• Biomolecules & Therapeutics
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• v.19 no.4
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• pp.438-444
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• 2011

The mushroom Coriolus versicolor contains biologically active polysaccharides, most of which belong to the ${\beta}$-glucan group. Diverse physicochemical properties, due to different sources and isolated types of ${\beta}$-glucans, can induce distinct biological activities. We investigated the effects of ${\beta}$-glucans from C. versicolor on phagocytic activity, nitric oxide (NO), TNF-${\alpha}$ production, and signaling of dectin-1, a well-known ${\beta}$-glucan receptor, in macrophages. ${\beta}$-Glucans increased phagocytic activity and TNF-${\alpha}$ and NO-iNOS/eNOS production. Laminarin, a specific inhibitor of dectin-1, showed strong inhibitory effects on phagocytosis and subsequent TNF-${\alpha}$, iNOS, and eNOS production increased by ${\beta}$-glucans, indicating that ${\beta}$-glucans reacts with dectin-1 receptors. We examined whether the aforementioned cytokines were involved in the signaling pathway from the dectin-1 receptor to phagocytosis, and found that the inhibition of iNOS, eNOS, and TNF-${\alpha}$ receptors significantly decreased ${\beta}$-glucan-induced phagocytosis. In conclusion, our study showed that dectin-1 signaling, triggered by ${\beta}$-glucans, subsequently elicited TNF-${\alpha}$ and NO-iNOS/eNOS production, and that these molecules seem to act as secondary molecules that cause eventual phagocytosis by macrophages. These findings suggest that C. versicolor could be used as a nutritional medicine that may be useful in the treatment of infectious disease.

• Biomolecules & Therapeutics
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• v.19 no.4
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• pp.419-424
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• 2011

Galla Rhois and its components are known to possess anti-infl ammatory properties. In the present study, we prepared equilibrium mixture of ethyl m-digallate and ethyl p-digallate isomers (EDG) from Galla Rhois and examined its effect on nitric oxide (NO) production in murine macrophage cell line. Treatment of RAW264.7 macrophages with EDG signifi cantly inhibited NO production and inducible nitric oxide synthase (iNOS) expression stimulated by LPS, as assessed by Western blot and quantitative RT-PCR analyses. We also demonstrated that EDG treatment led to an increase in heme oxygenase-1 (HO-1) mRNA and protein expression. EDG treatment also enhanced expression level of nuclear factor-erythroid 2-related factor 2 (Nrf2) in nucleus, which is critical for transcriptional induction of HO-1. Treatment with SnPP (tin protoporphyrin IX), a selective HO-1 inhibitor, reversed EDG-mediated inhibition of nitrite production, suggesting that HO-1 plays an important role in the suppression of NO production by EDG. Taken together, these results indicate that EDG isolated from Galla Rhois suppresses LPS-stimulated NO production in RAW 264.7 macrophages via HO-1 induction.

• Molecules and Cells
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• v.38 no.1
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• pp.26-32
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• 2015

Toll-like receptors (TLR) 7 and 9 transduce a cellular signal through the MyD88-dependent pathway and induce the production of inflammatory mediators against microbial nucleotide components. The repeated stimulation of TLR4 leads to endotoxin tolerance, but the molecular mechanisms of tolerance induced through the costimulation of individual TLR has not yet been established, although endosomal TLRs share signaling pathways with TLR4. In the present study, mouse macrophages were simultaneously stimulated with the TLR7 agonist, gardiquimod (GDQ), and the TLR9 agonist, CpG ODN 1826, to examine the mechanism and effector functions of macrophage tolerance. Compared with individual stimulation, the costimulation of both TLRs reduced the secretion of TNF-${\alpha}$ and IL-6 through the delayed activation of the NF-${\kappa}B$ pathway; notably, IL-10 remained unchanged in costimulated macrophages. This tolerance reflected the early induction of suppressor of cytokine signaling-1 (SOCS-1), according to the detection of elevated TNF-${\alpha}$ secretion and restored NF-${\kappa}B$ signaling in response to the siRNA-mediated abrogation of SOCS-1 signaling. In addition, the restimulation of each TLRs using the same ligand significantly reduced the expression of both TLRs in endosomes. These findings revealed that the costimulation of TLR7 and TLR9 induced macrophage tolerance via SOCS-1, and the restimulation of each receptor or both TLR7 and TLR9 downregulated TLR expression through a negative feedback mechanisms that protects the host from excessive inflammatory responses. Moreover, the insufficient and impaired immune response in chronic viral infection might also reflect the repeated and simultaneous stimulation of those endosomal TLRs.

• Journal of Nutrition and Health
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• v.45 no.5
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• pp.443-451
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• 2012

We compared the effects of grapefruit seed extract (GFSE), green tea extract (GT) and their microencapsulated extract on anti-inflammatory activities in murine RAW 264.7 macrophages cell line. In order to protect the bioactive compounds in the extracts, they were microencapsulated with maltodextrin and $H_2O$. Nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), inducible nitric oxide synthase (iNOS) protein expression and thiobarbiturate reactive substances (TBARS) were analyzed in LPS activated RAW 264.7 macrophages. The green tea extract at the range of $100-600{\mu}g/mL$ inhibited NO, PGE2 production and iNOS protein expression without cytotoxicity in a dose-dependent manner. Grapefruit seed extract had strong inhibitory effects on NO and PGE production and iNOS protein expression at the range of $5-20{\mu}g/mL$ without cytotoxicity. Microencapsulation of green tea extract had further inhibitory effects on NO and PGE2 production and on iNOS protein expression, whereas microencapsulated GFSE did not show any further inhibitory effects on these parameters. Taken together, our results suggest that GSFE might be a promising candidate for preventing inflammation related diseases, such as cardiovascular disease, cancer or diabetes, and the microencapsulation of green tea extract could improve its bioactivity.

• Journal of the Korean Society of Food Science and Nutrition
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• v.29 no.2
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• pp.342-348
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• 2000

In this experiment, we show the effects of herbal plant extracts on the production of nitric oxide (NO) and TNF-$\alpha$. The extracts of Angelica gigas, Astragalus membranaceus, Acanthopanax sessiliflorus and Houttuynia cordata had no effect on NO synthesis by itself in mouse macrophage cell line (RAW264.7). However, the stimulation with these extracts in the presence of murine interferon-${\gamma}$(mIFN-${\gamma}$) resulted in increased NO synthesis. When these extracts were used in combination with mIFN-${\gamma}$, there were a marked cooperative induction of NO and TNF-$\alpha$ synthesis in a dose-dependent manner. The same results were obtained in the mouse peritoneal macrophages used. The optimal concentration of these extracts on NO synthesis was shown at 100$\mu\textrm{g}$/mL with 100U/mL of mIFN-${\gamma}$. NO synthesis was inhibited by NG-monomethyl-L-arginine. When cell lines were treated with extracts, the expression of inducible NO synthetase (iNOS) was markedly increased in RT-PCR analysis. In addition, synergy between mIFN-${\gamma}$ and extracts was dependent on extracts-induced tumor necrosis factor-$\alpha$(TNF-$\alpha$). These results suggest that water extracts of herbal plants can induce iNOS, NO and TNF-$\alpha$ synthesis of mouse macrophage cell line (RAW264.7) and peritoneal macrophages in combination with mIFN-${\gamma}$.

• 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 Ginseng Research
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• v.43 no.2
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• pp.291-299
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• 2019

Background: Ginsenosides of Korean Red Ginseng extracts (RGE) and its saponin components suppress secretion of inflammasome-mediating cytokines, whereas the nonsaponin fraction (NS) of RGE oppositely stimulates cytokine secretion. Although direct exposure of NS to macrophages in mice induces cytokine production, oral administration of NS has not been studied in inflammasome-related disease in animal models. Methods: Mice were fed RGE or NS for 7 days and then developed peritonitis. Peritoneal cytokines were measured, and peritoneal exudate cells (PECs) were collected to assay expression levels of a set of toll-like receptors (TLRs) and cytokines in response to NS ingestion. In addition, the role of intestinal bacteria in NS-fed mice was assessed. The effect of preexposure to NS in bone marrow-derived macrophages (BMDMs) on cytokine production was further confirmed. Results: NS ingestion attenuated secretion of peritoneal cytokines resulting from peritonitis. In addition, the isolated PECs from NS-fed mice presented lower TLR transcription levels than PECs from control diet-fed mice. BMDMs treated with NS showed downregulation of TLR4 mRNA and protein expression, which was mediated by the $TLR4-MyD88-NF{\kappa}B$ signal pathway. BMDMs pretreated with NS produced less cytokines in response to TLR4 ligands. Conclusion: NS administration directly inhibits TLR4 expression in inflammatory cells such as macrophages, thereby reducing secretion of cytokines during peritonitis.

• Molecules and Cells
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• v.43 no.12
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• pp.989-1001
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• 2020

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes salmonellosis and mortality worldwide. S. Typhimurium infects macrophages and survives within phagosomes by avoiding the phagosome-lysosome fusion system. Phagosomes sequentially acquire different Rab GTPases during maturation and eventually fuse with acidic lysosomes. Lysophosphatidylcholine (LPC) is a bioactive lipid that is associated with the generation of chemoattractants and reactive oxygen species (ROS). In our previous study, LPC controlled the intracellular growth of Mycobacterium tuberculosis by promoting phagosome maturation. In this study, to verify whether LPC enhances phagosome maturation and regulates the intracellular growth of S. Typhimurium, macrophages were infected with S. Typhimurium. LPC decreased the intracellular bacterial burden, but it did not induce cytotoxicity in S. Typhimurium-infected cells. In addition, combined administration of LPC and antibiotic significantly reduced the bacterial burden in the spleen and the liver. The ratios of the colocalization of intracellular S. Typhimurium with phagosome maturation markers, such as early endosome antigen 1 (EEA1) and lysosome-associated membrane protein 1 (LAMP-1), were significantly increased in LPC-treated cells. The expression level of cleaved cathepsin D was rapidly increased in LPC-treated cells during S. Typhimurium infection. Treatment with LPC enhanced ROS production, but it did not affect nitric oxide production in S. Typhimurium-infected cells. LPC also rapidly triggered the phosphorylation of IκBα during S. Typhimurium infection. These results suggest that LPC can improve phagosome maturation via ROS-induced activation of NF-κB pathway and thus may be developed as a therapeutic agent to control S. Typhimurium growth.

• Animal Bioscience
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• v.34 no.3_spc
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• pp.463-470
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• 2021

Objective: This experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP. Methods: Nitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit. Results: P700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 μM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700. Conclusion: In animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.2
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• pp.139-146
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• 2021

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) produces NADPH, which is known to inhibit mitochondrial oxidative stress. Ureteral obstruction induces kidney inflammation and fibrosis via oxidative stress. Here, we investigated the role and underlying mechanism of IDH2 in unilateral ureteral obstruction (UUO)-induced kidney inflammation using IDH2 gene deleted mice (IDH2-/-). Eight- to 10-week-old female IDH2-/- mice and wild type (IDH2+/+) littermates were subjected to UUO and kidneys were harvested 5 days after UUO. IDH2 was not detected in the kidneys of IDH2-/- mice, while UUO decreased IDH2 in IDH2+/+ mice. UUO increased the expressions of markers of oxidative stress in both IDH2+/+ and IDH2-/- mice, and these changes were greater in IDH2-/- mice compared to IDH2+/+ mice. Bone marrow-derived macrophages of IDH2-/- mice showed a more migrating phenotype with greater ruffle formation and Rac1 distribution than that of IDH2+/+ mice. Correspondently, UUO-induced infiltration of monocytes/macrophages was greater in IDH2-/- mice compared to IDH2+/+ mice. Taken together, these data demonstrate that IDH2 plays a protective role against UUO-induced inflammation through inhibition of oxidative stress and macrophage infiltration.