• 대한임상검사과학회지
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• 제50권3호
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• pp.337-344
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• 2018

발생하는 간암 중 가장 주요한 형태인 간세포암은 강한 전이특성으로 인해 높은 재발율과 사망률을 보인다. Silymarin은 엉겅퀴에서 추출한 플라보노이드 성분으로 여러 암세포주에서 상피간엽전환(epithelial mesenchymal transition, EMT) 조절을 통해 항암효과를 보이는 것으로 보고되었다. 본 연구에서는 silymarin이 EMT의 조절을 통해 간세포암 세포주인 Huh7 cell의 침윤과 전이를 억제하는지를 분석하고자 하였다. Huh7 cell의 침윤과 전이 활성을 분석하기 위하여 wound healing assay와 in vitro invasion assay를 시행하였고 EMT 관련 유전자와 상위 신호전달물질의 발현 분석을 위해 Western blot assay를 실시하였다. 그 결과 silymarin은 농도 의존적으로 Huh7 cell의 침윤과 전이를 억제하였다. EMT 관련 유전자 중 세포 부착 단백질인 E-cadherin은 증가하였으나, 중간엽세포의 지표인 vimentin, 종양미세환경 조절에 관여하는 MMP-9의 발현은 억제되었고 이들의 활성에 관여하는 전사인자인 Snail과 nuclear factor $(NF)-{\kappa}B$ 또한 농도 의존적으로 활성이 감소하는 것을 확인할 수 있었다. 특히, 상위신호전달물질 중 silymarin은 phosphoinositide-3-kinase (PI3K)/Akt의 인산화 억제를 통해 EMT 관련 유전자들을 조절하는 것으로 나타났고 이것은 selective inhibitor인 LY294002의 처리 결과로 확인할 수 있었다. 결과적으로, silymarin은 PI3K/Akt 경로를 통해 EMT 관련 유전자의 발현을 조절함으로써 Huh7 cell의 침윤과 전이를 억제하는 것으로 생각된다. 이를 통해 silymarin이 간세포암의 전이 억제에 효과적인 항암물질의 후보가 될 수 있는 잠재력을 가진 후보물질이 될 수 있음을 보여주었다.

• IMMUNE NETWORK
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• 제3권3호
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• pp.201-210
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• 2003

Objective: The role of prostaglandin $E_2$ (PGE2) in the etiopathogenesis of immune and inflammatory diseases has become the subject of recent debate. To determine the role of PGE2 in rheumatoid arthritis (RA), we tested the effect of exogenous PGE2 on the production of cyclooxygenase-2 (COX-2) by rheumatoid synoviocytes. Methods: Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and cultured in the presence of PGE2. The COX-2 mRNA and protein expression levels were determined by RT-PCR and Western blot analysis, respectively. The PGE2 receptor subtypes in the FLS were analyzed by RT-PCR. Electrophoretic mobility shift assay (EMSA) was used to measure the NF-${\kappa}B$ binding activity for COX-2 transcription. The in vivoeffect of PGE2 on the development of arthritis was also tested in collagen induced arthritis (CIA) animals. Results: PGE2 ($10^{-11}$ to $10^{-5}M$) dose-dependently inhibited the expression of COX-2 mRNA and the COX-2 protein stimulated with IL-$1{\beta}$, but not COX-1 mRNA. NS-398, a selective COX-2 inhibitor, displayed an additive effect on PGE2-induced COX-2 downregulation. The FLS predominantly expressed the PGE2 receptor (EP) 2 and EP4, which mediated the COX-2 suppression by PGE2. Treatment with anti-IL-10 monoclonal antibodies partially reversed the PGE2-induced suppression of COX-2 mRNA, suggesting that IL-10 may be involved in modulating COX-2 by PGE2. Experiments using an inducer and an inhibitor of cyclic AMP (cAMP) suggest that cAMP is the major intracellular signal that mediates the regulatory effect of PGE2 on COX-2 expression. EMSA revealed that PGE2 inhibited the binding of NF-${\kappa}B$ in the COX-2 promoter via a cAMP dependent pathway. In addition, a subcutaneous injection of PGE2 twice daily for 2 weeks significantly reduced the incidence and severity of CIA as well as the production of IgG antibodies to type II collagen. Conclusion: Our data suggest that overproduced PGE2 in the RA joints may function as an autocrine regulator of its own synthesis by inhibiting COX-2 production and may, in part, play an anti-inflammatory role in the arthritic joints.

• International Journal of Oral Biology
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• 제34권2호
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• pp.87-95
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• 2009

Porphyromonas gingivalis is a major etiologic agent of chronic periodontitis and cytokines produced by macrophages play important roles in the pathogenesis of periodontal diseases. In this study we investigated the cytokine response of phorbol myristate acetatedifferentiated THP-1 cells exposed to P. gingivalis. Compared with the prominent cell wall components of P. gingivalis (lipopolysaccharide and the major fimbrial protein FimA), live P. gingivalis stimulated much higher levels of cytokine production. In addition, whereas low multiplicity of infection challenges (MOI=10) of P. gingivalis 381 stimulated high levels of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-6 (IL-6), and IL-1${\beta}$, high dose challenges with this bacterium (MOI = 100) resulted in a substantially diminished production of MCP-1 and IL-6. Moreover, high MOI P. gingivalis challenges achieved only low levels of induction of MCP-1 and IL-6 mRNA. The decreased production of MCP-1 and IL-6 appeared to be mediated by P. gingivalis proteases, because high MOI challenges with congenic protease mutant strains of this microorganism (MT10 and MT10W) did not result in a diminished production of MCP-1 and IL-6. Similar to its protease mutant strains, leupeptin (a protease inhibitor)- treated P. gingivalis at high doses induced high levels of MCP-1 production. To examine the mechanisms underlying the diminished production of MCP-1 by P. gingivalis proteases, the activation of mitogen-activated protein (MAP) kinases and NF-${\kappa}$B was compared between the 381 and MT10W strains. Whilst high doses of both 381 and MT10W similarly activated the three members of the MAP kinase family, the DNA binding activity of NF-${\kappa}$B, as revealed by gel shift assays, was greatly increased only by MT10W. Taken together, our data indicate that P. gingivalis stimulates the production of high levels of TNF-${\alpha}$, IL-1${\beta}$, IL-6, and MCP-1 but that high dose challenges with this bacterium result in a diminished production of MCP-1 and IL-6 via the protease-mediated suppression of NF-${\kappa}$B activation in THP-1 macrophagic cells.

• 대한본초학회지
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• 제32권2호
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• pp.77-85
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• 2017

Objectives : This study aimed to investigate the unknown mechanisms behind the anti- inflammatory activity of Hyeonto-dan(HT) 70% ethanol extract on LPS-stimulated RAW 264.7 cells. Methods : Cells were treated with Hyeonto-dan 1 h prior to addition of 200 ng/mL of LPS. Cell viability was measured by the MTS assay. Nitric oxide levels were determined by the Griess assay. $PGE_2$ were measured using EIA kit. Pro-inflammatory cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA). The expression of COX-2, iNOS, and MAPKs was investigated by Western blot, qRT-PCR. $NF-{\kappa}B$/p65 localization and interaction of the TLR-4 receptor with LPS was examined by immunofluorescence assays. Results : Hyeonto-dan had no cytotoxicity at the measured concentration. Hyeonto-dan inhibited NO production and pro-inflammatory cytokines such as IL-6, $TNF-{\alpha}$, and PGE2 as well as the protein and mRNA expression of iNOS and COX-2. Moreover, Hyeonto-dan inhibited the interaction between LPS and TLR-4 in murine macrophages. It suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2), c-jun N-terminal kinase (JNK 1/2) and p38. Finally, it inhibited translocation of $NF-{\kappa}B$ in response to competitive LPS. Conclusions : Based on the results of this study, Hyeonto-dan inhibited the binding of TLR-4 receptor to LPS and inhibited the phosphorylation of extracellular signaling pathway MAPKs. These inhibitory effects are thought that the amount of $NF-{\kappa}B$ delivered to the nucleus was decreased and the inflammatory reaction was prevented by decreasing the production of LPS-induced $PGE_2$, NO, IL-6 and $TNF-{\alpha}$.

• 대한한의학회지
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• 제40권3호
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• pp.35-58
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• 2019

Objectives: The aim of this study was to investigate the anti-inflammatory effects of Curcuma longa rhizoma extract in an experimental rat model of osteoarthritis. Methods: Osteoarthritis was induced in rats by injecting monosodium iodoacetate (MIA) into the knee joint cavity of rats. The rats were divided into 5 groups (Normal, Control, positive comparison, low (CL) and high (CH) concentration groups). Rats in the low concentration (CL) group had MIA-induced osteoarthritis; they were treated with Curcuma longa rhizoma extract at a dose of 50mg/kg body weight. Rats in the high concentration (CH) group had MIA-induced osteoarthritis; they were treated with Curcuma longa rhizoma extract at a dose of 100mg/kg body weight. Hind paw weight distribution and ROS levels were measured. At the end of all treatments, changes in alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine levels were analyzed. In addition, inflammatory protein levels were evaluated by western blot analysis. Results: In this study, hind paw weight distribution significantly improved in the CL and CH groups, while. Reactive oxygen species (ROS) production significantly decreased in both. The levels of ALT, AST, BUN, and creatinine did not significantly change in either group. The production of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), $p47^{phox}$, and Ras-related C3 botulinum toxin substrate 1 (RAC1) decreased in both. Catalase, heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) significantly increased in the CL and CH groups, respectively. Nuclear factor erythroid 2 (Nrf2) increased, but there were no significant differences between the experimental and control groups. Inflammatory cytokines, including nuclear factor-kappa Bp65 (NF-${\kappa}Bp65$), interleukin-1beta (IL-$1{\beta}$), and tumor necrosis factor-alpha (TNF-${\alpha}$), decreased significantly in both the CL and CH groups. Conclusions: Our results showed that Curcuma longa rhizoma extract has anti-inflammatory effects. Anti-inflammatory activity is regulated by the inhibition of inflammatory cytokines and mediators, such as NF-${\kappa}B$, therefore, it suppresses cartilage damage as well.

• Animal Bioscience
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• 제37권2호
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• pp.303-314
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• 2024

Objective: Rutin, also called vitamin P, is a flavonoids from plants. Previous studies have indicated that rutin can alleviate the injury of tissues and cells by inhibiting oxidative stress and ameliorating inflammation. There is no report on the protective effects of rutin on goat rumen epithelial cells (GRECs) at present. Hence, we investigated whether rutin can alleviate lipopolysaccharide (LPS)-induced damage in GRECs. Methods: GRECs were cultured in basal medium or basal medium containing 1 ㎍/mL LPS, or 1 ㎍/mL LPS and 20 ㎍/mL rutin. Six replicates were performed for each group. After 3-h culture, the GRECs were harvested to detect the relevant parameters. Results: Rutin significantly enhanced the cell activity (p<0.05) and transepithelial electrical resistance (TEER) (p<0.01) and significantly reduced the apoptosis rate (p<0.05) of LPS-induced GRECs. Rutin significantly increased superoxide dismutase, glutathione peroxidase, and catalase activity (p<0.01) and significantly decreased lactate dehydrogenase activity and reactive oxygen species and malondialdehyde (MDA) levels in LPS-induced GRECs (p<0.01). The mRNA and protein levels of interleukin 6 (IL-6), IL-1β, and C-X-C motif chemokine ligand 8 (CXCL8) and the mRNA level of tumor necrosis factor-α (TNF-α) and chemokine C-C motif ligand 5 (CCL5) were significantly increased in LPS-induced GRECs (p<0.05 or p<0.01), while rutin supplementation significantly decreased the mRNA and protein levels of IL-6, TNF-α, and CXCL8 in LPS-induced GRECs (p<0.05 or p<0.01). The mRNA level of toll-like receptor 2 (TLR2), and the mRNA and protein levels of TLR4 and nuclear factor κB (NF-κB) was significantly improved in LPS-induced GRECs (p<0.05 or p<0.01), whereas rutin supplementation could significantly reduce the mRNA and protein levels of TLR4 (p<0.05 or p<0.01). In addition, rutin had a tendency of decreasing the protein levels of CXCL6, NF-κB, and inhibitor of nuclear factor kappa-B alpha (0.05

• 대한통합의학회지
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• 제7권4호
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• pp.61-69
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• 2019

Purpose : Human gingival fibroblast cell is one of the the main cell types in periodontal tissue, which they can show anti-inflammatory activity through the production of numerous lines of inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and interleukins. Porphyromonas gingivalis, one of the oral pathogens, has reported to play a critical role in the development of periodontal diseases. This study aimed to investigate anti-inflammatory and antioxidative activities of Gracilaria textorii ethanol extract (GTEE) in P. gingivalis derived lipopolysaccharide (LPS-PG) stimulated human gingival fibroblast (HGF)-1 cell line. Methods : In order to analyze anti-inflammatory and antioxidative activities of GTEE in HGF-1 cell line, NOS enzyme activity, expression levels of iNOS, COX-2, NAD(P)H quinone dehydrogenase (NQO)1 and their transcription factors were estimated by Griess reaction and western hybridization. Results : LPS-PG induced overexpression of iNOS and COX-2, which was significantly attenuated by GTEE treatment in a dose-dependent manner without any cytotoxicity. In addition, intracellular NOS activity was in accordance with the result of iNOS expression. Due to important role in the regulation of inflammatory responses, phosphorylated status of p65 and c-jun, each subunit of nuclear factor (NF)-κB and activator protein (AP)-1, was also dose-dependently ameliorated by GTEE treatment. One of phase II enzymes, NQO1, and its transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), were analyzed since elevated phase II enzyme expression inhibited inflammatory response, which was significantly elevated by GTEE treatment in HGF-1 cell line. Conclusion : In conclusion, GTEE mitigated LPS-PG-stimulated inflammatory responses by attenuating NF-κB and AP-1 activation as well as accelerating NQO1 and Nrf2 expression in HGF-1 cell line. These results indicate that GTEE might be utilized a promising strategy for potential anti-inflammatory agent in periodontal diseases.

• The Korean Journal of Physiology and Pharmacology
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• 제25권5호
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• pp.395-401
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• 2021

Extended inflammation and cytokine production pathogenically contribute to a number of inflammatory disorders. Formosanin C (FC) is the major diosgenin saponin found in herb Paris formosana Hayata (Liliaceae), which has been shown to exert anti-cancer and immunomodulatory functions. In this study, we aimed to investigate anti-inflammatory activity of FC and the underlying molecular mechanism. RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) or pretreated with FC prior to being stimulated with LPS. Thereafter, the macrophages were subjected to analysis of the expression levels of pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, as well as two relevant enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). The analysis revealed that FC administration blunted LPS-induced production of NO and PGE in a dose-dependent manner, while the expression of iNOS and COX-2 at both mRNA and protein levels was inhibited in LPS-stimulated macrophages pre-treated with FC. Moreover, LPS stimulation upregulated mRNA expression and medium release of TNF-α, IL-1β, and IL-6, whereas this effect was blocked upon FC pre-administration. Mechanistic studies showed that inhibitory effects of FC on LPS-induced inflammation were associated with a downregulation of IκB kinase, IκB, and p65/NF-κB pathway. Taken together, these data suggest that FC possesses an inflammation-suppressing activity, thus being a potential agent for the treatment of inflammation-associated disorders.

• 대한한방소아과학회지
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• 제31권1호
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• pp.43-51
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• 2017

Objectives Hataedock method is a Korean medical therapy which removes fetal toxin by orally administering herbal decoction to neonates. This study was to observe skin damage and anti-inflammatory effect via regulating IL-4 activity in NC/Nga mice which were induced atopic dermatitis (AD)-like skin lesion by Dermatophagoides (D.) farinae after applying Douchi Hataedock method. Methods NC/Nga mice with 3 weeks of gestational age were used. Each 10 mice were allocated to the control group (Ctrl), the AD-induced group (AE), and the group which induced AD after administering Douchi extract (GT). After 4 weeks from administering Douchi extract to the mice, the primary AD was induced by applying D. farinae extract 6 times per week for 3 weeks and then the secondary AD was induced by the same method after 1 week from the primary AD induction. To identify the skin damage and anti-inflammatory effect, we observed LxR, IL-4, Fc ${\varepsilon}$ receptor, substance P, and $NF-{\kappa}B$. Results The GT group showed alleviation of skin injury and decrease in capillary angiogenesis. Stratum corneum damage, epithelial cell hyperplasia, lymphocyte infiltration, and capillary distribution relatively decreased in the GT group. LxR-positive reaction in the GT group were increased by 53% than that of the AE group. IL-4 production, $Fc{\varepsilon}$ receptor activity, and substance P-positive reaction in the GT group were decreased by 82%, 42%, and 82% respectively compare to those of the AE group. $NF-{\kappa}B$-positive reaction in the GT group were decreased by 15% compare to that of the AE group. Conclusions Hataedock method with Douchi extract alleviated AD via reducing inflammatory cytokines secreted at the early stage of AD. Thus, Douchi Hataedock method has a beneficial effect for the prevention and treatment of AD.

• 동의생리병리학회지
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• 제22권3호
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• pp.678-683
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• 2008

Aconitum pseudo-laeve var. erectum has traditionally been used for the treatment of water retention in the body. Administration of the aqueous extract of Aconitum pseudo-laeve var. erectum has the efficiency of anti-inflammatory activity and modulates the intestinal immune system. However, the mechanism of anti-inflammatory action of Aconitum pseudo-laeve var. erectum has not been clarified yet. In the present study, the effect of Aconitum pseudo-laeve var. erectum against LPS-stimulated expressions of COX-2 and iNOS in cells of the murine RAW 264.7 macrophages was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcription- polymerase chain reaction (RT-PCR), PGE2 immunoassay, and NO detection. The results of the present study indicate that Aconitum pseudo-laeve var. erectum is a potent inhibitor of the LPS-induced NO and $PGE_{2}$ production by blocking iNOS and $NF{\kappa}B$ activation in RAW 264.7 macrophages. These findings suggest that Aconitum pseudo-laeve var. erectum is a potential therapeutic for the treatment of inflammatory syndrome.