• The Korea Journal of Herbology
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• v.25 no.2
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• pp.89-98
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• 2010

Objective : The aim of this study was to determine whether methanol extract of Keum-Ryung-Ja-San (KRJS) inhibit production of NO, $PGE_2$, iNOS, COX-2 and pro-inflammatory cytokines in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Methods : Cytotoxic activity of extracts on RAW 264.7 cells was measured using 5-(3-caroboxymeth-oxyphenyl)-2H-tetra-zolium inner salt (MTS) assay. The nitric oxide (NO) production was measured by Griess reagent system. And proinflammatory cytokines and PGE2 were measured by ELISA kit. The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2), $I{\kappa}-B-\alpha$ and nuclear NF-${\kappa}B$ p65 expression were detected by western blot. Results : Our results indicated that methanol extract of KRJS significantly inhibited the LPS-induced NO, $PGE_2$ production and iNOS, COX-2 expression accompanied by an attenuation of TNF-$\alpha$, IL-$1{\beta}$ and IL-6 production in RAW 264.7 cells. Moreover, methanol extract of KRJS treatment also blocked LPS-induced NF-${\kappa}B$ activation. Conclusion : These findings indicate that methanol extract of KRJS inhibits the production of pro-inflammatory mediators and cytokines via suppression of NF-${\kappa}B$ activation. Take together, these results indicate that methanol extract of KRJS has the potential for use as an agent of anti-chronic inflammatory diseases.

• The Journal of Internal Korean Medicine
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• v.37 no.4
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• pp.591-600
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• 2016

Objective: This study aimed to examine the relaxation effects and underlying mechanisms of Cynomorii herba (CH) extract in isolated rabbit corpus cavernous tissues.Methods: We experimented with CH extract (0.01-3.0 mg/mL). Nω-nitro-L-arginine (L-NNA) was experimented before the CH extract to contracted strips induced by phenylephrine (PE, 1 μM)and compared with nonexperimented. In addition, we experimented with calcium chloride (Ca²⁺, 1 mM) after pretreatment of the CH extract in Ca²⁺-free Krebs-Ringer solution to contracted strips induced by PE. The cell viability and nitric oxide (NO) concentration of human umbilical vein endothelial cells (HUVECs) were measured by an methylthiazol-2-yl-2, 5-diphenyl tetrazoliumbromide (MTT) assay and Griess reagent system. The ratio of smooth muscles to collagen fibers, in addition to eNOS- and PDE-5-positive reactions, was examined by histochemical and immunohistochemical staining.Results: The CH extract significantly induced the relaxation of the cavernous strips, and the pretreatment with L-NNA inhibited CH extract-induced relaxation. The L-NNA pretreatment reduced the increased contraction induced by the addition of Ca²⁺in Ca²⁺-free solution. Furthermore, the NO concentration of the HUVECs increased. When the CH extract was applied to the corpus cavernosum of the penis (CCP) of Sprague Dawley rats, the ratio of smooth muscles to collagen fibers by PE and the formation of eNOS around the helicine artery increased. However, the CH extract treatment decreased PDE-5 positive reactions.Conclusions: These results show that the relaxation effects induced by the CH extract are associated with the suppression of the influx of extracellular Ca²⁺ via the production of NO and eNOS and inhibition of PDE-5.

• Journal of Physiology & Pathology in Korean Medicine
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• v.25 no.2
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• pp.294-299
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• 2011

The purpose of this study is to investigate effects of Red Ginseng-Ejung-tang (RE) on nitric oxide (NO) and hydrogen peroxide production in RAW 264.7 mouse macrophages induced by lipopolysaccharide (LPS). Cell viability was measured by modified MTT assay. NO production was measured by Griess reagent assay. Hydrogen peroxide production was measured by dihydrorhodamine 123 (DHR) assay. RE did not show cell toxicity against RAW 264.7 for 24 hr incubation at the concentrations of 10, 25, 50, 100, and $200{\mu}g/mL$ in RAW 264.7. RE significantly inhibited NO production for 24 hr incubation at the concentrations of 10, 25, 50, and $100{\mu}g/mL$ in RAW 264.7 (P < 0.05). RE significantly inhibited the LPS-induced production of NO for 24 hr incubation at the concentrations of 10, 25, 50, and $100{\mu}g/mL$ in RAW 264.7 (P < 0.05). RE significantly inhibited the LPS-induced production of hydrogen peroxide for 16, 24, 40, 48, 64, and 72 hr incubation at the concentrations of 50, 100, and $200{\mu}g/mL$ in RAW 264.7 (P < 0.05). These results suggest that RE has anti-inflammatory property related with its inhibition of NO and hydrogen peroxide production in LPS-induced macrophages.

• Journal of Society of Preventive Korean Medicine
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• v.16 no.3
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• pp.155-165
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• 2012

Objective : The object of this study is to confirm the immune enhancement effect of Rubus coreanus Miquel (RC) (30% EtOH extract) on RAW264.7 mouse macrophage cell line. Methods : RAW264.7 cells were treated with $10-500{\mu}g/mL$ RC for 24 hours. Cell viability was then measured using WST assays. Levels of intracellular NO and ROS were measured by Griess reagent and DCFH-DA staining respectively. Levels of iNOS and COX-2 mRNA was determined by RT-PCR. Secretion levels of IL-$1{\beta}$ and IL-6 cytokines were evaluated by sandwich ELISA assay. Western blot analysis was performed to measure the levels of intracellular molecules related to MAPKs pathways. Results : RC suppressed the growth of RAW264.7 cells. RC increased the production of NO and ROS. RC increased the mRNA and protein levels of COX-2 and iNOS. RC augmented the levels of secreted IL-$1{\beta}$ and IL-6 cytokines. RC increased MAPKs phosphorylation. Conclusion : In summary, our result shows that RC has inflammatory effect increasing the levels of NO, ROS and secreted cytokines and activating MAPKs. Hence, RC seems to have an immune enhancement effect.

• Journal of dental hygiene science
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• v.15 no.6
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• pp.753-760
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• 2015

Although nuclear factor of activated T cell (NFAT) plays a key role in inflammation, its anti-inflammatory effects and mechanism of action in periodontitis are still unknown. This study aimed to identify the effects of NFAT on the proinflammatory mediators activated by lipopolysaccharide (LPS) plus nicotine stimulation in human periodontal ligament cells (hPDLCs). The production of nitric oxide (NO) and prostaglandin $E_2(PGE_2)$ was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and NFAT proteins was evaluated by Western blot analysis. LPS plus nicotine synergistically induced the production of NO and $PGE_2$ and increased the protein expression of iNOS, COX-2 and NFAT. Treatment with an NFAT inhibitor blocked the LPS plus nicotine-stimulated NO and $PGE_2$ release as well as the expression of iNOS and COX-2. Our data suggest that the LPS plus nicotine-induced inflammatory effects on hPDLCs may act through a novel mechanism involving the action of NFAT. Thus, NFAT may provide a potential therapeutic target for the treatment of periodontal disease associated with smoking and dental plaque.

• Journal of Cancer Prevention
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• v.21 no.3
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• pp.144-151
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• 2016

Background: Immunoregulatory elements have emerged as useful immunotherapeutic agents against cancer. In traditional medicine, Mori folium, the leaf of Morus alba L. (Moraceae), has been used for various medicinal purposes; however, the immunomodulatory effects have not been fully identified. We evaluated the immunoenhancing potential of water extract of Mori folium (WEMF) in murine RAW264.7 macrophages. Methods: RAW264.7 cells were treated with WEMF for 24 hours and cell viability was detected by an MTT method. Nitric oxide (NO) levels in the culture supernatants were assayed using Griess reagent. The productions of prostaglandin $E_2$ ($PGE_2$) and immune-related cytokines was measured using ELISA detection kits. The mRNA and protein expression levels of Inducible NO synthase, COX-2, and cytokines were assayed by reverse transcription-PCR and Western blotting, respectively. The effect of WEMF on phagocytic activity was measured using a Phagocytosis Assay Kit. Results: WEMF significantly stimulated the production of NO and $PGE_2$ as immune response parameters at noncytotoxic concentrations, which was associated with the increased expression of inducible NO synthase and COX-2. The release and expression of cytokines, such as $TNF-{\alpha}$, interleukin $(IL)-1{\beta}$, IL-6, and IL-10, were also significantly increased in response to treatment with WEMF. Moreover, WEMF promoted the macrophagic differentiation of RAW264.7 cells and the resulting phagocytosis activity. Conclusions: WEMF has the potential to modulate the immune function by regulating immunological parameters. Further studies are needed to identify the active compounds and to support the use of WEMF as an immune stimulant.

• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1200-1209
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• 2021

Sepsis is an acute inflammatory response that leads to life-threatening complications if not quickly and adequately treated. Cytolysin, hemolysin, and pneumolysin are toxins produced by gram-positive bacteria and are responsible for resistance to antimicrobial drugs, cause virulence and lead to sepsis. This work assessed the effects of aloe-emodin (AE) and photodynamic therapy (PDT) on sepsis-associated gram-positive bacterial toxins. Standard and antibiotic-resistant Enterococcus faecalis, Staphylococcus aureus, and Streptococcus pneumonia bacterial strains were cultured in the dark with varying AE concentrations and later irradiated with 72 J/cm^-2 light. Colony and biofilm formation was determined. CCK-8, Griess reagent reaction, and ELISA assays were done on bacteria-infected RAW264.7 cells to determine the cell viability, NO, and IL-1β and IL-6 pro-inflammatory cytokines responses, respectively. Hemolysis and western blot assays were done to determine the effect of treatment on hemolysis activity and sepsis-associated toxins expressions. AE-mediated PDT reduced bacterial survival in a dose-dependent manner with 32 ㎍/ml of AE almost eliminating their survival. Cell proliferation, NO, IL-1β, and IL-6 cytokines production were also significantly downregulated. Further, the hemolytic activities and expressions of cytolysin, hemolysin, and pneumolysin were significantly reduced following AE-mediated PDT. In conclusion, combined use of AE and light (435 ± 10 nm) inactivates MRSA, S. aureus (ATCC 29213), S. pneumoniae (ATCC 49619), MDR-S. pneumoniae, E. faecalis (ATCC 29212), and VRE (ATCC 51299) in an AE-dose dependent manner. AE and light are also effective in reducing biofilm formations, suppressing pro-inflammatory cytokines, hemolytic activities, and inhibiting the expressions of toxins that cause sepsis.

• The Korea Journal of Herbology
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• v.37 no.1
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• pp.41-50
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• 2022

Objectives : The aim of this study was to investigate the effect of water extract of Agastache rugosa (AR) on productions of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophages. Methods : Cell viabilities were measured with MTT assay. The production of nitric oxide (NO) from RAW 264.7 cells was measured with Griess reagent assay. The production of cytokines in RAW 264.7 cells was measured with multiplex cytokine assay. Results : AR showed no cytotoxicity on RAW 264.7 cells. AR at concentrations of 25, 50, 100, and 200 ㎍/mL significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. AR at concentrations of 50, 100, and 200 ㎍/mL significantly inhibited productions of TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells; AR at concentrations of 50 and 200 ㎍/mL significantly inhibited productions of RANTES (CCL5) in LPS-stimulated RAW 264.7 cells; AR at concentrations of 100 ㎍/mL significantly inhibited productions of macrophage inflammatory protein-1β in LPS-stimulated RAW 264.7 cells; AR at concentrations of 50, 100, and 200 ㎍/mL significantly increased productions of IP-10 (CXCL10) in LPS-stimulated RAW 264.7 cells; AR at concentrations of 100 and 200 ㎍/mL significantly increased MCP-1 (CCL-2) in LPS-stimulated RAW 264.7 cells; AR at concentrations of 50 and 100 ㎍/mL significantly increased productions of IL-10 in LPS-stimulated RAW 264.7 cells. Conclusions : AR might have immunomodulatory effects on productions of NO, cytokines, and chemokines in LPS-stimulated RAW 264.7 mouse macrophages.

• The Korea Journal of Herbology
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• v.32 no.2
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• pp.17-24
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• 2017

Objective : This study was designed to evaluate candidate materials as anti-inflammation agent from extracts of various Korean Compositae herbs in Hwaak mountain. Among Korea medicinal herbs, Ainsliaea acerifolia (AA) belongs to the Compositae family, has been used for the treatment of rheumatic arthritis. However, AA has not been previously reported to have an anti-inflammatory effect. Therefore, we investigated the anti-inflammatory effects of AA and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Methods : Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in RAW 264.7 macrophages. Nitric oxide (NO) was measured with Griess reagent and pro-inflammatory cytokines were detected by enzyme immunoassay (EIA) kits in LPS-stimulated RAW 264.7 macrophages. Protein expressions of inducible nitric oxide synthase, and cyclooxygenase-2 (COX-2) and p65 subunit of nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) were determined by Western blot analysis. Results : Among 8 extracts of Korean Compositae herbs tested, AA showed the inhibition of NO production without cytotoxicity. Consistent with the observation, AA reduced the expression levels of iNOS and COX-2 proteins in LPS-simulated RAW 264.7 macrophages in dose-dependent manner. In addition, AA inhibited the productions of $TNF-{\alpha}$ and IL-6 in LPS-simulated RAW 264.7 macrophages. However, AA did not inhibit activation of p65 $NF-{\kappa}B$ in LPS-simulated RAW 264.7 macrophages. Conclusion : These results suggest that down-regulation of iNOS, COX-2 protein expression and $TNF-{\alpha}$ and IL-6 production by AA are responsible for its anti-inflammatory effects.

• The Korea Journal of Herbology
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• v.34 no.4
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• pp.9-18
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• 2019

Objectives : It has been known to be correlated between phlegm and dementia from the perspective of oriental medicine, but it is unexplored whether herbal medicines to treat phlegm have pharmacological actions on Alzheimer's disease (AD). The aim of this study was to evaluate and to compare effects of herbal medicines to treat phlegm against AD in vitro. Methods : We selected 11 herbal medicines which treat phlegm and obtained each extract by boiling in 10-fold distilled water for 2 h. And we performed the assay of acetylcholinesterase (AChE) inhibitory effects of 11 herbal extracts. Next, we evaluated neuroprotective effects of them against amyloid $beta_{25-35}$ ($A{\beta}_{25-35}$) plaque-induced toxicity in HT22 mouse hippocampal neuronal cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To investigate whether they show the anti-inflammatory effects against lipopolysaccharide (LPS), we also measured the levels of nitric oxide (NO) in BV2 microglia cells using griess reagent assay. Results : We found that Gamiyeongsin-hwan (GYH) and Cheonghunhwadam-tang (CHT) exhibited remarkable AChE inhibitory effects. In HT22 cells, Arisaematis Rhizoma, Trichosanthis Semen and Fritillariae Thunbergii Bulbus suppressed $A{\beta}_{25-35}$ plaque-induced neuronal cell death. In BV2 cells, Cheongung-hwan significantly inhibited the increase of NO contents induced by LPS and GYH and CHT showed a tendency to inhibit LPS-induced NO generation. Conclusions : These results suggest that several herbal medicines to treat phlegm showed the significant effects on AChE inhibition, neuroprotection against $A{\beta}_{25-35}$ plaque-induced toxicity, and inhibition of NO generation. Therefore, we demonstrate the possibility that herbal medicines with treating phlegm has effects against AD.