• Title/Summary/Keyword: iNOS assay

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Lonicera Japonioa Inhibits the Production of NO through the Suppression of NF-kB Activity in LPS-stimulated Mouse Peritoneal Macrophages

  • Kim Young-hee;Kim Han-do
    • The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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    • v.17 no.1
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    • pp.163-171
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    • 2004
  • The flowers of Lonicera japonica Thunb. (Caprifoliaceae) has been used as anti-inflammatory drug in the folk medicine recipe and been proved its anti-inflammatory effect in the oriental medicine. However, the action mechanism of Lonicera japonica that exhibits anti-inflammatory effects has not been determined. Since nitric oxide (NO) is one of the major inflammatory parameter, we studied the effect of aqueous extracts of Lonicera japonica (AELJ) on NO production in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. NO and inducible NO synthase (iNOS) level were significantly reduced in LPS-stimulated macrophages by AELJ compared to those without Electrophoretic mobility shift assay (EMSA) indicated that AELJ blocked the activation of nuclear factor kappa B (NF-kB), which was considered to be a potential transcription factor for the iNOS expression. AELJ also blocked the phosphorylation and degradation of inhibitor of kappa B-alpha (IkB-${\alpha}$). Furthermore, IkB kinase alpha (IKK${\alpha}$), which is known to phosphorylate serine residues of IkB directly, is inhibited by AELJ in vivo and in vitro. These results suggest that AELJ could exert its anti-inflammatory actions by suppressing the synthesis of NO through inhibition of NF-kB activity.

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The Anti-inflammatory Effects of Hataedock Taken Douchi Extracts on Atopic Dermatitis-like Skin Lesion of NC/Nga Mouse (두시(豆豉) 추출물을 이용한 하태독법(下胎毒法)이 NC/Nga 생쥐에서 유발된 아토피 유도 피부염에 미치는 항염증 효과)

  • Aum, Sun Ho;Ahn, Sang Hyun;Park, Sun Young;Cheon, Jin Hong;Kim, Ki Bong
    • The Journal of Pediatrics of Korean Medicine
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    • v.30 no.2
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    • pp.1-9
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    • 2016
  • Objectives Hataedock is a Korean herbal medical oral treatment that removes fetal toxic heat and meconium from new born babies. The purpose of this study is to evaluate whether Hataedock treatment of Duchi extracts has anti-inflammation effects on AD (Atopic Dermatitis)-induced NC/Nga mice. Methods After Hataedock treatment of Duchi extracts on days 0, 3-week-old NC/Nga mice were sensitized on days 28, 35, 42 by exposure of DNFB (dinitrochlorobenzene) and were induced to have AD. Immunohistochemistry of NF-${\kappa}B$ p65, iNOS, COX-2 and TUNEL assay of apoptotic body was used to identify changes of skin damages and anti-inflammation effects. Results The alleviate effect of the skin damage and angiogenesis was observed in DT group. The damage of stratum corneum, hyperplasia, edema, infiltration of lymphocytes and distribution of capillary were decreased in DT group. Also, the study results suggested that Hataedock treatment of Duchi extracts in DT group remarkably downregulated levels of NF-${\kappa}B$ p65 by 70% (p < 0.001), as well as COX-2 by 51%, iNOS by 62% (p < 0.001). Additionally, Hataedock treatment of Duchi extracts in DT group up-regulated apoptosis of inflammatory cells by 68% in atopic dermatitis-like skin lesion. Conclusions From the study results, we observed that Hataedock treatment of Duchi extracts alleviates AD through diminishing various inflammatory cytokines in the skin lesions, which are involved in the initial steps of AD development. It is anticipated to have potential applications for prevention and treatment of atopic dermatitis.

Anti-inflammatory Effect of Ribes fasciculatum in IFN-${\gamma}$/LPS-stimulated Mouse Peritoneal Macrophage

  • Kim, Jin Kyu;Kim, Ha Na;Kang, Chung Sik;Seo, Je Han;Seo, Hyun Won;Im, Jun Sang;Kim, Bong Seok;Cha, Dong Seok;Kwon, Jin;Oh, Chan Ho;Ma, Sang Yong;Nam, Jung Il;Jeon, Hoon
    • Natural Product Sciences
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    • v.20 no.2
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    • pp.113-118
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    • 2014
  • Ribes fasciculatum which belongs to Saxifragaceae has been widely used as a traditional medicine for the treatment of symptoms associated with lacquer poison. However, pharmacological studies on the R. fasciculatum are extremely limited until now. Thus, in this study, we evaluated the possible anti-inflammatory effects of ethyl acetate fraction of R. fasciculatum (ERF) using IFN-${\gamma}$/LPS-stimulated peritoneal macrophage model. We investigated the change in nitrite level in the absence or presence of ERF after LPS stimulation, and we found that ERF effectively attenuates the NO production in a dose dependent manner without notable toxicity. To determine the mechanism of the inhibitory action of ERF on NO production, we performed iNOS enzyme activity assay and Western blotting. Here we showed that both of iNOS enzyme activities and iNOS expressions were significantly down-regulated by ERF, indicating that these dual activities of ERF are responsible for ERF-mediated NO suppression. In addition, ERF inhibitied the expression of cyclooxygenase-2 (COX-2), an another key enzyme in inflammation through suppression of NF-${\kappa}B$ activation. We also tested anti-inflammatory properties of ERF not only in vitro, but in vivo using trypsin-induced paw edema model in mice. Our results revealed that the increased paw volume in response to trypsin injection was recovered by ERF supplement dose dependently.

Inhibition of LPS induced iNOS, COX-2 and cytokines expression by salidroside through the $NF{-\kappa}B$ inactivation in RAW 264.7 cells (Salidroside의 RAW 264.7 세포에서 $NF{-\kappa}B$ 불활성화를 통한 LPS에)

  • Won, So-Jung;Park, Hee-Juhn;Lee, Kyung-Tae
    • Korean Journal of Pharmacognosy
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    • v.39 no.2
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    • pp.110-117
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    • 2008
  • In this study, we investigated the anti-inflammatory effects of salidroside (SAL) isolated from the MeOH extract of Acer tegmentosum Maxim heartwood in RAW 264.7 macrophage cells. SAL pretreatment significantly inhibited nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) productions in the lipopolysaccharide (LPS)-induced RAW 264.7 cells. Western blot and RT-PCR analyses revealed that SAL inhibited the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in a concentration-dependent manner. In addition, SAL reduced the release and the mRNA expressions of tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$) and interleukin-6 (IL-6). Furthermore, nuclear factorkappa B ($NF{-\kappa}B$) luciferase reporter assay was performed to know the involvement of SAL in the production of pro-inflammatory cytokines, we confirmed that LPS-induced transcription activity of $NF{-\kappa}B$ was inhibited by SAL. Taken together, our data indicate that anti-inflammatory property of salidroside might be the result from the inhibition of iNOS, COX-2, $TNF-{\alpha}$ and IL-6 expressions via the down-regulation of $NF{-\kappa}B$ activity.

Inhibition of Inducible Nitric Oxide Synthase and Cyclooxygenase-2 by Gamijihwang-tang Via Suppression of Nuclear Factor-B Activation in RAW 264.7 cells

  • Jang Du-Hyun;Kim Ji-Young;Han Eun-Hee;Park Hee-Ok;Kim Dong-Hee;Jeong Hye-Gwang;Yoo Dong-Yeol
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.19 no.5
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    • pp.1405-1410
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    • 2005
  • Asthma is recognized today as an inflammatory disease of the lung characterized by acute non-specific airway hypersensitiveness in association with chronic pulmonary inflammation. Gamijihwang-tang(GJT), a fortified prescription of YMJHT, is applied for the treatments of chronic coughing and asthma, and post-delivery coughing and asthma in the gynecology. Also in the clinical practice, GJT is known to be very effective for controlling coughing and asthma as a cold sequoia. In this study, we investigated the effects of GJT on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) production, and on the level of inducible nitric oxide synthase (iNOS) and Cyclooxygenase-2 expression in murine macrophage RAW 264.7 cells. We found that GJT inhibited LPS-induced NO and $PGE_2$ production in a dose dependent manner. Furthermore, GJT inhibited the expression of LPS-induced iNOS and COX-2 protein and mRNA expression in RAW 264.7 macrophages. Treatment with GJT of RAW 264.7 cells transfected with a reporter construct indicated a reduced level of LPS-induced nuclear factor-KB (NF-kB) activity and effectively lowered NF-kB binding as measured by transient transfection assay. These results suggest that the main inhibitory mechanism of the GJT may be the reduction of iNOS and COX-2 gene expression through blocking of NF-kB activation.

Study on Anti-inflammatory Effect of Ethanol Extract of Gentianae sino-ornata (용담화 에탄올 추출물의 항염증 효과에 관한 연구)

  • Choi, Hyung-Wook;In, Myung-Hee;Lee, Shun-Hua;Kim, Eun-Joo;Lim, Kyu-Sang;Woo, Won-Hong
    • The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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    • v.28 no.4
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    • pp.51-61
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    • 2015
  • Objective : This research has been conducted to comparative analysis of anti-inflammatory effects to suggest a usefulness of ethanol extracts fromGentianae sino-ornata(GSO) as a functional material for inflammatory activities.Methods : Cell viability was measured by neutral red (NR) assay, and nitric oxide (NO) production in RAW264.7 cells was monitored by measuring the nitrite content in culture medium. The expressions of cyclooxigenase-2 (COX-2) was determined by western blot analysis, and Inducible nitric oxide syntase (iNOS) and cytokine were determined by reverse transcriptase-polymerase chain reaction (RT-PCR).Results : When the GSO extract was added the concentration of 5-20 ㎍/㎕, the viability of cells was maintained 90% or more at all levels. NO production was suppressed by the treatment of GSO in LPS-stimulated RAW264.7 cells. GSO inhibited the expression of iNOS, COX-2, IL-1βand IL-6 in LPS-stimulated RAW264.7 cells.Conclusions: From this results, we consider that GSO can be a useful therapeutic and preventive approach to various inflammatory diseases as a functional material for inflammatory activities.

Inhibition of TNF-α-mediated NF-κB Transcriptional Activity in HepG2 Cells by Dammarane-type Saponins from Panax ginseng Leaves

  • Song, Seok-Bean;Tung, Nguyen Huu;Quang, Tran Hong;Ngan, Nguyen Thi Thanh;Kim, Kyoon-Eon;Kim, Young-Ho
    • Journal of Ginseng Research
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    • v.36 no.2
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    • pp.146-152
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    • 2012
  • Panax ginseng (PG) is a globally utilized medicinal herb. The medicinal effects of PG are primarily attributable to ginsenosides located in the root and leaf. The leaves of PG are known to be rich in various bioactive ginsenosides, and the therapeutic effects of ginseng extract and ginsenosides have been associated with immunomodulatory and anti-inflammatory activities. We examined the effect of PG leaf extract and the isolated ginsenosides, on nuclear factor (NF)-${\kappa}B$transcriptional activity and target gene expression by applying a luciferase assay and reverse transcription polymerase chain reaction in tumor necrosis factor (TNF)-${\alpha}$-treated hepatocarcinoma HepG2 cells. Air-dried PG leaf extract inhibited TNF-${\alpha}$-induced NF-${\kappa}B$transcription activity and NF-${\kappa}B$-dependent cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) gene expression more efficiently than the steamed extract. Of the 10 ginsenosides isolated from PG leaves, Rd and Km most significantly inhibited activity in a dose-dependent manner, with $IC_{50}$ values of $12.05{\pm}0.82$ and $8.84{\pm}0.99\;{\mu}M$, respectively. Furthermore, the ginsenosides Rd and Km inhibited the TNF-${\alpha}$-induced expression levels of the COX-2 and iNOS gene in HepG2 cells. Air-dried leaf extracts and their chemical components, ginsenoside Rd and Km, are involved in the suppression of TNF-${\alpha}$-induced NF-${\kappa}B$ activation and NF-${\kappa}B$-dependent iNOS and COX-2 gene expression. Consequently, air-dried leaf extract from PG, and the purified ginsenosides, have therapeutic potential as anti-inflammatory.

Aqueous extract of Lycii fructus suppresses inflammation through the inhibition of nuclear factor kappa B signal pathway in murine raw 264.7 macrophages

  • Kim, Beum-Seuk;Lim, Hyung-Ho;Song, Yun-Kyung;Sung, Yun-Hee;Kim, Sung-Eun;Chang, Hyun-Kyung;Shin, Mal-Soon;Kim, Chang-Ju;Lee, Hye-Jung;Kim, Dong-Hee
    • Advances in Traditional Medicine
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    • v.10 no.3
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    • pp.155-164
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    • 2010
  • Lycii fructus is the fruit of Lycium chinense Miller and is part of the Solanaceae family. Lycii fructus produces various effects such as hypotensive, hypoglycemic, anti-pyretic, and anti-stress activities. Lycii fructus is known to contain betaine, carotene, nicotinic acid, zeaxanthin, and cerebroside. In the present study, the effects of Lycii fructus aqueous extract on lipopolysaccharide (LPS)-induced inflammation in murine raw 264.7 macrophage cells were investigated. In this study we utilized the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcriptionpolymerase chain reaction (RT-PCR), Western blotting, and nitric oxide (NO) detection. Lycii fructus aqueous extract suppressed NO production by inhibiting the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-$\alpha$) mRNA and iNOS protein in murine raw 264.7 macrophage cells. Also, Lycii fructus aqueous extract suppressed the activation of nuclear factor-kappa B (NF-${\kappa}B$) in the nucleus. These results demonstrated that Lycii fructus aqueous extract causes an anti-inflammatory effect that was likely produced by the suppression of iNOS expression through the down-regulation of NF-$\hat{e}B$ binding activity.

NF-kB and AP-1-regulatory Mechanism of Buthus Martensi Karsch Herbal Acupuncture Solution on Inflammatory Cytokine-induced Human Chondrocytes Dysfunction

  • Cho, Jae-Yong;Kim, Kyung-Ho;Cho, Hyun-Seok;Lim, Dae-Jung;Hwang, Ji-Hye;Kim, Kap-Sung
    • Journal of Acupuncture Research
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    • v.23 no.2
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    • pp.61-72
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    • 2006
  • Objectives : Human chondrocytes co-treated with Buthus martensi Karsch herbal acupuncture solution(BMK-HAS) extract produced significantly less NO compared with chondrocytes stimulated with $IL-1{\beta}$ alone Methods : Activation and translocation of and NF-kB DNA binding activity were determined by Western blotting and specific enzyme-linked immunosorbent assay. Results : The inhibition of NO production correlated with the suppression of induction and expression of nuclear factor-kB (NF-kB) and activation protein-1 (AP-1)-dependent gene. BMK-HAS inhibited the activation and translocation of NF-kB to the nucleus, indicating that BMK-HAS inhibits the $IL-1{\beta}-induced$ production of NO in human chondrocytes by interfering with the activation of NF-kB through a novel mechanism. In addition, BMK-HAS reduced prostaglandin E2 (PGE2)production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) or cyclooxygenase-1 (COX-1) was observed. My data, therefore, suggest that BMK-HAS may be a therapeutically effective inhibitor of $IL-1{\beta}-induced$ inflammatory effects that are dependent on NF-kB activation in human OA chondrocytes. Conclusion : The results indicate that BMK-HAS exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and PGE2 production, which could be due to a decreased expression of iNOS and COX-2 through the transcription factors NF-kB and AP-1.

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2-Nonadecanone Alleviates Depression through Inflammation Relief in SD Rat

  • Lee, Gil-Hyun;Hyun, Kyung-Yae
    • Biomedical Science Letters
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    • v.24 no.3
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    • pp.206-212
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    • 2018
  • Depression is a type of mood disorder characterized by hypochondriasis, decreased appetite, and insomnia. Depression is a disease that affects more than 100 million people worldwide. 2-Nonadecanone (NAC) is a bioactive substance that constitutes Fomes fomentarius, and NAC is expected to have an antidepressant effect. By using the forced swimming test (FST), we investigated the effects of treatment with NAC on immobility subacutely in rats after oral dosing once a day for 2 days. Serum levels of cytokine interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were determined by enzyme-linked immunosorbent assay (ELISA). Nuclear factor-2 (Nrf-2) and inducible nitric oxide synthases (iNOS) were analyzed by western blot method. NAC dose-dependently decreased immobility in the FST. NAC dosedependently decreased FST-induced increase of cytokine levels, as manifested by significantly stronger effects on $IL-1{\beta}$ and $TNF-{\alpha}$ levels at higher doses than the lowest dose of NAC. Western blot analysis showed that Nrf-2 was significantly lower in the NAC-treated group than in the disease-induced group. The iNOS results were also significantly lower in the NAC-treated group than in the other groups. Considering FST results, the antidepressant effect of NAC is effective. Considering the results of cytokine and protein expression, this anti-depressant effect may be related to the anti-inflammatory effect. Therefore, it can be said that the anti-inflammatory effect of NAC increases the antidepressant effect in the FST experiment.