• The Journal of Korean Medicine
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• v.30 no.1
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• pp.51-63
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• 2009

Objectives: Peroxynitrite ($ONOO^-$), superoxide anion radical (${\cdot}{O_2}^-$ and nitric oxide (NO) are cytotoxic because they can oxidize several cellular components such as proteins, lipids and DNA. They have been implicated in the aging processes, and age-related diseases such as Alzheimer's disease, rheumatoid arthritis, cancer, diabetes, obesity and atherosclerosis. The aim of this study was to investigate the $ONOO^-$, NO, ${\cdot}{O_2}^-$ scavenging and NF-${\kappa}B$ related anti-inflammatory activities of Sotosaja-hwan in ob/ob mice. Methods: Mice were grouped and treated for 5 weeks as follows. Both the normal lean (C57/BL6J black mice) and control obese (ob/ob mice) groups have received standard chow. The experimental groups were fed with a diet of chow supplemented with 30 and 90 mg Sotosaja-hwan per 1 kg of body weight for 14 days. For this study, the fluorescent probes, namely 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), 4,5-diaminofluorescein (DAF-2) and dihydrorhodamine 123 (DHR 123) were used. Western blotting was performed using anti-phospho-$I{\kappa}B$-${\alpha}$, anti-IKK-${\alpha}$, anti-NF-${\kappa}B$ (p50, p65), anti-COX-2, anti-iNOS, anti-YCAM-1 and anti-MMP-9 antibodies, respectively. Results: Sotosaja-hwan inhibited the generation of $ONOO^-$, NO and ${\cdot}{O_2}^-$ in the lipopolysaccharide (LPS)-treated mouse kidney postmitochondrial fraction in vitro. The generation of $ONOO^-$, NO, ${\cdot}{O_2}^-$ and PGE2 were inhibited in the Sotosaja-hwan-administered ob/ob mice groups. The GSH/GSSG ratio was decreased in the ob/ob mice, whereas the ratio was improved in the Sotosaja-hwan-administered groups. Sotosaja-hwan inhibited the protein expression levels of phospho-$I{\kappa}B$-${\alpha}$, IKK-${\alpha}$, NF-${\kappa}B$ (p50, p65), COX-2, iNOS, YCAM-1 and MMP-9 genes. Conclusions: These results suggest that Sotosaja-hwan is an effective $ONOO^-$, ${\cdot}{O_2}^-$ and NO scavenger and has NF-kB related anti-inflammatory activity in ob/ob mice. Therefore, Sotosaja-hwan might be a potential therapeutic drug against the inflammation process and inflammation-related diseases.

• Proceedings of the PSK Conference
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• 2005.11a
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• pp.231-232
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• 2005

Bacteria of Shigella spp. are responsible for shigellosis in humans, a disease characterized by destruction of the colonic epithelium that is induced by the inflammatory response elicited by invasive bacteria. They use a type III secretion system injecting effector proteins into host cells to induce their entry into epithelial cells and triggers apoptosis in macrophages. We present evidence that the effector OspG is a protein kinase that binds various ubiquitinylated ubiquitin-conjugating enzymes (E2s) and blocks degradation of phospho-$I{\kappa}B{\alpha}$ induced upon entry of bacteria into epithelial cells. Transfection experiments confirmed that OspG interferes with the $NF-{\kappa}B$ activation patway by preventing phospho-$I{\kappa}B{\alpha}$ degradation, suggesting that OspG inactivates a component of the $SCF^{{\beta}-TrCP}$ ubiquitin ligase complex (E3) involved in phospho-$I{\kappa}B{\alpha}$ ubiquitination. Upon infection of ileal loops in rabbits, the ospG mutant induced a stronger inflammatory response compared with the wild-type strain, indicating that OspG down-regulates the host innate response induced by invasive bacteria.

• Journal of Applied Biological Chemistry
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• v.53 no.3
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• pp.147-153
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• 2010

$\beta$-glycyrrhetinic acid (GA), the active principle of licorice (Glycyrrhiza glabra L.) has been reported to exhibit anti-inflammatory properties in different animal models. In this study, the effects of GA on the production of inflammatory mediators including tumor necrosis factor (TNF)-$\alpha$, interleukin (IL)-6, nitric oxide (NO), and prostaglandin E (pGE)-2 were examined in RAW 264.7 cells in vitro. Furthermore, to elucidate a possible mechanism for the inhibitory effect of GA on the production of TNF-$\alpha$, it was investigated whether the treatment of GA affects the I-${\kappa}B{\alpha}$ degradation and subsequent nuclear translocation of NF-${\kappa}B$. Various inflammatory responses were induced in the culture system by treating with a lipopolysaccharide (LPS). GA showed anti-inflammatory activities in dose-dependant manner with $IC_{50}$ of $5.4{\mu}M$ by inhibiting the production of TNF-$\alpha$ in RAW 264.7 cells. In addition, the treatment of GA blocked both I-${\kappa}B{\alpha}$ degradation and the nuclear translocation of NF-${\kappa}B$ from cytosol to nucleus. However, it did not affect the production of IL-6, NO, and PGE-2, implying the direct blocking of the production of TNF-$\alpha$ resulting from both the I-${\kappa}B{\alpha}$ degradation and the nuclear translocation of NF-${\kappa}B$. This finding might provide the underlying mechanism to explain the reported anti-inflammatory activities of GA in animal models.

• The Journal of Internal Korean Medicine
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• v.25 no.1
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• pp.28-45
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• 2004

Objectives : The main purpose of this study is to evaluate the effect of Injinchunggan-tang on $TNF-{\alpha}$ signal transmission system. Materials and Methods : We analyzed the following with quantitative RT-PCR method; the effect of Injinchunggan-tang on secretion of $TNF-\alpha$ mRNA/protein and stability, the effect on gene revelation that consists of signal transmission system (TRAIL, NIK, A20, TRADD, RAIDD, RIP TNFR-I, TNFR-II, TRAF1, TRAF2, FADD), the one on activation of p38, Erk1/2 MAPK and the rate of nuclear $NF-{\kappa}B/cytosolic\;NF-{\kappa}B$ in HepG2 cell. We also analyzed the inhibitory effect of Injinchunggan-tang on the apoptosis of HepG2 cell that $TNF-{\alpha}$ induces and the $NF-{\kappa}B$ restraint effected by transfection of $I{\kappa}B{\Delta}N$ through tryphan blue exclusion assay. Results : Injinchunggan-tang prohibits revelation of $TNF-{\alpha}$ mRNA in HepG2 cell and the creation of protein. However, it has no effect on the stability of $TNF-{\alpha}$ mRNA. While it did not have any effect on the generation of TRAIL, NIK, A20, TRADD, RAIDD and RIP genes, Injinchunggan-tang reduces the revelation of TNFR-I, TNFR-II, TRAF1, TRAF2 and FADD genes. It has been confirmed that Injinchunggan-tang restraints the revelation of $TNF-{\alpha}$ mRNA that is promoted by ethanol, acetaldehyde, lipopolysaccharide, in proportion to the treatment density and time. It activated $NF-{\kappa}B$ of HepG2 cell and promoted activation of $NF-{\kappa}B$ that is occurred by $TNF-{\alpha}$. It has been observed that the restraint effect against the $TNF-{\alpha}$ inducing apoptosis is lost when it is intercepted the function of $NF-{\kappa}B$ in HepG2 cell. Conclusion: It has been confirmed that Injinchunggan-tang has restraining effect against the revelation of $TNF-{\alpha}$ and mRNA that is constituent element of TNF-a signal transmission system. It also has been revealed that it restraints the activation of p38, Erk1/2 by $TNF-{\alpha}$. Through this prohibiting effect, it is inferred that it restraints signal transmission among various cells that are related to inflammation reaction. Meanwhile, Injinchunggan-tang protects liver cell from apoptosis that is caused by $TNF-{\alpha}$, by maintaining the activating function for $NF-{\kappa}B$.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.78-78
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• 2003

Inulin, an active component of Chicorium intybus root, has been shown to stimulate the growth of bifidobacteria, and inhibit colon carcinogenesis. NO mediates a number of the host-defense functions of activated macrophages, including antimicrobial and tumoricidal activity. We examined the effect of inulin on the synthesis of NO in RAW 264.7 cells. Inulin alone had no effect, whereas inulin with IFN-ν synergistically increased the NO production and inducible NO synthase (iNOS) expression in RAW 264.7 cells. Synergy between IFN-ν and inulin was mainly dependent on inulin-induced TNF-${\alpha}$ secretion. Also, protein kinase C (PKC)-${\alpha}$ was involved in the inulin-induced NO production. Inulin-mediated NO production was inhibited by the protein tyrosine kinase (PTK) inhibitor, tyrphostin AG126. Since iNOS gene transcriptions have been shown to be under the control of the NF -$\kappa$B/Rel family of transcription factors, we assessed the effect of inulin on NF -$\kappa$B/Rel using an EMSA. Inulin produced strong induction of NF-$\kappa$B/Rel binding, whereas AP-l binding was slightly induced in RAW 264.7 cells. Inulin stimulated phosphorylation and degradation of I$\kappa$B-${\alpha}$. These results suggest that in IFN-ν-primed RAW 264.7 cells inulin might stimulate NO synthesis via activation of PKC-${\alpha}$ and PTK, resulting in the activation of NF-$\kappa$B.

• The Journal of Internal Korean Medicine
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• v.30 no.2
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• pp.288-297
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• 2009

Objective : Inflammatory cytokines have a close relationship to insulin dependent diabetes mellitus (IDDM). The inhibitory effect of Smilacis Glabrae Rhizoma (SGR) were examined on production of nitric oxide (NO), prostaglandin $E_2$ $(PGE_2)$, synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and NF-${\kappa}$B activation in Raw 264.7 cells. Methods: Raw 264.7 cells were pretreated with SGR(20, 50, 100 ${\mu}g$/ml), and then cultured with lipopolysaccharides (LPS). Cell viability was measured by MTT assay; inhibition of NO, $PGE_2$, and TNF-${\alpha}$ production were measured by Griess reagent and enzyme-linked immunosorbent assay(ELISA). Induction of COX-2 and iNOS were determined by western blotting analysis. Inhibition of NF-${\kappa}$B was measured by immunofluorescence assay (IFA). Results: SGR inactivated NF-${\kappa}$B, and inhibited the production of NO, iNOS, and $PGE_2$. Inhibition of COX-2 and TNF-${\alpha}$ could not be confirmed. Conclusions: From the above result. SGR was found to have an anti-inflammatory effect of inhibition of NO, iNOS, and $PGE_2$ production via inhibition of NF-${\kappa}$B.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.26 no.4
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• pp.70-80
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• 2013

Objectives : Haliotidis Concha has been used to treat various human diseases such as liver dysfunction and inflammatory disorder. Although it has been shown the effects of Haliotidis Concha on the various diseases, it has almost not been studied about the anti-inflammatory effects of the Haliotidis Concha and its mechanisms. Methods : This research investigated the effects of the Haliotidis Concha ethanol extract (HCE) on the production of nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) as well as tumor necrosis factor-alpha (TNF-${\alpha}$). The protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were assayed by immunoblot analyses, and the productions of NO, $PGE_2$ and TNF-${\alpha}$ were assessed by ELISA. Results : Haliotidis Concha decreased the production of NO and $PGE_2$, and inhibited the expression iNOS and COX-2 proteins in a concentration-dependent manner in LPS-treated Raw 264.7 cells. HCE suppressed the ability of LPS to activate the signaling pathways of nuclear factor kappa B (NF-${\kappa}B$) as indicated by HCE inhibited nuclear NF-${\kappa}B$ level and I-${\kappa}B{\alpha}$ phosphorylation. Also, HCE inhibited mitogen-activated protein kinases (MAPKs). Conclusions : HCE repressed the production of LPS-inducible NO, $PGE_2$ and TNF-${\alpha}$, which may be mediated by inhibition of NF-${\kappa}B$ translocation. This study suggest the use for the treatment of acute inflammatory disorders.

• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.190-195
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• 2011

Nuclear factor ${\kappa}B$ (NF-${\kappa}B$) is a nuclear transcription factor that modulates the expression of inflammatory cytokines such as tumor necrosis factor (TNF)-${\alpha}$. trans-10, cis-12 (t10c12)-conjugated linoleic acid (CLA) participates in the inhibition of TNF-${\alpha}$ production upon lipopolysaccharide (LPS)-stimulation. However, in our previous study, t10c12-CLA enhanced the production of TNF-${\alpha}$ by LPS-unstimulated porcine peripheral blood mononuclear cells (PBMCs) and RAW 264.7 macrophages in vitro. To resolve this apparent contradiction, we hypothesized that the effect of t10c12-CLA on TNF-${\alpha}$ production depends on NF-${\kappa}B$ activation induced by LPS stimulation. To test this hypothesis, we assessed the in vitro effect of t10c12-CLA on TNF-${\alpha}$ production and NF-${\kappa}B$ p65 activity in LPS-stimulated and LPS-unstimulated porcine PBMCs. t10c12-CLA treatment resulted in increased TNF-${\alpha}$ production by LPS-unstimulated PBMCs but decreased TNF-${\alpha}$ production by LPS-stimulated PBMCs. t10c12-CLA increased the degradation of inhibitory ${\kappa}B$ ($I{\kappa}B$)-${\alpha}$ protein and activated NF-${\kappa}B$ p65 in LPS-unstimulated PBMCs, but had the opposite effect in LPS-stimulated PBMCs. Notably, t10c12-CLA enhanced NF-${\kappa}B$ p65 binding activity in LPS-unstimulated PBMCs exposed to caffeic acid phenethyl ester (CAPE), a NF-${\kappa}B$ inhibitor. Conversely, it inhibited NF-${\kappa}B$ p65 binding activity in LPS-stimulated PBMCs exposed to CAPE. These results suggest that t10c12-CLA may have different actions under different physiological conditions, and that its effect may be associated with a change in NF-${\kappa}B$ p65 activity.

• Tuberculosis and Respiratory Diseases
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• v.48 no.5
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• pp.682-698
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• 2000

Glucocorticoid receptor (GR) functions as a suppressor of inflammation by inhibiting the expression of many cytokine genes activated by NF-${\kappa}B$. The goal of this study is to investigate the mechanism by which GR repress NF-${\kappa}B$ activation in lung epithelial cells. We used A549 and BEAS-2B lung epithelia! cell lines. Using Ig$G{\kappa}$-NF-${\kappa}B$ luciferase reporter gene construct, we found that dexamethasone significantly suppressed TNF-$\alpha$-induced NF-${\kappa}B$ activation and the overexpression of GR showed dose-dependent reduction of TNF-$\alpha$-induced NF-${\kappa}B$ activity in both cell lines. However, DNA binding of NF-${\kappa}B$ induced by TNF-$\alpha$ in electromobility shift assay was not inhibited by dexamethasone. Super shift assay with anti-p65 antibody demonstrated the existence of p65 in NF-${\kappa}B$ complex induced by $\alpha$ Western blot showed that $I{\kappa}B{\alpha}$ degradation induced by TNF-$\alpha$ was not affected by dexamethasone and $I{\kappa}B{\kappa}$ was not induced by dexamethasone, neither. To evaluate p65 specific transactivation, we adopted co-transfection study of Gal4-p65TA1 or TA2 fusion protein expression system together with 5xGal4-luciferase vector. Co-transfection of GR with Gal4-p65TA1 or TA2 repressed luciferase activity profoundly to the level of 10-20% of p65TA1- or TA2-induced transcriptional activity. And this transrepressional effect was abolished by co-transfection of CBP of SRC-1 expression vectors. These results suggest that GR-mediated transrepression of NF-${\kappa}B$ in lung epithelial cells is through competing for binding to limiting amounts of transcriptional coactivators, CBP or SRC-1.

• IMMUNE NETWORK
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• v.10 no.5
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• pp.145-152
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• 2010

Background: The flowers of Inula japonica (Inulae Flos) have long been used in traditional medicine for the treatment of inflammatory diseases. In the present study, we investigated the anti-inflammatory properties of Inulae Flos Extract (IFE). Methods: The anti-inflammatory effects of IFE against nitric oxide (NO), $PGE_2$, TNF-${\alpha}$, and IL-6 release, as well as NF-${\kappa}B$ and MAP kinase activation were evaluated in RAW 264.7 cells. Results: IFE inhibited the production of NO and the expression of inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW264.7 cells. In addition, IFE reduced the release of pro-inflammatory cytokines, such as TNF-${\alpha}$ and IL-6. Furthermore, IFE inhibited the NF-${\kappa}B$ activation induced by LPS, which was associated with the abrogation of $I{\kappa}B-{\alpha}$ degradation and subsequent decreases in nuclear p65 and p50 levels. Moreover, the phosphorylation of ERK, JNK, and p38 MAP kinases in LPS-stimulated RAW 264.7 cells was suppressed by IFE in a dose-dependent manner. Conclusion: These results suggest that the anti-inflammation activities of IFE might be attributed to the inhibition of NO, iNOS and cytokine expression through the down-regulation of NF-${\kappa}B$ activation via suppression of $I{\kappa}B{\alpha}$ and MAP kinase phosphorylation in macrophages.