCA) in mice. With regard to its mechanism of action, CTE suppressed the activating phosphorylation of spleen tyrosine kinase (Syk), a key enzyme in mast cell signaling processes and that of LAT, a downstream adaptor molecule of Syk in $Fc{\varepsilon}RI$-mediated signal pathways. CTE also suppressed the activating phosphorylation of mitogen-activated protein (MAP) kinases and Akt. The present results strongly suggest that the anti-allergic activity of CTE is mediated through inhibiting degranulation and allergic cytokine secretion by inhibition of Syk kinase in mast cells. Therefore, CTE may be useful for the treatment of allergic diseases.

• Biomolecules & Therapeutics
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• v.20 no.1
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• pp.43-49
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• 2012

Stimulation of mast cells through the high affinity IgE receptor (Fc${\varepsilon}$RI) induces degranulation, lipid mediator release, and cytokine secretion leading to allergic reactions. Although various signaling pathways have been characterized to be involved in the Fc${\varepsilon}$RI-mediated responses, little is known about the precious mechanism for the expression of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) in mast cells. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), reduces the expression of TNF-${\alpha}$ in rat basophilic leukemia (RBL-2H3) cells. IgE or specific antigen stimulation of RBL-2H3 cells increases the expression of TNF-${\alpha}$ and activates various signaling molecules including S6K1, Akt and p38 MAPK. Rapamycin specifically inhibits antigeninduced TNF-${\alpha}$ mRNA level, while other kinase inhibitors have no effect on TNF-${\alpha}$ mRNA level. These data indicate that mTOR signaling pathway is the main regulation mechanism for antigen-induced TNF-${\alpha}$ expression. TNF-${\alpha}$ mRNA stability analysis using reporter construct containing TNF-${\alpha}$ adenylate/uridylate-rich elements (AREs) shows that rapamycin destabilizes TNF-${\alpha}$ mRNA via regulating the AU-rich element of TNF-${\alpha}$ mRNA. The antigen-induced activation of S6K1 is inhibited by specific kinase inhibitors including mTOR, PI3K, PKC and $Ca^{2+}$chelator inhibitor, while TNF-${\alpha}$ mRNA level is reduced only by rapamycin treatment. These data suggest that the effects of rapamycin on the expression of TNF-${\alpha}$ mRNA are not mediated by S6K1 but regulated by mTOR. Taken together, our results reveal that mTOR signaling pathway is a novel regulation mechanism for antigen-induced TNF-${\alpha}$ expression in RBL-2H3 cells.

• Journal of Life Science
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• v.17 no.3 s.83
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• pp.350-355
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• 2007

The rat RBL-2H3 mast cells contain various Src-family kinases. Previous reports with this cell line indicated that Lyn activation is an important initial signaling for the activation of the cells. However, the role and location of other Src-family kinase isoforms which are expressed in the cells are not clear. In this study, we now show that isoforms of Src-family kinases, Lyn, fyn, Fgr, c-Src, and Yes are differentially expressed and located differently in the cells as indicated by RT-PCR, immunoblotting analysis, and confocal microscopy. Lyn and Fgr were located on plasma membrane but on the other hand c-Src and Yes were located on intracellular organelle. All of Src-family kinases were cloned and overexpressed for investigating the roles of the isoforms. Overexpression of Fyn and Fgr, not Lyn and c-Src, stimulated Ag-induced degranulation in the cells. Our findings strongly suggest for the first time that each of Src-family kinase isoform can regulate differentially $Fc{\varepsilon}RI$-mediated signaling in RBL-2H3 mast cells.