• 한방안이비인후피부과학회지
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• 제25권3호
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• pp.1-12
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• 2012

Objectives : Fagopyrum esculentum(FE) has been used for removal of inflammation of internal organs and treatment of sore and ulcer by heat toxin in Korean herbal medicines. In this study, To investigated the protective effect of FE on allergic response, we determined whether FE inhibits allergic response. Methods : The effect of FE was analyzed by ELISA, RT-PCR and Western blot in RBL-2H3 cells. We investigated cell viability, ${\beta}$-hexosaminidase, as a marker of degranulation, cytokne, and intracellular ROS and MAPK and NF-${\kappa}B$ signaling. Results : We found that FE suppressed ${\beta}$-hexosaminidase release, the production of IL-4 and TNF-${\alpha}$ and intracellular ROS level in RBL-2H3 by the anti-DNP IgE plus DNP-HSA stimulation. FE also significantly inhibited cytokine mRNA expressions, such as IL-$1{\beta}$, IL-2, IL-3, IL-4, IL-5, IL-6, IL-13, TNF-${\alpha}$ and GM-CSF in RBL-2H3. In addition, PF suppressed the phospholyation of ERK1/2, JNK1/2, p38 and $I{\kappa}B{\alpha}$ and NF-${\kappa}B$ signal transduction pathway. Conclusions : Our results indicate that FE protects against allergic response and exerts an anti-inflammatory effect through the inhibition of degranulation and production of cytokines and ROS via the suppression MAPK and NF-${\kappa}B$ of signal transduction. Abbrevations : FE, Fagopyrum esculentum; RBL-2H3, rat basophilic leukemia cell line; ROS, reactive oxygen species; MAPK, Mitogen-activated protein kinase; $NF{\kappa}B$, nuclear factor ${\kappa}B$; $TNF{\alpha}$, Tumor necrosis factor alpha; GM-CSF, Granulocyte macrophage colony-stimulating factor; ERK, extracellular-signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; p38, p38 MAP kinase; $I{\kappa}B{\alpha}$, inhibitory-kappa B alpha.

• Molecules and Cells
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• 제21권1호
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• pp.42-51
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• 2006

We investigated the mechanism of contraction induced by S1P in esophageal smooth muscle cells. Western blot analysis demonstrated that $S1P_1$, $S1P_2$, $S1P_3$, and $S1P_5$ receptors existed in the cat esophagus. Only penetration of EDG-5 ($S1P_2$) antibody into permeabilized cells inhibited S1P-induced contraction. Pertussis toxin (PTX) also inhibited contraction, suggesting that it was mediated by $S1P_2$ receptors coupled to a PTXsensitive $G_i$ protein. Specific antibodies to $G_{i2}$, $G_q$ and $G_{\beta}$ inhibited contraction, implying that the S1P-induced contraction depends on PTX-insensitive $G_q$ and $G_{\beta}$ dimers as well as the PTX-sensitive $G_{i2}$. Contraction was not affected by the phospholipase $A_2$ inhibitor DEDA, or the PLD inhibitor ${\rho}$-chloromercuribenzoate, but it was abolished by the PLC inhibitor U73122. Incubation of permeabilized cells with $PLC{\beta}3$ antibody also inhibited contraction. Contraction involved the activation of a PKC pathway since it was affected by GF109203X and chelerythrine. Since $PKC{\varepsilon}$ antibody inhibited contraction, $PKC{\varepsilon}$ may be required. Preincubation of the muscle cells with the MEK inhibitor PD98059 blocked S1P-induced contraction, but the p38 MAP kinase inhibitor SB202190 did not. In addition, co-treatment of cells with GF 109203X and PD98059 did not have a synergistic effect, suggesting that these two kinases are involved in the same signaling pathway. Our data suggest that S1P-induced contraction in esophageal smooth muscle cells is mediated by $S1P_2$ receptors coupled to PTX-sensitive $G_{i2}$ proteins, and PTX-insensitive $G_q$ and $G_{\beta}$ proteins, and that the resulting activation of the $PLC{\beta}3$ and $PKC{\varepsilon}$ pathway leads to activation of a p44/p42 MAPK pathway.

• International Journal of Oral Biology
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• 제37권3호
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• pp.137-145
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• 2012

Aggregatibacter actinomycetemcomitans is the most important etiologic agent of aggressive periodontitis and can interact with endothelial cells. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are chemokines, playing important roles in periodontal pathogenesis. In our current study, the effects of A. actinomycetemcomitans on the production of MCP-1 and IL-8 by human umbilical vein endothelial cells (HUVEC) were investigated. A. actinomycetemcomitans strongly induced the gene expression and protein release of both MCP-1 and IL-8 in a dose- and time-dependent manner. Dead A. actinomycetemcomitans cells were as effective as live bacteria in this induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, did not affect the mRNA up-regulation of MCP-1 and IL-8 by A. actinomycetemcomitans. However, genistein, an inhibitor of protein tyrosine kinases, substantially inhibited the MCP-1 and IL-8 production by A. actinomycetemcomitans, whereas pharmacological inhibition of each of three members of mitogen-activated protein (MAP) kinase family had little effect. Furthermore, gel shift assays showed that A. actinomycetemcomitans induces a biphasic activation (early at 1-2 h and late at 8-16 h) of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and an early brief activation (0.5-2 h) of activator protein-1 (AP-1). Activation of canonical NF-${\kappa}B$ pathway ($I{\kappa}B$ kinase activation and $I{\kappa}B-{\alpha}$ degradation) was also demonstrated in these experiments. Although lipopolysaccharide from A. actinomycetemcomitans also induced NF-${\kappa}B$ activation, this activation profile over time differed from that of live A. actinomycetemcomitans. These results suggest that the expression of MCP-1 and IL-8 is potently increased by A. actinomycetemcomitans in endothelial cells, and that the viability of A. actinomycetemcomitans and bacterial internalization are not required for this effect, whereas the activation of protein tyrosine kinase(s), NF-${\kappa}B$, and AP-1 appears to play important roles. The secretion of high levels of MCP-1 and IL-8 resulting from interactions of A. actinomycetemcomitans with endothelial cells may thus contribute to the pathogenesis of aggressive periodontitis.

• KSBB Journal
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• 제20권1호
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• pp.21-25
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• 2005

흔하게 사용되어온 제초제인 paraquat는 파킨슨병의 원인이 될 수 있는 유력한 위험 요소이다. 헴산화효소-1(HO-1)은 산화적 스트레스와 소포체 스트레스의 marker인데, 여러 가지 자극에 의해 heme을 분해하여 biliverdin, 일산화탄소, 철 성분으로 전환시킨다 본 연구에서는 뇌의 흑색질 유래의 도파민 세포주 SN4741에서 paraquat가 시간별, 농도별로 HO-1을 활성화시키는 기작을 조사하였다. HO-1이 Paraquat에 의해 활성화되는 것은 주로 유전자 전사 수준에서 조절되었다. HO-1 유전자의 promoter와 5' enhancer인 El, E2를 결실시킨 실험에서, E2 enhancer가 도파민 세포에서 paraquat에 의한 HO-1 유전자 발현을 유도하는 핵심 부위로 판명되었다 E2 enhancer 부위를 돌연변이 시킨 실험 결과는 전사인자 활성 단백질-1 (AP-1) 결합부위를 통해 HO-1 발현이 유도됨을 밝히게 되었다. 또한, 도파민 세포에서 HO-1 유전자 발현의 조절과 신호전달 과정의 관계를 조사하기 위해 MAP kinase들의 특이적 저해제를 처리하고 paraquat로 자극을 준 결과, JNK 저해제인 SP600125가 가장 현저하게 paraquat에 의한 HO-1 발현을 억제하였다. 결론적으로, 도파민 세포에서 paraquat가 HO-1을 유도하는 데는 E2 enhancer가 중요하게 작용하고, AP-1과 JNK 경로를 통해 HO-1 발현이 조절된다는 사실을 처음으로 밝히게 되었다.

• 대한영양사협회학술지
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• 제25권3호
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• pp.217-228
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• 2019

There have been no published studies concerning the anti-inflammatory effects of corn silk on colon cancer cells. Thus, this study was conducted to investigate the effect of corn silk extract containing high levels of maysin on inflammation and its mechanism of action in colon cancer cells. SW 480 human colon cancer cells were treated with $1{\mu}g/mL$ of lipopolysaccharide (LPS) to induce inflammation, and next they were treated with different concentrations of corn silk extract (0, 5, 10 and $15{\mu}g/mL$). The concentrations of nitric oxide (NO) were determined. The mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor ${\alpha}$ ($TNF-{\alpha}$), interleukin-1beta ($IL-1{\beta}$) and interleukin-6 (IL-6), were determined. Western blot analysis was performed to determine the protein expressions of nuclear factor-kappa B ($NF-{\kappa}B$) and mitogen-activated protein kinases, and the latter consists of extracellular signal-related kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 MAP kinase (p38). The concentration of NO and the mRNA expression of iNOS were significantly and dose-dependently decreased in the corn silk-treated groups (P<0.05). The mRNA expression of $TNF-{\alpha}$, $IL-1{\beta}$ and IL-6 were significantly increased in the LPS-treated group (P<0.05), but these expressions were significantly and dose-dependently decreased in the corn silk treated groups (P<0.05). The protein expressions of $NF-{\kappa}B$ (in a dose-dependent fashion), ERK (at 10 and $15{\mu}g/mL$), JNK (at $15{\mu}g/mL$) and p38 (at 10 and $15{\mu}g/mL$) were significantly decreased with corn silk treatments (P<0.05). In conclusion, corn silk extract containing high levels of maysin seems to inhibit the LPS-induced inflammatory responses in SW480 colon cancer cells via the $NF-{\kappa}B$ pathway.

• Archives of Pharmacal Research
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• 제23권6호
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• pp.605-612
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• 2000

Green tea polyphenols (GTP) have been demonstrated to suppress tumorigenesis in several chemical-induced animal carcinogenesis models, and predicted as promising chemopreventive agents in human. Recent studies of GTP extracts showed the involvement of mitogen-activated protein kinases (MAPKs) in the regulation of Phase II enzymes gene expression and induction of apoptosis. In the current work we compared the biological actions of five green tea catechins: (1) induction of ARE reporter gene, (2) activation of MAP kinases, (3) cytotoxicity in human hepatoma HepG2-C8 cells, and (4) caspase activation in human cervical squamous carcinoma HeLa cells. For the induction of phase IIgene assay, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) potently induced antioxidant response element (ARE)-mediated luciferase activity, with induction observed at 25 $\mu\textrm{m}$with EGCG. The induction of ARE reporter gene appears to be structurally related to the 3-gallate group. Comparing the activation of MAPK by the five polyphenols, only EGCG showed potent activation of all three MAPKs (ERK, JNK and p38) in a dose- and time-dependent manner, whereas EGC activated ERK and p38. In the concentration range of 25 $\mu\textrm{m}$ to 1 mM, EGCG and ECG strongly suppressed HepG2-ARE-C8 cell-growth. To elucidate the mechanisms of green tea polyphenol-induced apoptosis, we measured the activation of an important cell death protein, caspase-3 induced by EGCG, and found that caspase-3 was activated in a dose- and time-dependent manner. Interestingly, the activation of caspase-3 was a relatively late event (peaked at 16 h), whereas activation of MAPKs was much earlier (peaked at 2 h). It is possible, that at low concentrations of EGCG, activation of MAPK leads to ARE-mediated gene expression including phase II detoxifying enzymes. Whereas at higher concentrations of EGCG, sustained activation of MAPKs such as JNK leads to apoptosis. These mechanisms are currently under investigation in our laboratory. As the most abundant catechin in GTP extract, we found that EGCG potently induced ARE-mediated gene expression, activated MAP kinase pathway, stimulated caspase-3 activity, and induced apoptosis. These mechanisms together with others, may contribute to the overall chemopreventive function of EGCG itself as well as the GTP.

• IMMUNE NETWORK
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• 제2권2호
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• pp.72-78
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• 2002

Background: The precise mechanism by which CTLA-4 regulates T cell immune responses is still not fully understood. Previously we proposed that CTLA-4 could downregulate T cell function by modulating a signaling cascade initiated from the T cell receptor complex. The evidence for this notion comes from our findings that CTLA-4 associated with the T cell receptor zeta (TCR zeta) chain, and hence regulated TCR zeta phosphorylation by co-associated SHP-2 tyrosine phosphatase (1). In this report, we investigated whether any other signaling molecules could be involved in the CTLA-4 signaling pathway. Methods: We have taken biochemical approaches, such as immunoprecipitation followed by autoradiography or immunoblotting, to identify the molecules associated with CTLA-4. To perform these assays, we used activated primary T cells and ectopically transfected 293 cells. Various truncation mutants of CTLA-4 were used to map the interaction site on CTLA-4. Results: We found that in addition to TCR zeta and SHP-2, a recently cloned small adaptor molecule, SAP (SLAM-associated protein), was also able to associate with CTLA-4. We identified the domain of SAP association in CTLA-4 being a motif involving GVYVKM. This motif has been previously found to bind SHP-2 through its phosphorylated tyrosine interaction with SH-2 domain of SHP-2. Indeed, co-expression of SAP and SHP-2 reduced their binding to CTLA-4 significantly, suggesting that SAP and SHP-2 compete for the common binding site, GVYVKM. Thus, by blocking SHP-2 recruitment SAP could function as a negative regulator of CTLA-4. Conclusion: Taken together, our data suggest the existence of complicate signaling cascade in regulating CTLA-4 function, and further provide evidence that SAP can act either as a positive or negative regulator depending on the nature of the associating receptors.

• The Korean Journal of Physiology and Pharmacology
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• 제19권2호
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• pp.141-149
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• 2015

"G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic ${\beta}$-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with $30{\mu}M$ linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus.

• International Journal of Oral Biology
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• 제39권1호
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• pp.15-22
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• 2014

Aggregatibacter actinomycetemcomitans is an important pathogen in the development of localized aggressive periodontitis. Lipopolysaccharide (LPS) is a virulent factor of periodontal pathogens that contributes to alveolar bone loss and connective tissue degradation in periodontal disease. Our present study was designed to investigate the cytokine expression and signaling pathways regulated by A. actinomycetemcomitans LPS (Aa LPS). Cytokine gene expression profiling in RAW 264.7 cells was performed by microarray analyses. The cytokine mRNA and protein levels and related signaling pathways induced by Aa LPS were measured by RT-PCR, ELISA and western blotting. Microarray results showed that Aa LPS strongly induced the expression of NF-${\kappa}B$, NF-${\kappa}B$-related genes, inflammatory cytokines, TNF-${\alpha}$ and IL-$1{\beta}$ in RAW 264.7 cells. NF-${\kappa}B$ inhibitor pretreatment significantly reduced the levels of TNF-${\alpha}$ and IL-$1{\beta}$ mRNA and protein. In addition, the Aa LPS-induced TNF-${\alpha}$ and IL-$1{\beta}$ expression was inhibited by p38/JNK MAP kinase inhibitor pretreatment. These results show that Aa LPS stimulates TNF-${\alpha}$ and IL-$1{\beta}$ expression through NF-${\kappa}B$ and p38/JNK activation in RAW 264.7 cells, suggesting the essential role of this pathway in the pathogenesis of localized aggressive periodontitis.

• KSBB Journal
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• 제29권1호
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• pp.50-57
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• 2014

NF-${\kappa}B$ is a transcriptional factor which is involved in many biological processes including immunity, inflammation, and cell survival. Many investigators studied on the mechanism involved in activation of NF-${\kappa}B$ signalling pathway via ubiquitination and degradation of $I{\kappa}B$ regarding skin disease. Some specific molecules including Akt, MEK, p38 MAP Kinase, Stat3, et al. represent convergence points and key regulatory proteins in signaling pathways controlling cellular events such as growth and differentiation, energy homeostasis, and the response to stress and inflammation. Ultraviolet (UV) irradiation has many adverse effects on skin, including inflammation, alteration in the extracellular matrix, cellular senescence, apoptosis and skin cancer. Prunus mume, a naturally derived plant extract, has beneficial biological activities as blood fluidity improvement, anti-fatigue action, antioxidative and free radical scavenging activities, inhibiting the motility of Helicobacter pyolri. Previous reports on various beneficial function prompted us to investigate UVB-induced or other immunostimulated biological marker regarding P. mume extract. P. mume extract suppresses UVB-induced cyclooxygenase-2 (COX-2) expression in mouse skin epidermal JB6 P+ cells. The activation of activator protein-1 and nuclear factor-${\kappa}B$ induced by UVB was dose-dependently inhibited by P. mume extract treatment. This results suggest that P. mume extracts might be used as a potential agents for protection of inflammation or UVB induced skin damage.