• Journal of Ginseng Research
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• v.41 no.1
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• pp.43-51
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• 2017

Background: BIOGF1K, a compound K-rich fraction prepared from the root of Panax ginseng, is widely used for cosmetic purposes in Korea. We investigated the functional mechanisms of the anti-inflammatory and antioxidative activities of BIOGF1K by discovering target enzymes through various molecular studies. Methods: We explored the inhibitory mechanisms of BIOGF1K using lipopolysaccharide-mediated inflammatory responses, reporter gene assays involving overexpression of toll-like receptor adaptor molecules, and immunoblotting analysis. We used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to measure the antioxidative activity. We cotransfected adaptor molecules, including the myeloid differentiation primary response gene 88 (MyD88) and Toll/interleukin-receptor domain containing adaptor molecule-inducing interferon-${\beta}$ (TRIF), to measure the activation of nuclear factor (NF)-${\kappa}B$ and interferon regulatory factor 3 (IRF3). Results: BIOGF1K suppressed lipopolysaccharide-triggered NO release in macrophages as well as DPPH-induced electron-donating activity. It also blocked lipopolysaccharide-induced mRNA levels of interferon-${\beta}$ and inducible nitric oxide synthase. Moreover, BIOGF1K diminished the translocation and activation of IRF3 and NF-${\kappa}B$ (p50 and p65). This extract inhibited the upregulation of NF-${\kappa}B$-linked luciferase activity provoked by phorbal-12-myristate-13 acetate as well as MyD88, TRIF, and inhibitor of ${\kappa}B$ ($I{\kappa}B{\alpha}$) kinase ($IKK{\beta}$), and IRF3-mediated luciferase activity induced by TRIF and TANK-binding kinase 1 (TBK1). Finally, BIOGF1K downregulated the NF-${\kappa}B$ pathway by blocking $IKK{\beta}$ and the IRF3 pathway by inhibiting TBK1, according to reporter gene assays, immunoblotting analysis, and an AKT/$IKK{\beta}$/TBK1 overexpression strategy. Conclusion: Overall, our data suggest that the suppression of $IKK{\beta}$ and TBK1, which mediate transcriptional regulation of NF-${\kappa}B$ and IRF3, respectively, may contribute to the broad-spectrum inhibitory activity of BIOGF1K.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.1
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• pp.28-35
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• 2008

Purpose: The nitric oxide (NO) release by inducible nitric oxide synthase (iNOS) is the key events in macrophage response to lipopolysaccharide (LPS) which is suggested to be a crucial mediator for inflammatory and innate immune responses. NO is an important mediator involved in many host defense action and may also lead to a harmful host response to bacterial infection. However, given the importance of iNOS in a variety of pathophysiological conditions, control of its expression and signaling events in response to LPS has been the subject of considerable investigation. Materials and Methods: The Raw264.7 macrophage cell line was used to observe LPS-stimulated iNOS expression. The expression of iNOS is observed by Western blot analysis and real-time RT-PCR. Protein kinase C $(PKC)-{\alpha}$ overexpressing Raw264.7 cells are established to determine the involvement of $PKC-{\alpha}$ in LPS-mediated iNOS expression. $NF-{\kappa}B$ activity is measured by $I{\kappa}B{\alpha}$ degradation and $NF-{\kappa}B$ luciferase activity assay. Results: We found that various PKC isozymes regulate LPS-induced iNOS expression at the transcriptional and translational levels. The involvement of $PKC-{\alpha}$ in LPS-mediated iNOS induction was further confirmed by increased iNOS expression in $PKC-{\alpha}$ overexpressing cells. $NF-{\kappa}B$ dependent transactivation by LPS was observed and $PKC-{\alpha}$ specific inhibitory peptide abolished this activation, indicating that $NF-{\kappa}B$ activation is dependent on $PKC-{\alpha}$. Conclusion: Our data suggests that $PKC-{\alpha}$ is involved in LPS-mediated iNOS expression and that its downstream target is $NF-{\kappa}B$. Although $PKC-{\alpha}$ is a crucial mediator in the iNOS regulation, other PKC isozymes may contribute LPS-stimulated iNOS expression. This finding is needed to be elucidated in further study.

• The Journal of Korean Medicine
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• v.29 no.1
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• pp.47-59
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• 2008

Objective : Anti-inflammatory effects of the extract of Lycii Fructus on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells were investigated. Method : In order to assess the cytotoxic effect of Lycii Fructus on the raw 264.7 macrophages 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was performed. Reverse transcription-polymerase chain reaction(RT-PCR) analysis of the mRNA levels of tumor necrosis factor-$\alpha$(TNF-$\alpha$) and inducible nitric oxide synthase(iNOS) was performed in order to provide an estimate of the relative level of expression of these genes. The protein level of the inhibitor of nuclear factor-${\kappa}B(I{\kappa}B)$ and nuclear factor-${\kappa}B$(NF-${\kappa}B$) activity was investigated by Western blot assay. NO production was investigated by NO detection. Result : Lycii Fructus suppressed NO production by inhibiting the LPS-induced expressions of iNOS and TNF-$^-\alpha$ mRNA and iNOS protein in RAW 264.7 macrophage cells. Also, Lycii Fructus suppressed activation of NF-${\kappa}B$ in the nucleus. Conclusion : These results show that the extract of Lycii Fructus has anti-inflammatory effect probably by suppressing iNOS expressions through the down-regulation of NF-${\kappa}B$ binding activity.

• Journal of Veterinary Clinics
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• v.31 no.6
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• pp.469-476
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• 2014

The aims of this study were to explore the effects of conjugated linoleic acid (CLA) on reactive oxygen species (ROS) production in lipopolysaccharide (LPS)-naïve and LPS-stimulated RAW 264.7 macrophages and to examine whether these effects affect the regulation of tumor necrosis factor-alpha (TNF-${\alpha}$) production, and nuclear factor-kappa B (NF-${\kappa}B$) and peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$) activation. Trans-10, cis-12(t10c12)-CLA increased the production of ROS, as well as TNF-${\alpha}$ in LPS-naïve RAW 264.7 cells. The CLA-induced TNF-${\alpha}$ production was suppressed by treatment of diphenyleneiodonium chloride (DPI), a NADPH oxidase inhibitor. In addition, CLA enhanced the activities of NF-${\kappa}B$ and $PPAR{\gamma}$ in LPS-naïve RAW 264.7 cells, and this effect was abolished with DPI treatment. LPS treatment increased ROS production, whereas CLA reduced LPS-induced ROS production. LPS increased both TNF-${\alpha}$ production and NF-${\kappa}B$ activity, whereas t10c12-CLA reduced TNF-${\alpha}$ production and NF-${\kappa}B$ activity in LPS-stimulated RAW 264.7 cells. DPI treatment suppressed LPS-induced ROS production and NF-${\kappa}B$ activity. Moreover, DPI enhanced the inhibitory effects of t10c12-CLA on TNF-${\alpha}$ production and NF-${\kappa}B$ activation in LPS-stimulated RAW 264.7 cells. However, neither t10c12-CLA nor DPI affected $PPAR{\gamma}$ activity in LPS-stimulated RAW 264.7 cells. Taken together, these data indicate that t10c12-CLA induces TNF-${\alpha}$ production by increasing ROS production in LPS-naïve RAW 264.7 cells, which is mediated by the enhancement of NF-${\kappa}B$ activity via $PPAR{\gamma}$ activation. By contrast, t10c12-CLA suppresses TNF-${\alpha}$ production by inhibiting ROS production and NF-${\kappa}B$ activation via a $PPAR{\gamma}$-independent pathway in LPS-stimulated RAW 264.7 cells. These results suggest that t10c12-CLA can modulate TNF-${\alpha}$ production and NF-${\kappa}B$ activation through formation of ROS in RAW 264.7 macrophages.

• Toxicological Research
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• v.24 no.1
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• pp.17-22
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• 2008

Sopungsan (SS) is a traditional Korean decoction used for the treatment of dermatitis. The aim of this study is to confirm whether or not SS has a preventive effect on the development of atopic dermatitis in dinitrochlorobenzene-applied Nc/Nga mice. SS was administered orally to Nc/Nga mice, which led to the remarkable suppression of the development of dermatitis, as determined by a histological examination and the serum IgE levels. Moreover, SS inhibited the production of thymus- and activation-regulated chemokine (TARC) and its mRNA expression in a keratinocyte cell line, HaCaT, which had been stimulated with tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interferon-${\gamma}$ (IFN-${\gamma}$). Activation of the nuclear factor-${\kappa}B$ (NF-${\kappa}B$) or activator protein-1 (AP-1) is one of key steps in the signaling pathways mediating induction of TARC. In this study, SS selectively suppressed NF-${\kappa}B$ activation which may be essential for TARC expression in $TNF-{\alpha}/IFN-{\gamma}$ treated keratinocytes. The inhibitory effect of SS on NF-${\kappa}B$ activation and TARC production might be associated with the anti-dermatitic effects of SS.

• Korean Journal of Microbiology
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• v.54 no.3
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• pp.208-213
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• 2018

Activation of nuclear factor kappa B ($NF{\kappa}B$) leads to the inflammatory process. During this $NF{\kappa}B$-dependent inflammation process, inducible nitric oxide synthase (iNOS) are expressed in the inflammatory cells. Our previous data indicated that a specific septapeptide (GVAWWMY) from the soybean extract fermented by Bacillus licheniformis B1 inhibited iNOS mRNA expression and NO production in cultured macrophage cells. Our further experiments revealed that treatment of same septapeptide resulted in inhibition of LPS-induced $NF{\kappa}B$ activation by reversing degradation of $I{\kappa}B{\alpha}$, an inhibitory protein for $NF{\kappa}B$. The molecular docking indicated that the septapeptide binds to $I{\kappa}B$ kinase ${\beta}$ ($IKK{\beta}$), and thus it can inhibit phosphorylation of $I{\kappa}B{\alpha}$. Supporting this, the binding site for the septapeptide has the highest affinity (-8.7 kcal/mol) and the site was located at the kinase domain (KD) of $IKK{\beta}$, which can significantly affect the kinase activity of $IKK{\beta}$.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.6
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• pp.521-527
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• 2001

Diclofenac, a phenylacetic acid derivative, is a widely used non-steroidal anti-inflammatory drug (NSAID) to provide effective relief of inflammation and pain. Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation. We examined the inhibitory effects of diclofenac on the induction of iNOS in RAW 264.7 macrophages which were activated with lipopolysaccharide (LPS) plus interferon-gamma $(IFN-{\gamma}).$ Treatment of RAW 264.7 cells with diclofenac and other NSAIDs (aspirin and indomethacin) significantly inhibited NO production and iNOS protein expression induced by LPS plus $IFN-{\gamma}.$ Also, diclofenac but not aspirin and indomethacin, inhibited iNOS mRNA expression and nuclear factor-kappa B $(NF-{\kappa}B)$ binding activity concentration-dependently. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a CAT reporter gene revealed that only diclofenac inhibited the iNOS promoter activity induced by LPS plus $IFN-{\gamma}$ through the $NF-{\kappa}B$ sites of iNOS promoter. Taken together, these suggest that diclofenac may exert its anti-inflammatory effect by inhibiting iNOS gene expression at the transcriptional level through suppression of $NF-{\kappa}B$ activation.

• Biomolecules & Therapeutics
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• v.16 no.4
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• pp.385-393
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• 2008

As an attempt to search for bioactive natural products exerting anti-inflammatory activity, we evaluated the effects of the methanol extract of Polytrichum commune Hedw (PCM) (Polytrichaceae) on lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin $E_2$ ($PGE_2$) and pro-inflammatory cytokines release in murine macrophage cell line RAW 264.7. PCM potently inhibits the production of NO, $PGE_2$, tumor necrosis factor (TNF)-$\alpha$ and interleukin (IL)-6. Consistent with these results, PCM also concentration-dependently inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygase (COX)-2 at the protein levels, and iNOS, COX-2, TNF-$\alpha$ and IL-6 at the mRNA levels without an appreciable cytotoxic effect on RAW 264.7 macrophag cells. Furthermore, PCM inhibited LPS-induced nuclear factor-kappa B (NF-$\kappa$B) activation as determined by NF-$\kappa$B reporter gene assay, and this inhibition was associated with a decrease in the nuclear translocation of p65 and p50 NF-$\kappa$B. Taken together, these results suggest that PCM may play an anti-inflammatory role in LPS-stimulated RAW 264.7 macrophages through the inhibitory regulation of iNOS, COX-2, TNF-$\alpha$ and IL-6 via NF-$\kappa$B inactivation.

• Molecules and Cells
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• v.39 no.5
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• pp.403-409
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• 2016

NME1 is a well-known metastasis suppressor which has been reported to be downregulated in some highly aggressive cancer cells. Although most studies have focused on NME1, the NME1 gene also encodes the protein (NME1L) containing N-terminal 25 extra amino acids by alternative splicing. According to previous studies, NME1L has potent anti-metastatic activity, in comparison with NME1, by interacting with $IKK{\beta}$ and regulating its activity. In the present study, we tried to define the role of the N-terminal 25 amino acids of NME1L in $NF-{\kappa}B$ activation signaling. Unfortunately, the sequence itself did not interact with $IKK{\beta}$, suggesting that it may be not enough to constitute the functional structure. Further construction of NME1L fragments and biochemical analysis revealed that N-terminal 84 residues constitute minimal structure for homodimerization, $IKK{\beta}$ interaction and regulation of $NF-{\kappa}B$ signaling. The inhibitory effect of the fragment on cancer cell migration and $NF-{\kappa}B$-stimulated gene expression was equivalent to that of whole NME1L. The data suggest that the N-terminal 84 residues may be a core region for the anti-metastatic activity of NME1L. Based on this result, further structural analysis of the binding between NME1L and $IKK{\beta}$ may help in understanding the anti-metastatic activity of NME1L and provide direction to NME1L and $IKK{\beta}$-related anti-cancer drug design.

• Journal of Life Science
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• v.21 no.10
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• pp.1377-1384
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• 2011

Astaxanthin (ATX) is a red-orange carotenoid pigment that occurs naturally in a wide variety of living organisms. In this study we investigated the inhibitory effects of ATX on the induction of inducible nitric oxide synthase (iNOS), nitric oxide (NO), proinflammatory cytokines, nuclear factor-kappa B(NF-${\kappa}B$) and reactive oxygen species (ROS) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In addition, we tested the superoxide radical scavenging activity of ATX by scavenging assay. iNOS and NF-${\kappa}B$ expressions were determined by immunoblot analysis. Interleukin (IL)-6 and tumour necrosis factor-${\alpha}$ (TNF-${\alpha}$) were assayed by ELISA. NO production was monitored by measuring the amount of nitrite. ROS was examined by using the 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA) method. At a concentration of 100 ${\mu}M$, ATX inhibited the expression level of LPS-induced NF-${\kappa}B$, as well as the production of LPS-induced NO and proinflammatory cytokines (IL-6 and TNF-${\alpha}$), by suppressing iNOS expression. In particular, the maximal inhibition rate of IL-6 and TNF-${\alpha}$ production by ATX (100 ${\mu}M$) was 65.2----- and 21.2-----, respectively. In addition, ATX inhibited the LPS-induced transcriptional activity of NF-${\kappa}B$, and this was associated with suppressing the translocations of NF-${\kappa}B$ from the cytosol to the nucleus. Moreover, at various concentrations (25-100 ${\mu}M$), ATX inhibited the intracellular level of ROS. At a concentration of 5 mg/ml, the superoxide radical scavenging activity of ATX was 1.33 times higher than ${\alpha}$-tocopherol of the same concentration. These results showed that ATX inhibited the expression of iNOS and the production of NO and proinflammatory cytokines resulting from ROS production and NF-${\kappa}B$ activation in macrophages. Furthermore, ATX was found to be more effective in superoxide radical scavenging activities compared to ${\alpha}$-tocopherol. These findings are expected to strengthen the position of ATX as anti-inflammatory medicine and antioxidant.