• Journal of Applied Biological Chemistry
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• v.61 no.2
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• pp.205-211
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• 2018

Rebaudioside A is a natural sweetener isolated from Stevia rebaudiana Bertoni, one of the glycosides based on steviol. Recent studies have shown that rebaudioside A inhibits the inflammatory response by inhibiting cytokines secretion such as interleukin-$1{\alpha}/1{\beta}$ in activated RAW264.7 mouse macrophage cells by LPS. However, the inhibitory mechanism of inflammation by rebaudioside A in the presence of LPS has not been fully elucidated. Therefore, in this study, we tried to investigate the anti-inflammatory activity of rebaudioside A at the protein level when RAW264.7 cells were stimulated by LPS. The inducible nitric oxide synthase protein expression level was reduced in the group treated with $250{\mu}M$ rebaudioside A compared to the LPS-treated group. In addition, the mRNA expression level of $NF-{\kappa}B$, which is a representative nuclear transcription factor by inflammatory signal, was also decreased as compared with that of LPS-treated group. In addition, $NF-{\kappa}B$ and inhibitor-${\kappa}B$ ($I-{\kappa}B$) complexes that are known to be dissociated by $I-{\kappa}B$ phosphorylation and ubiquitination were less phosphorylated than LPS treated group in the presence rebaudioside A. Finally, we could find that rebaudioside A was involved in the $NF-{\kappa}B$ pathway through reducing extracellular signal-regulated kinase1/2 phosphorylation in a concentration-dependent manner. These results suggest that rebaudioside A might suppress inflammatory reaction through MAPK and $NF-{\kappa}B$ regulation in LPS-stimulated RAW264.7.

• Korean Journal of Food Science and Technology
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• v.51 no.1
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• pp.70-80
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• 2019

The aim of this study was to investigate the effects of red ginseng-derived non-saponin fraction (NSF) on inflammatory responses and monocyte-to-macrophage differentiation in RAW264.7 and THP-1. NSF effectively inhibited inflammatory responses by downregulating nitric oxide (NO) production and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). NSF ($2000{\mu}g/mL$) decreased the levels of NO, iNOS, and COX-2 by 33, 83, and 64%, respectively. NSF inhibited the differentiation of monocyte-to-macrophage by decreasing cell adherence along with downregulation of the cluster of differentiation molecule $11{\beta}$ ($CD11{\beta}$) and CD36. In addition, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin 6, and monocyte chemoattractant protein 1 (MCP-1), were significantly reduced with NSF treatment. The NSF-mediated inhibition of inflammatory responses was due to the regulation of nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). NSF effectively suppressed the translocation of $NF-{\kappa}B$ into the nucleus, while nuclear Nrf2 and its target protein, heme oxygenase-1, levels were significantly increased.

• Fisheries and Aquatic Sciences
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• v.14 no.4
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• pp.275-282
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• 2011

Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been implicated in various inflammatory diseases. In this study, we investigated the anti-inflammatory activities of Chondria crassicaulis ethanolic extract (CCE) by measuring its effects on the expression of iNOS and COX-2 proteins in lipopolysaccharide (LPS)-treated RAW 264.7 murine macrophages. CCE significantly and dose-dependently inhibited the LPS-induced release of nitric oxide and prostaglandin $E_2$, and suppressed the expression of iNOS and COX-2 proteins in LPS-stimulated RAW 264.7 cells, without causing any cytotoxicity. It also inhibited the production of the pro-inflammatory cytokines such as interleukin (IL)-$1{\beta}$, IL-6, and tumor necrosis factor (TNF)-${\alpha}$ in LPS-stimulated RAW 264.7 cells. Moreover, treatment with CCE strongly suppressed nuclear factor-${\kappa}B$ (NF-${\kappa}B$) promoter-driven expression in LPS-treated RAW 264.7 cells. CCE treatment blocked nuclear translocation of the p65 subunit of NF-${\kappa}B$ by preventing proteolytic degradation of inhibitor of ${\kappa}B-{\alpha}$. These results indicate that CCE regulates iNOS and COX-2 expression through NF-${\kappa}B$-dependent transcriptional control, and identifies potential candidates for the treatment or prevention of inflammatory diseases.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.91-99
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• 2016

(E)-3-(3-methoxyphenyl)-1-(2-pyrrolyl)-2-propenone (MPP) is an aldol condensation product resulting from pyrrole-2-carbaldehyde and m- and p- substituted acetophenones. However, its biological activity has not yet been evaluated. Since it has been reported that some propenone-type compounds display anti-inflammatory activity, we investigated whether MPP could negatively modulate inflammatory responses. To do this, we employed lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells and examined the inhibitory levels of nitric oxide (NO) production and transcriptional activation, as well as the target proteins involved in the inflammatory signaling cascade. Interestingly, MPP was found to reduce the production of NO in LPS-treated RAW264.7 cells, without causing cytotoxicity. Moreover, this compound suppressed the mRNA levels of inflammatory genes, such as inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-${\alpha}$. Using luciferase reporter gene assays performed in HEK293 cells and immunoblotting analysis with nuclear protein fractions, we determined that MPP reduced the transcriptional activation of nuclear factor (NF)-${\kappa}B$. Furthermore, the activation of a series of upstream signals for NF-${\kappa}B$ activation, composed of Src, Syk, Akt, and $I{\kappa}B{\alpha}$, were also blocked by this compound. It was confirmed that MPP was able to suppress autophosphorylation of overexpressed Src and Syk in HEK293 cells. Therefore, these results suggest that MPP can function as an anti-inflammatory drug with NF-${\kappa}B$ inhibitory properties via the suppression of Src and Syk.

• Journal of Acupuncture Research
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• v.23 no.2
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• pp.139-157
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• 2006

In the present study, I have investigated the bee venom (BV) and melittin (a major component of BV) -mediated anti-proliferative effects, and defined its mechanisms of action in cultured rat aortic vascular smooth muscle cells (VSMCs). BV and melittin $(0.4{\sim}0.8\;{\mu}g/ml)$ effectively inhibited 50 ng/ml platelet derived growth factor BB (PDGF-BB)-induced VSMCs proliferations. The regulation of apoptosis has attracted much attention as a possible means of eliminating excessively proliferating VSMCs. In the present study, the treatment of BV and melittin strongly induced apoptosis of VSMCs. I examined the effects on $NF-{\kappa}B$ activation to investigate a possible mechanism for anti-proliferative effects of BV and melittin, the PDGF-BB-induced $I{\kappa}B{\alpha}$ phosphorylation and its degradation were potently inhibited by melittin, and DNA binding activity and nuclear translocation of $NF-{\kappa}B$ p50 subunit in response to the action of PDGF-BB were potently attenuated by melittin. In further investigations, melittin markedly inhibited the PDGF-BB-induced phosphorylation of Akt but not ERK1/2, upstream signals of $NF-{\kappa}B$. Treatment of melittin also potently induced pro-apoptotic protein p53, Bax, and caspase-3 expression, but decreased anti-apoptotic protein Bcl-2 expression. These results suggest that the anti-proliferative effects of BV and melittin in VSMCs through induction of apoptosis via suppressions of $NF-{\kappa}B$ and Akt activation, and enhancement of apoptotic signal pathway. Based on these results, BV acupuncture can be a candidate as a therapeutic method for restenosis and atherosclerosis.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.4
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• pp.384-392
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• 2013

An ethanolic extract of Undaria pinnatifida was fractionated using several solvents. Of the fractions, the ethyl acetate fraction had the greatest inhibitory effect on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells. Using this fraction (U. pinnatifida ethyl acetate extract, UPE), we investigated the molecular mechanism underlying its inhibitory effect on LPS-stimulated RAW 264.7 cells. Pretreatment of the cells with up to $100{\mu}g/mL$ UPE significantly inhibited NO production and inducible nitric oxide synthase (iNOS) expression, in a dose-dependent manner. Similarly, UPE treatment markedly reduced the production of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6 and tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), while it strongly suppressed the nuclear translocation of nuclear factor-kappa B (NF-${\kappa}B$) by preventing proteolytic degradation of inhibitor of nuclear factor ${\kappa}B$ $(I{\kappa}B)-{\alpha}$. Moreover, UPE treatment significantly reduced the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) in LPS-stimulated cells. These results indicate that UPE contains anti-inflammatory compounds and suggest that it might be used as a functional food material that assists in prevention of inflammatory diseases.

• The Journal of Korean Obstetrics and Gynecology
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• v.21 no.2
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• pp.166-183
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• 2008

Purpose: This study was performed to determine anti-imflammatory effects of Haedoksamultang. Methods: In this study, I examined the effects of Haedoksamultang on the production of nitric oxide(NO), tumor necrosis factor-${\alpha}$(TNF-${\alpha}$), and interlukin-1${\beta}$(IL-1${\beta}$) as well as the expression of inducible NO synthase(iNOS), cyclooxygenase-2(COX-2), TNF-${\alpha}$, and IL-1${\beta}$ in RAW 264.7 cells. Haedoksamultang inhibited LPS-stimulated NO production. Western blotting and RT-PCR analysis showed that Haedoksamultang suppressed LPS-induced iNOS and COX-2 protein and mRNA expression in RAW 264.7 cells. Haedoksamultang also suppressed the expression and production of LPS-stimulated TNF-${\alpha}$ and IL-1${\beta}$ in RAW 264.7 cells. Haedoksamultang inhibited NF-${\kappa}B$ activation in LPS-treated RAW 264.7 cells. Moreover, this compound blocked $I{\kappa}B-{\alpha}$ phosphorylation and nuclear translocation of the cytosolic NF-${\kappa}B$ p65 subunit, which highly correlated with the production and expression of inflammatory mediators. Results: Haedoksamultang suppresses that inflammation-associated gene expression by blocking NF-${\kappa}B$ activation. Conclusion: These results suggest that Haedoksamultang may be beneficial for treating inflammatory disease.

• The Journal of Korean Obstetrics and Gynecology
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• v.21 no.4
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• pp.36-48
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• 2008

Purpose: The purpose of this study is to investigate anti-inflammatory effect and immune responses of aqueous extract from Fructus Chaenomelis (FC). Methods: We studied anti-inflammatory effect by means of examining the production of NO(nitric oxide) and expressions of pro-inflammatory cytokine (TNF-$\alpha$(tumor necrosis factor-alpha), IL(Interleukin)-6, IL-12) in the LPS-induced peritoneal macrophages of mice. Also, The western blot analysis has been done to look into the mechanism of anti-inflammatory effect. Results: 1. The FC extract did not have any cytotoxicity in the peritoneal macrophages. 2. The FC extract inhibits the productions of NO, IL-6. IL-12 in the LPS-stimulated peritoneal macrophages of mice, but not of TNF-$\alpha$. 3. The FC extract inhibits the activation of NF-${\kappa}B$(nuclear factor-kappa B) by keeping $I{\kappa}B-\alpha$(inhibitory kappa B-alpha) from degradating, but not of MAPKs(mitogen-activated protein kinases) such as ERK(extracelluar signa 1-regulated kinase), JNK(c-Jun N-terminal kinase), p38. Conclusion: These results show that FC extract inhibits the production of pro-inflammatory cytokines such as IL-6. IL-12. NO by inhibiting NF-${\kappa}B$ activation in the peritoneal macrophages of mice. In conclusion, this experiment suggests that FC extract may be effective for the treatment of acute and chronic inflammation including genitourinary infection.

• The Journal of Korean Medicine
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• v.24 no.2
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• pp.138-147
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• 2003

Objectives : In this study, we investigated the mechanism by which Chelidonium majus (CM) regulates nitric oxide (NO) production. Methods : Using mouse peritoneal macrophages, the mechanism by which CM regulates NO or tumor necrosis $factor-{\alpha}(TNF-{\alpha})$ production was examined. NO release was measured by the Griess method. $TNF-{\alpha}$ production was measured by the ELISA method. The protein extracts were prepared and samples were analyzed for the inducible NOS(iNOS) expression and nuclear factor kappa $B(NF-{\kappa}B)$ activation by Western blotting. Results : When CM was used in combination with recombinant $interferon-{\gamma}{\;}(rIFN-{\gamma})$, there was a marked cooperative induction of NO production. CM had an effect on NO production by itself. The expression of the iNOS gene was increased in $rIFN-{\gamma}$ plus CM-stimulated peritoneal macrophages and almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of $NF-{\kappa}B$. The $NF-{\kappa}B$ activation was increased in rIFN-{\gamma} plus CM-induced peritoneal macrophages. The increased production of NO from $rIFN-{\gamma}$ plus CM-stimulated peritoneal rnacrophages was decreased by the treatment with $N^{G}-monomethyl-{_L}-arginine{\;}(N^{G}MMA){\;}N^{\alpha}-Tosyl-Phe$ chloromethyl ketone (TPCK) , and was almost completely inhibited by pre-treatment with PDTC. Furthermore, treatment with CM alone or rIFN-{\gamma} plus CM in peritoneal macrophages caused a significant increase in $TNF-{\alpha}$ production. PDTC decreased CM-induced $TNF-{\alpha}$ production significantly. After CM treatment in HT-29 or AGS cells, cell viability decreased. Conclusions : These findings demonstrate that CM increases the production of NO and $TNF-{\alpha}{\;}by{\;}rIFN-{\gamma}-primed$ macrophages and suggest that NF-B plays a critical role in mediating these effects of CM.

• Journal of the Korean Society of Food Science and Nutrition
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• v.43 no.10
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• pp.1527-1534
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• 2014

In this study, we investigated the anti-inflammatory effects of Lactobacillus rhamnosus fermented black garlic (FBG) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. FBG did not show cytotoxicity in RAW 264.7 cells at concentrations less than $800{\mu}g/mL$, and cell viability increased with FBG concentration. Nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) production as well as tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), interleukin-$1{\beta}$ (IL-$1{\beta}$) and IL-6 formation decreased in an FBG concentration-dependent manner, in LPS-induced RAW 264.7 cells. Furthermore, activation of LPS-inducible nitric synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-${\kappa}B$), and inhibitory kappa B ($I{\kappa}B$) protein expression was effectively inhibited by FBG treatment in LPS-induced RAW 264.7 cells. In contrast, heme oxygenase-1 (HO-1) protein expression significantly increased. These results indicate that the anti-inflammatory activity of FBG was due to activation of NF-${\kappa}B$, inhibition of cytokine production, and expression of iNOS and COX-2. From these results, we expect that FBG could contribute to the prevention and improvement of inflammatory disease.