Min-Sup Lee

Phlorofucofuroeckol A inhibits the LPS-stimulated iNOS and COX-2 expressions in macrophages via inhibition of NF-κB, Akt, and p38 MAPK

We have recently reported that phlorofucofuroeckol A isolated from the edible brown algae Ecklonia stolonifera showed potential antioxidative and anti-inflammatory properties in macrophage stimulated by LPS treatments. In this study, we further investigated the pharmacological characteristic of phlorofucofuroeckol A in regulations of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 through regulatory and signaling pathways using LPS-treated RAW 264.7 cells. Treatment with 20 μM of phlorofucofuroeckol A significantly decreased levels of iNOS and COX-2 mRNA induced by LPS stimulation. As results, levels of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were significantly reduced by treatments of phlorofucofuroeckol A in LPS-stimulated RAW 264.7 cells. Phlorofucofuroeckol A inhibited promoter activities of inflammatory-mediators (iNOS and COX-2) and transcriptional factors (nuclear factor-κB, NF-κB, and AP-1) in LPS-treated RAW 264.7 cells. Moreover, phlorofucofuroeckol A inhibited activation of Akt and p38 MAPK in LPS-treated RAW 264.7 cells. These results indicate that the phlorofucofuroeckol A regulates iNOS and COX-2 expressions through the NF-κB-dependent transcriptional control associated with inhibition of multiple signaling proteins, suggesting potential candidates of phloroglucinol derivatives for treatments of inflammatory diseases.

Anti-inflammatory Activities of an Ethanol Extract of Ecklonia stolonifera in Lipopolysaccharide-Stimulated RAW 264.7 Murine Macrophage Cells

Ecklonia stolonifera is a brown alga that was shown to have antioxidant, anti-inflammatory, tyrosinase inhibitory, and chemopreventive activities. However, the molecular mechanisms underlying its anti-inflammatory activity remain unclear. In this study, we investigated the molecular mechanism of the anti-inflammatory action of E. stolonifera ethanolic extracts (ESE) using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. ESE inhibited LPS-induced nitric oxide (IC(50) = 72 ± 1.9 μg/mL) and prostaglandin E(2) (IC(50) = 98 ± 5.3 μg/mL) production in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells. ESE also reduced the production of pro-inflammatory cytokines in LPS-stimulated RAW 264.7 cells. LPS-induced nuclear factor-κB (NF-κB) transcriptional activity and NF-κB translocation into the nucleus were significantly inhibited by ESE treatment through the prevention of the degradation of inhibitor κB-α. Moreover, ESE inhibited the activation of Akt, ERK, JNK1/2, and p38 MAPK in LPS-stimulated RAW 264.7 cells. The main components with anti-inflammatory activity in ESE were identified as phlorofucofuroeckol A and B based on the inhibition of NO production. Our results indicate that ESE can be considered as a potential source of therapeutic agents for inflammatory diseases.

Dieckol enhances the expression of antioxidant and detoxifying enzymes by the activation of Nrf2–MAPK signalling pathway in HepG2 cells

Dieckol was previously reported to exhibit antioxidant and anticancer activities in vitro studies. In this study, we characterised the mechanism underlying the dieckol-mediated expression of antioxidant and detoxifying enzymes. Dieckol suppressed the production of intracellular reactive oxygen species in the presence or absence of H2O2 and increased glutathione level in HepG2 cells. Dieckol enhanced the activities of antioxidant enzymes, and the expression of detoxifying enzymes including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO1), and glutathione S-transferase (GST) in HepG2 cells. Enhanced expression of antioxidant and detoxifying enzymes by dieckol was presumed to be the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2) demonstrated by its nuclear translocation and transcriptional activity via activation of mitogen-activated protein kinases in HepG2 cells. Furthermore, we demonstrated dieckol induced the expression of HO-1 in mouse liver. These results demonstrate that the dieckol-mediated cytoprotection in HepG2 cells is mediated through a ROS-independent up-regulation of antioxidant and detoxifying enzymes via Nrf2 activation as well as its intrinsic antioxidant activity, suggesting that dieckol may be used as a natural cytoprotective agent.

Phlorofucofuroeckol A Suppresses Expression of Inducible Nitric Oxide Synthase, Cyclooxygenase-2, and Pro-inflammatory Cytokines via Inhibition of Nuclear Factor-κB, c-Jun NH2-Terminal Kinases, and Akt in Microglial Cells

Microglial activation has been implicated in many neurological disorders for its inflammatory and neurotrophic effects. In this study, we investigated the effects of phlorofucofuroeckol A isolated from Ecklonia stolonifera Okamura on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated microglia. Pre-treatment of phlorofucofuroeckol A attenuated the productions of nitric oxide, prostaglandin E2, and pro-inflammatory cytokines in LPS-stimulated microglia. Profoundly, phlorofucofuroeckol A treatment showed inactivation of nuclear factor-κB (NF-κB) by preventing the degradation of inhibitor κB-α and the nuclear translocation of p65 NF-κB subunit. Moreover, phlorofucofuroeckol A inhibited the activation of c-Jun NH2-terminal kinases (JNKs), p38 mitogen-activated protein kinase (MAPK), and Akt, but not that of extracellular signal-regulated kinase. These results indicate that phlorofucofuroeckol A inhibits the LPS-induced expression of inflammatory mediators through inactivation of NF-κB, JNKs, p38 MAPK, and Akt pathways. These findings suggest that phlorofucofuroeckol A can be considered as a nutraceutical candidate for the treatment of neuroinflammation in neurodegenerative diseases.

Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells

BackgroundExcessive pro-inflammatory cytokine production from activated microglia contributes to neurodegenerative diseases, thus, microglial inactivation may delay the progress of neurodegeneration by attenuating the neuroinflammation. Among 5 selected brown algae, we found the highest antioxidant and anti-neuroinflammatory activities from Myagropsis myagroides ethanolic extract (MME) in lipopolysaccharide (LPS)-stimulated BV-2 cells.MethodsThe levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunesorbent assay. The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blot. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunefluorescence and reporter gene assay, respectively.ResultsMME inhibited the expression of iNOS and COX-2 at mRNA and protein levels, resulting in reduction of NO and PGE2 production. As a result, pro-inflammatory cytokines were reduced by MME. MME also inhibited the activation and translocation of NF-κB by preventing inhibitor κB-α (IκB-α) degradation. Moreover, MME inhibited the phosphorylation of extracellular signal regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). Main anti-inflammatory compound in MME was identified as sargachromenol by NMR spectroscopy.ConclusionsThese results indicate that the anti-inflammatory effect of sargachromenol-rich MME on LPS-stimulated microglia is mainly regulated by the inhibition of IκB-α/NF-κB and ERK/JNK pathways.

eckol enhances heme oxygenase-1 expression through activation of Nrf2/JNK pathway in HepG2 cells.

Eckol isolated from Ecklonia stolonifera was previously reported to exhibit cytoprotective activity with its intrinsic antioxidant activity in in vitro studies. In this study, we characterized the mechanism underlying the eckol-mediated the expression of heme oxygenase-1 (HO-1). Eckol suppressed the production of intracellular reactive oxygen species and increased glutathione level in HepG2 cells. Eckol treatment enhanced the expression of HO-1 at the both level of protein and mRNA in HepG2 cells. Enhanced expression of HO-1 by eckol was presumed to be the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2) demonstrated by its nuclear translocation and increased transcriptional activity. c-Jun NH2-terminal kinases (JNKs) and PI3K/Akt contributed to Nrf2-mediated HO-1 expression. These results demonstrate that the eckol-mediated expression of HO-1 in HepG2 cells is regulated by Nrf2 activation via JNK and PI3K/Akt signaling pathways, suggesting that eckol may be used as a natural antioxidant and cytoprotective agent.

Hexane fraction from Laminaria japonica exerts anti-inflammatory effects on lipopolysaccharide-stimulated RAW 264.7 macrophages via inhibiting NF-kappaB pathway.

PurposeLaminaria japonica is a representative marine brown alga used as a culinary item in East Asia. L. japonica extract was shown to exert various biological activities; however, its anti-inflammatory activity has not been reported. The aim of this study is to investigate the molecular mechanisms underlying its anti-inflammatory action.MethodsAnti-inflammatory mechanisms of L. japonican-hexane fraction (LHF) were assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. An anti-inflammatory compound isolated from LHF by reverse-phase chromatography was identified using nuclear magnetic resonance (NMR) spectroscopy.ResultsOur results indicate that LHF significantly inhibited LPS-stimulated nitric oxide (NO) and prostaglandin E2 (PGE2) secretion in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) with no cytotoxicity. As results, levels of pro-inflammatory cytokines were significantly reduced by pretreatment of LHF in LPS-stimulated RAW 264.7 cells. Treatment of LHF strongly suppressed nuclear factor-κB (NF-κB) promoter-driven expression and nuclear translocation of NF-κB by preventing proteolytic degradation of inhibitor of κB (IκB)-α in LPS-stimulated RAW 264.7 cells. Moreover, LHF inhibited the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW 264.7 cells. One of the anti-inflammatory compounds was isolated from LHF and identified as fucoxanthin.ConclusionsThese results indicate that the LHF-mediated inhibition of NO and PGE2 secretion in LPS-stimulated macrophages is regulated by NF-κB inactivation through inhibition of IκB-α, MAPKs, and Akt phosphorylation. LHF may be considered as a functional food candidate for the prevention or treatment of inflammatory diseases.

Anti-inflammatory effect of ethanolic extract from Myagropsis myagroides on murine macrophages and mouse ear edema

BackgroundThis study aims to investigate anti-inflammatory effect of ethanolic extract of Myagropsis myagroides (EMM) in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and the phorbol 12-myristate 13-acetate (PMA)-induced ear edema in mice, and to clarify its underlying molecular mechanisms.MethodsThe levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blotting. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunocytochemistry and reporter gene assay, respectively. PMA-induced mouse ear edema was used as the animal model of inflammation. Anti-inflammatory compounds in EMM were isolated using high-performance liquid chromatography and identified by nuclear magnetic resonance.ResultsEMM significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. EMM strongly suppressed nuclear translocation of NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. EMM reduced ear edema in PMA-induced mice. One of the anti-inflammatory compounds in EMM was identified as 6,6’-bieckol.ConclusionsThese results suggest that the anti-inflammatory properties of EMM are associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines through the inhibition of NF-κB pathway in LPS-stimulated macrophages.

Anthocyanins Inhibit Lipogenesis During Adipocyte Differentiation of 3T3-L1 Preadipocytes

Anthocyanins have been shown to suppress body weight and fat mass in animal studies. However, the effect of anthocyanins on the process of lipid accumulation during adipocyte differentiation is not fully understood and the lipogenic transcription factors regulated by anthocyanins have not been identified. We investigated the effects of anthocyanins on lipogenesis pathways during adipocyte differentiation in 3T3-L1 cells. Anthocyanins reduced triglyceride (TG) accumulation in a dose-dependent manner during adipocyte differentiation. Accumulation of TG was rapidly reversed by anthocyanin withdrawal. Anthocyanins markedly reduced gene and protein expression levels of lipogenic transcription factors such as liver X receptor α, sterol regulatory element-binding protein-1c, peroxisome proliferators-activated receptor-γ, and CCAAT enhancer-binding protein-α. In addition, the target gene and protein expression of these lipogenic transcription factors such as fatty acid synthase, stearoyl-CoA desaturase-1, and acetyl-CoA carboxylase α were markedly suppressed by anthocyanins. Thus, anthocyanins suppress lipid accumulation in adipocytes due to broad inhibition of the transcription factors regulating lipogenesis. This may partially explain the mechanism by which anthocyanins exert their anti-obesity effect.

Anti-inflammatory effects of phlorofucofuroeckol B-rich ethyl acetate fraction obtained from Myagropsis myagroides on lipopolysaccharide-stimulated RAW 264.7 cells and mouse edema.

Myagropsis myagroides has been used as a Chinese medicine and its extract has shown various biological activities, however, its anti-inflammatory mechanism remains unknown. In this study, we investigated the inhibitory effects of the ethyl acetate fraction of M. myagroides (EFM) on the production of inflammatory mediators and pro-inflammatory cytokines in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. EFM significantly inhibited LPS-induced production of nitric oxide (NO), prostaglandin E(2), and pro-inflammatory cytokines in a dose-dependent manner and suppressed the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 in RAW 264.7 cells. Inhibitory effect of EFM on iNOS expression and NO production was further confirmed using LPS-activated mouse peritoneal macrophages. EFM treatment strongly suppressed the activation of nuclear factor-kappa B (NF-κB) by suppressing phosphorylation of Akt and extracellular signal-regulated kinases (ERKs). EFM as well as phlorofucofuroeckol B (PFF-B), a major compound isolated from EFM, reduced ear edema induced by phorbol 12-myristate 13-acetate in mice. These results indicate that the anti-inflammatory effect of EFM, rich in PFF-B, on LPS-stimulated macrophages is regulated by the inhibition of NF-κB pathway through the inhibition of ERKs and Akt phosphorylation in LPS-stimulated macrophage cells.