• Title/Summary/Keyword: iNOS assay

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Anti-inflammatory Effect of Oyster Shell Extract in LPS-stimulated Raw 264.7 Cells

  • Lee, Se-Young;Kim, Hak-Ju;Han, Ji-Sook
    • Preventive Nutrition and Food Science
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    • v.18 no.1
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    • pp.23-29
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    • 2013
  • This study was designed to investigate the anti-inflammatory effect of oyster shell extract on the production of pro-inflammatory factors [NO, reactive oxygen species (ROS), nuclear factor-kappa B (NF-${\kappa}B$), inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2)] and pro-inflammatory cytokines [Interleukin-$1{\beta}$ (IL-$1{\beta}$), Interleukin-6 (IL-6) and TNF-${\alpha}$] in the lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Cell viability, as measured by the MTT assay, showed that oyster shell extract had no significant cytotoxicity in Raw 264.7 cells. The treatment with oyster shell extract decreased the generation of intracellular reactive oxygen species dose dependently and increased antioxidant enzyme activities, such as SOD, catalase, GSH-px in LPS-stimulated macrophage cells. Oyster shell extract significantly suppressed the production of NO and also decreased the expressions of iNOS, COX-2 and NF-${\kappa}B$. Additionally, oyster shell extract significantly inhibited the production of IL-$1{\beta}$, IL-6, and TNF-${\alpha}$ in LPS-stimulated Raw 264.7 cells. Thus, these results showed that the oyster shell extract had an anti-inflammatory effect on LPS-stimulated Raw 264.7 cells.

Neuroprotective Effect of Cirsium japonicum and Silibinin on Lipopolysaccharide-induced Inflammation in BV2 Microglial Cells (대계와 실리비닌의 Mouse BV2 Microglial Cells에서 Lipopolysaccharide에 의해 유발된 염증반응에 대한 신경보호 효과)

  • Yeo, Hyun-Soo;Kim, Dong-Woo;Jun, Chan-Yong;Choi, You-Kyung;Park, Chong-Hyeong
    • The Journal of Internal Korean Medicine
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    • v.28 no.1
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    • pp.166-175
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    • 2007
  • Objectives : This study was designed to evaluate the neuroprotective effect of Cirsium japonicum and Silibinin on lipopolysaccharide-induced inflammation in BV2 microglial cells. Methods : We studied on the neuroprotective effect of lipopolysaccharide-induced inflammation using MTS assay, western blot, and nitric oxide detection on mouse BV2 microglial cells. Results : Cirsium japonicum dose-dependently (50${\mu}g/ml$${\sim}$$250{\mu}g/ml$) inhibited nitrite production and iNOS expression in lipopolysaccharide-induced BV2 microglia and also significantly reduced lipopolysaccharide-induced COX-2 activation in western blot. Silibinin dose-dependently (10${\mu}M$${\sim}$$100{\mu}M$) inhibited nitrite production and iNOS expression in lipopolysaccharide-induced BV2 microglial cells. Silibinin also significantly reduced lipopolysaccharide-induced COX-2 activation in western blot. Conclusion : These effects of neuroprotection related to anti-inflammation suggest that Cirsium japonicum and Silibininmay be useful candidates for the development of a drug for related neurodegenerative diseases.

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MOK, a Pharmacopuncture Medicine, Reduces Inflammatory Response through Inhibiting the Proinflammatory Cytokine Production in LPS-stimulated Mouse Peritoneal Macrophages

  • Hwang, Ji Hye;Hwang, Min Sub;Park, Yong-ki
    • Journal of Acupuncture Research
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    • v.34 no.1
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    • pp.11-21
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    • 2017
  • Objectives : In this study, we investigated the anti-inflammatory and anti-oxidative effects of MOK, a pharmacopuncture medicine, in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. Methods : Peritoneal macrophages were isolated from ICR mice. Primary macrophages were treated with MOK extract (1.25, 2.5, 5, 10, and 20 mg/ml) for 30 min and then stimulated with LPS ($1{\mu}g/ml$) for the indicated times. Cytotoxicity was measured using MTT and LDH assays. Nitric oxide (NO) production in culture supernatants was measured using the Griess assay. The mRNA expression of iNOS, COX-2, proinflammatory cytokines (TNF-${\alpha}$, IL-$1{\beta}$, and IL-6) and antioxidant enzymes (HO-1 and MnSOD) was measured by RT-PCR. Results : Treatment with MOK extract (2.5, 5, and 10 mg/ml) significantly decreased LPS-induced NO production in peritoneal macrophages through inhibition of iNOS expression. The expression of COX-2, TNF-${\alpha}$, IL-$1{\beta}$, and IL-6 mRNA was also decreased in LPS-stimulated macrophages upon treatment with MOK extract. MOK treatment also increased the expression of HO-1 and MnSOD mRNA in macrophages. Conclusion : These results indicate that MOK exerts anti-inflammatory and antioxidant effects by regulating the transcription levels of inflammatory mediators and antioxidant proteins in activated macrophages.

Anti-inflammatory Effect of Mugi-hwan Water Extract in RAW 264.7 Cells (무기환(戊己丸)의 RAW 264.7 세포에 대한 항염증작용 연구)

  • Kim, Ilhyun;Choi, Chonghwan;Lee, Sewon;Song, Yungsun
    • Journal of Korean Medicine Rehabilitation
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    • v.23 no.3
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    • pp.27-35
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    • 2013
  • Objectives The aim of this study was to investigate anti-inflammatory activity of Mugi-hwan (MGH) Water Extract. Methods Cells were treated with 2 ug/ml of LPS 1 hour prior to the addition of MGH. Cell viability was measured by MTS assay. The production of NO was determined by reacting cultured medium with Griess reagent. The expression of COX-2, iNOS and MAPKs was investigated by Western blot, RT-PCR. The content of level of cytokines ($PGE_2$, IL-6, in media from LPS-stimulated Raw 264.7 cells was analyed by ELISA kit. Results MGH inhibited the production of NO, $PGE_2$, IL-6 as well as the expressions of iNOS, COX-2 in the murine macrophage, RAW 264.7 cells. MGH also had suppression effects of LPS induced MAPKs activation. Conclusions These results suggest that MGH has an anti-inflammatory therapeutic potential, which may result from inhibition of MAPK phosphorylation, thereby decreasing the expression of pro-inflammatory genes.

Effects of Kochiae Fructus Extracts on the Expression of Angiogeneis and Inflammation Related Proteins (지부자(地膚子)의 신생혈관 및 염증매개 단백질 발현에 미치는 영향)

  • Na, Sang-Hyuk;Shin, Yong-Cheol;Ko, Seong-Gyu
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.20 no.3
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    • pp.557-562
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    • 2006
  • Hypoxia induced angiogenesis and inflammation are essential processes for metastasis and progress of solid tumors. We examined the anti-angiogenic and inflammation related activity of Kochiae Fructus (KF) extract. To investigate the roles of the KF extract, we performed MTS assay, western blots using HaCaT cells and $HepG_2$ cells. The results are as follows. The protein level of $HIF-1{\alpha}$ was reduced when induced by $CoCl_2$ in $HepG_2$ cells treated with KF extract and induced by IGF-11 in HaCaT cells treated with KF extract. KF extract reduced the mRNA level of VEGF in HaCaT cells and KF extract reduced the protein level of iNOS in HaCaT cells. These results suggest that KF extract contributes to the anti-angiogenic and anti-inflammatory activities and also we could assume that KF extract act as antioxidant or anti-inflammatory agents via reduction of $HIF-1{\alpha}$.

Anti-inflammatory Effect of Imyosan Extract is more potent than that of its Component Herb Extracts in Murine Macrophages (마우스 대식세포인 RAW 264.7에 대한 이묘산(二妙散)의 메탄올 추출물의 항염증 효과)

  • Yun, Hyun-Jeong;Park, Sun-Dong
    • The Korea Journal of Herbology
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    • v.23 no.3
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    • pp.163-173
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    • 2008
  • Objectives : Imyosan (IMS), a drug preparation comprised of Phellodendri Cortex (PC) and Atractylodis Rhizoma (AR), is commonly used as a traditional herbal medicine in Korea and China for the treatment of various inflammatory diseases. However, little is known about the effect of IMS and its component herbs on inflammatory mediators in RAW 264.7 cells. Therefore, in this study, methanol extracts of IMS and its component herbs were examined to determine if they inhibited inflammatory effects in RAW 264.7 cells. Methods : Cytotoxic activity of IJHT and its components on RAW 264.7 cells was using 5-(3-carboxymethoxyphenyl)-2H-tetrazolium inner salt (MTS) assay. The nitric oxide (NO) production was measured by Griess reagent system. And proinflammatory cytokines were measured by ELISA kit. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were detected by western blot. Results : Methanol extract of IMS and its component herbs were significantly reduced iNOS and COX-2 expression as well as NO, PGE2, $IL-1{\beta}$ and IL-6 production in RAW 264.7 cells. Conclusions : The results of this study indicated that the anti-inflammatory effect of Imyosan extract is more potent than that of extracts of its component herbs in macrophages.

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Anti-inflammatory effect of new calcium hydroxide paste containing silicon-substituted hydroxyapatite in lipopolysaccharide-stimulated macrophages

  • Roh, Ji-Yeon;Kim, Ki-Rim
    • Journal of Korean society of Dental Hygiene
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    • v.18 no.4
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    • pp.423-432
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    • 2018
  • Objectives: Calcium hydroxide, a root canal temporary sealer, has long been used and it has anti-bacterial and anti-inflammatory activity. To investigate the properties of a newly developed calcium hydroxide paste comprising silicon-substituted hydroxyapatite (Si-HA), we examined the anti-inflammatory activity of the new calcium hydroxide paste in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS), which causes infection of the root canal. Methods: The test materials, including Calcipex II as control group and the newly developed TRC paste, were extracted from cell culture media and then diluted for experiment. In LPS-stimulated RAW 264.7 cells, the cytotoxicity and nitric oxide (NO) production of test materials were measured by MTT assay and Griess reagents, respectively. Also, the expression of the inducible nitric oxide synthase (iNOS) was assessed by western blotting. Results: The IC50 values of Calcipex II and TRC paste were $17.6mg/m{\ell}$ and $13.5mg/m{\ell}$, respectively. The level of NO, increased by LPS, was dose-dependently inhibited more by TRC paste than Calcipex II treatment. In addition, iNOS expression was decreased by 71% and 92% at concentrations of $2mg/m{\ell}$ and $20mg/m{\ell}$ of TRC paste, respectively. Conclusions: We demonstrated that the Si-HA calcium hydroxide paste has a slightly improved anti-inflammatory property and further studies are needed before clinical recommendations are proposed.

Antiinflammatory and Antiangiogenic Activities of Flavonoids Isolated from Belamcandae Rhizoma

  • Jung, Sang-Hoon;Ahn, Kwang-Seok;Lee, Yeon-Sil;Shin, Kuk-Hyun;Kim, Yeong-Shik
    • Proceedings of the PSK Conference
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    • 2003.10b
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    • pp.202.4-203
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    • 2003
  • The present study was carried out to clarify whether isoflavonoids isolated from Belamcandae Rhizoma (Iridaceae) inhibit inflammation and angiogenesis by the experimental methods in vitro and in vivo. Among the isolated isoflavonoids, such as irigenin, irisflorentine, and iristectorene B inhibited nitric oxide (NO) production, as measured by nitrite formation at 3-30 ${\mu}M$. Also these compounds reduced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzyme expression in a concentration dependent manner, when measured by western blotting, at 3-30 ${\mu}M$. Irigenin, irisflorentine and iristectoren B decreased angiogenesis of chick embryos in the chorioallantoic membrane assay. These compounds also reduced the proliferation of calf pulmonary arterial endothelial (CPAE) cells and found to possess relatively weak gelatinase/collagenase inhibitory activity in vitro. These compounds, when administered subcataneously at the dose of 30mg/kg for 20 days to mice implanted with murine Lewis lung carcinoma (LLC), caused a significant inhibition of tumor volume. Therefore, antiangiogenic activities of isoflavonoids from Belamcandae Rhizoma might be due to antiproliferative activities under inhibition the induction of COX-2 and iNOS enzyme.

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Effect of Actinidia polygama on LPS-induced Inflammation in Mouse BV2 Microglial cells (목천료자(木天蓼子)가 LPS로 유되된 Mouse BV2 Microglial cells의 염증반응에 미치는 영향)

  • Kim, Kitae
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.36 no.4
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    • pp.120-124
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    • 2022
  • Actinidia polygama has long been used in traditional Korean medicine to treat rheumatoid arthritis and gout. Although numerous chemical compounds in the fruit extracts of A. polygama have been characterized and their role in inhibiting nitric oxide (NO) production has been reported, the anti-inflammatory properties of A. polygama extracts remain to be explored. In this study, we investigated the in-vivo effect of A. polygama extract on lipopolysaccharide (LPS)-induced inflammation in BV-2 microglial cell lines. We discovered that 100% ethyl alcohol extract of A. polygama effectively attenuates the release of NO and is superior to both water extract and 50% ethanol extract. Using MTT assay, western blot, and ELISA on LPS-induced BV-2 microglial cells lines, we established the ability of A. polygama extract to markedly suppress the expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines, such as tumor necrosis factor alpha and interleukin-6. These results reveal that the anti-inflammatory property of A. polygama in BV-2 microglial cells is due to the downregulation of iNOS, COX-2, MAPK protein, and pro-inflammatory cytokines.

Astilboides tabularis (Hemsl.) Engl. Exerts Anti-inflammatory Effects through NF-κB Signaling Pathway in Lipopolysaccharide (LPS)-induced RAW 264.7 Cells

  • Hye-Jeong Park;So-Yeon Han;Da-Yoon Lee;Seo-Yoon Park;Jun-Hwan Jeong;Yoon-Jae Kwon;Tae-Won Jang;Jae-Ho Park
    • Proceedings of the Plant Resources Society of Korea Conference
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    • 2022.09a
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    • pp.109-109
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    • 2022
  • Astilboides tabularis (A. tabularis) has been thought to be material for functional food and could help to prevent diabetes for centuries. A. tabularis has been reported to contain phytochemicals (catechin, chlorogenic acid, and rutin), which have antioxidant and anti-inflammatory effects. We focused on anti-inflammatory effects of ethyl acetate fraction of A. tabularis (EAT) through NF-κB signaling pathway. Phytochemicals of EAT were analyzed by total flavonoid and phenolics assay, its content was 219.2 ± 2.01 and 524.7 ± 0.80 mg/g respectively. EAT significantly reduced the expression of iNOS and COX-2, which caused the production of nitric oxide (NO). EAT suppressed mRNA levels of iNOS, COX-2, and cytokines (IL-6, IL-1β, and TNF-α). EAT suppressed the expression of p-p65 through the inhibition of phosphorylation of IkB-a, and the result of immunofluorescence showed that inhibited the translocation of p65 from the cytoplasm to the nucleus.

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