• Molecular & Cellular Toxicology
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• v.1 no.3
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• pp.196-202
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• 2005

Nitric oxide (NO) is a small, diffusible, and highly reactive molecule, which plays dichotomous regulatory roles under physiological and pathological conditions. NO promotes apoptosis in some cells, and inhibits apoptosis in other cells. In the present study, we attempted to characterize the NO signaling pathway and cellular response in PC12 cells treated with cytokines. $IFN{\gamma}\;and\;TNF{\alpha}$ treatment resulted in a synergistic increase of nitrite accumulation, with the induction of inducible nitric oxide synthase (iNOS) in the PC12 cells. Moreover, as nitrite concentration increased, cell viability decreased. In order to explore MAP kinase involvement in nitric oxide production resultant from $IFN{\gamma}\;and\;TNF{\alpha}$ stimulation, we measured the activation of MAP kinase using specific MAP kinase inhibitors. PC12 cells pretreated with SB203580, a p38 MAP kinase-specific inhibitor, resulted in the inhibition of iNOS expression and NO production. However, PD98059, an ERK/MAP kinase-specific inhibitor, was not observed to exert such an effect. In addition, Stat1 activated by $IFN{\gamma}\;and\;TNF{\alpha}$ was interacted with p38 MAPK. These data suggest that p38 MAP kinase mediates cytokine-mediated iNOS expression in the PC12 cells, and Jak/Stat pathway interferes with p38 MAPK signaling pathway.

• The Korea Journal of Herbology
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• v.22 no.2
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• pp.147-153
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• 2007

Objectives : Sophora flavescens (SF) is widely used in traditional herbal medicine in Korea and is well recognized for its anti-inflammatory effect. However, its effect on Tumornecrosis factor alpha (Tnf) production in BV2 microglial cell is not yet known. Methods : We investigated the effect of SF on the production and expression of Tnf, a well known inflammatory mediator, in lipopolysaccaride (LPS)-activated BV2 microglial cells. Results : The LPS-induced Tnf production was markedly reduced by treatment with SF (50 ${\mu}g/ml$). In reverse transcription polymerase chain reaction (RT-PCR) analysis, SF suppressed the LPS activated expression of Tnf mRNA. In addition, Western blot analysis confirmed that SF suppressed the expression of Tnf. Sophora flavescens also inhibited the LPS-induced phosphylation of extracellular signal-regulated kinases (ERK), which mediate the Tnfproduction signaling pathway whereas LPS-induced phosphylation of p38 mitogen activated protein kinase (p38 MAPK), and c-Jun NH2-terminal kinases (JNK) was not inhibited by SF, which implies that SF suppresses LPS-induced Tnf production via the ERK mediated pathway. Conclusion : Taken together, these findings indicated that SF inhibits LPS-induce Tnf production, and that this inhibitory effect is mediated via the ERK pathway.

• YAKHAK HOEJI
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• v.42 no.3
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• pp.296-301
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• 1998

To investigate the effects of ginsenosides from Panax ginseng on mitogenic responses in macrophages and splenocytes from murine, we examined the effects of representative protopanaxadiol and protopanaxatriol ginsenosides ($Rb_1,\;Rb_2,\;Re\;and\;Rg_1$) on tumor necrosis factor-${\alpha}$ (TNF-(${\alpha}$) production in murine macrophage cell line (RAW264.7 cells) stimulated by lipopolysaccharide (LPS) and T cell proliferation in splenocytes stimulated by concanavalin A (Con A). Among the ginsenosides tested, protopanaxadiol ginsenosides ($Rb_1\;and\;Rb_2$) significantly inhibited TNF-${\alpha}$ production in a dose-dependent manner. However, protoppanaxatriol ginsenosides (Re and $Rg_1$) showed little inhibitory activity. The molar concentrations of $Rb_1\;and\;Rb_2$ producing 50% inhibition ($IC_{50}$) of TNF-${\alpha}$ production were $55.8{\mu}g/ml\;(48.0{\mu}M)\;and\;31.8{\mu}g/ml (27.9{\mu}M)$, respectively. As a positive control, prednisolone also exhibited inhibitory activity with an $IC_{50}$ value of $21.7{\mu}M$. In T cell proliferation, $Rg_1$, was not effective but $Rb_1$ and Re or $Rb_2$ significantly increased or inhibited at high concentration, 75 and $100{\mu}g/ml$. In contrast, prednisolone showed potent inhibitory activity with an $IC_{50}$ value of 6.1nM. These results suggest that ginsenosides may take part in the mitogen-induced signaling pathway for TNF-${\alpha}$ production and T cell proliferation from macrophages and splenocytes.

• Tuberculosis and Respiratory Diseases
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• v.75 no.5
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• pp.205-209
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• 2013

Background: We investigated whether prunetin significantly affects tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$)-induced MUC5AC mucin gene expression, production, inhibitory kappa B ($I{\kappa}B$) degradation and nuclear factor kappa B (NF-kB) p65 translocation in human airway epithelial cells. Methods: Confluent NCI-H292 cells were pretreated with prunetin for 30 minutes and then stimulated with TNF-${\alpha}$ for 24 hours or the indicated periods. MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The effect of prunetin on TNF-${\alpha}$-induced degradation of $I{\kappa}B$ and translocation of NF-${\kappa}B$ p65 was investigated by western blot analysis. Results: We found that incubation of NCI-H292 cells with prunetin significantly inhibited mucin production and down-regulated the MUC5AC gene expression induced by TNF-${\alpha}$. Prunetin inhibited TNF-${\alpha}$-induced degradation of $I{\kappa}B$ and translocation of NF-${\kappa}B$ p65. Conclusion: This result suggests that prunetin inhibits the NF-${\kappa}B$ signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production regulated by the NF-${\kappa}B$ signaling pathway.

• Journal of Life Science
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• v.29 no.5
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• pp.530-536
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• 2019

Glioblastoma (GBM) is the most incurable brain cancer derived from the transformed glial cells. Standard anti-GBM treatment, including surgery and chemoradiotherapy, does not ensure good prognosis for the patients with GBM, because successful therapy is often impeded by presence of glioma stem cells (GSCs). GSCs, which is generally divided into proneural (PN) and mesenchymal (MES) subtype, are understood as subpopulation of cancer cells responsible for GBM initiation, progression and recurrence after standard treatments. In the present study, we demonstrate that PN subtype GSCs differentially transit to MES subtype GSCs by specific cytokines. The expression of CD44, a marker of MES subtype GSCs, was observed when GSC11 PN subtype GSCs were exposed to tumor necrosis factor alpha ($TNF-{\alpha}$) cytokine and GSC23 PN subtype GSCs were treated to transforming growth factor beta 1 ($TGF-{\beta}1$) cytokine. Ivy glioblastoma atlas project (Ivy GAP) bioinformatics database showed that $TNF-{\alpha}$ and $TGF-{\beta}1$ were highly expressed in necrotic region and perivascular region, respectively. In addition, $TNF-{\alpha}$ signaling was relatively upregulated in necrotic region, while $TGF-{\beta}$ signaling was increased in perivascular region. Taken together, our observations suggest that MES subtype GSCs can be derived from various PN subtype GSCs by multimodal cytokine stimuli provided by neighboring tumor microenvironment.

• Biomolecules & Therapeutics
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• v.23 no.2
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• pp.110-118
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• 2015

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$), and tumor necrosis factor-${\kappa}B$ (TNF-${\alpha}$) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-${\alpha}$ in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-${\alpha}$, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, $PGE_2$, and TNF-${\alpha}$ in LPS-treated BV2 microglial cells by suppressing ROS and NF-${\kappa}B$. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-${\kappa}B$ signaling pathway.

• Biomolecules & Therapeutics
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• v.23 no.1
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• pp.39-44
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• 2015

Tolfenamic acid (TA) is a traditional non-steroid anti-inflammatory drug (NSAID) and has been broadly used for the treatment of migraines. Nuclear factor kappa B (NF-${\kappa}B$) is a sequence-specific transcription factor and plays a key role in the development and progression of inflammation and cancer. We performed the current study to investigate the underlying mechanisms by which TA suppresses inflammation focusing on NF-${\kappa}B$ pathway in TNF-${\alpha}$ stimulated human normal and cancer cell lines and lipopolysaccharide (LPS)-stimulated mouse macrophages. Different types of human cells (HCT116, HT-29 and HEK293) and mouse macrophages (RAW264.7) were pre-treated with different concentrations of TA and then exposed to inflammatory stimuli such as TNF-${\alpha}$ and LPS. Transcriptional activity of NF-${\kappa}B$, $l{\kappa}B-{\alpha}$-degradation, p65 translocation and mitogen-activated protein kinase (MAPK) activations were measured using luciferase assay and Western blots. Pre-treatment of TA repressed TNF-${\alpha}$- or LPS-stimulated NF-${\kappa}B$ transactivation in a dose-dependent manner. TA treatment reduced degradation of $l{\kappa}B-{\alpha}$ and subsequent translocation of p65 into nucleus. TA significantly down-regulated the phosphorylation of c-Jun N-terminal kinase (JNK). However, TA had no effect on NF-${\kappa}B$ signaling and JNK phosphorylation in HT-29 human colorectal cancer cells. TA possesses anti-inflammatory activities through suppression of JNK/NF-${\kappa}B$ pathway in different types of cells.

• The Korea Journal of Herbology
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• v.27 no.1
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• pp.59-64
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• 2012

Objective : This study was performed to evaluate the effect of Forsythiae Fructus (FF) ethanol extract on inflammatory cytokine production and its underlying mechanisms in mouse macrophages. Methods : Peritoneal macrophages from thioglycollate medium-injected mice were cultured and stimulated with lipopolysaccharide(LPS) or LPS/interferon(IFN)-${\gamma}$ for cytokine measurement and cellular signaling molecule analysis. Results : FF ethanol extract decreased the levels of secreted tumor necrosis factor(TNF)-${\alpha}$ and interleukin(IL)-6 in IFN-${\gamma}$/LPS-stimulated cells in a concentration-dependent manner. FF extract reduced IFN-${\gamma}$/LPS-induced STAT1 phosphorylation and LPS-induced p38 and JNK activation, but not ERK1/2 activity. The extract also inhibited LPS-induced $I{\kappa}B{\alpha}$ degradation through suppression of $I{\kappa}B{\alpha}$ kinase. Conclusions : These results suggest that FF ethanol extract affects the production of TNF-${\alpha}$ and IL-6 through inhibition of activation of STAT-1, $I{\kappa}B{\alpha}$, p38, and JNK.

• Biomedical Science Letters
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• v.18 no.3
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• pp.181-192
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• 2012

Toll-like receptors (TLRs) induce innate immune responses that are essential for host defense against invading microbial pathogens. In general, TLRs have two major downstream signaling pathways; myeloid differential factor 88 (MyD88) and Toll/IL-1R domain-containing adaptor inducing IFN-${\beta}$ (TRIF) leading to the activation of NF-${\kappa}B$ and IRF3. Numerous studies demonstrated that certain phytochemicals possessing anti-inflammatory effects inhibit NF-${\kappa}B$ activation induced by pro-inflammatory stimuli including lipopolysaccharide and tumor necrosis factor-${\alpha}$ ($TNF{\alpha}$). However, the direct molecular targets for such anti-inflammatory phytochemicals are not fully identified. In this paper, we will discuss about the molecular targets of phytochemicals in TLRs signaling pathways. These results present a novel anti-inflammatory mechanism of phytochemicals in TLRs signaling.

• Molecules and Cells
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• v.38 no.1
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• pp.26-32
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• 2015

Toll-like receptors (TLR) 7 and 9 transduce a cellular signal through the MyD88-dependent pathway and induce the production of inflammatory mediators against microbial nucleotide components. The repeated stimulation of TLR4 leads to endotoxin tolerance, but the molecular mechanisms of tolerance induced through the costimulation of individual TLR has not yet been established, although endosomal TLRs share signaling pathways with TLR4. In the present study, mouse macrophages were simultaneously stimulated with the TLR7 agonist, gardiquimod (GDQ), and the TLR9 agonist, CpG ODN 1826, to examine the mechanism and effector functions of macrophage tolerance. Compared with individual stimulation, the costimulation of both TLRs reduced the secretion of TNF-${\alpha}$ and IL-6 through the delayed activation of the NF-${\kappa}B$ pathway; notably, IL-10 remained unchanged in costimulated macrophages. This tolerance reflected the early induction of suppressor of cytokine signaling-1 (SOCS-1), according to the detection of elevated TNF-${\alpha}$ secretion and restored NF-${\kappa}B$ signaling in response to the siRNA-mediated abrogation of SOCS-1 signaling. In addition, the restimulation of each TLRs using the same ligand significantly reduced the expression of both TLRs in endosomes. These findings revealed that the costimulation of TLR7 and TLR9 induced macrophage tolerance via SOCS-1, and the restimulation of each receptor or both TLR7 and TLR9 downregulated TLR expression through a negative feedback mechanisms that protects the host from excessive inflammatory responses. Moreover, the insufficient and impaired immune response in chronic viral infection might also reflect the repeated and simultaneous stimulation of those endosomal TLRs.