The dried root of Anthriscus sylvestris (Umbelliferae) is used in Korean traditional medicine as an antipyretic, an analgesic, and a cough remedy. Deoxypodophyllotoxin (DPT; Fig. 1A), widely distributed in higher plants, is a major lignan in this plant. DPT has antiproliferative, 1) antitumor, 2,3) and antiviral activities. 4) Recently, we reported that DPT inhibits the passive cutaneous anaphylaxis (PCA) reaction, 5) has strong dual cyclooxygenase (COX-2) selective/5-lipoxygenase (LOX) inhibitory activity, 6) and has anti-asthmatic effects.7) However, the underlying mechanisms of its inhibition of nitric oxide (NO) generation have not been elucidated. Therefore, we examined the effect of DPT on NO generation to better understand its anti-inflammatory effects.NO generation plays an important role in atherosclerosis, inflammation, and carcinogenesis. 8,9) NO is generated by NO synthase (NOS), and three isoforms of NOS have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS).10) The suppression of NO synthesis might be useful for the treatment of inflammatory diseases. NO produced by constitutive NOS (cNOS) is critical in the maintenance of cellular function, whereas NO produced by iNOS is an important mediator of acute and chronic inflammation.11) If the inflammation is chronic, then healthy host cells may be killed by NO, contributing to inflammatory pathology. 12)The transcription factor, nuclear factor (NF)-kB, is activated by a variety of stimuli and regulates diverse gene expression and biological responses. The stimuli include bacterial endotoxin, lipopolysaccharide (LPS), ionizing radiation, and carcinogens that are often associated with inflammatory diseases and tumorigenesis. 13,14) The binding of NF-kB to kB sites is important for iNOS induction by LPS. NF-kB exists in the cytoplasm of unstimulated cells in a quiescent form bound to its inhibitor, IkB.15) External stimuli for macrophage activation increase phosphorylation of IkBa, which causes its degradation, allowing active NF-kB to translocate to the nucleus and bind its cognate DNA binding sites in the regulatory regions of a variety of genes. p38 and p44/42 mitogen-activated protein kinase (MAPK) are also involved in iNOS expression.16) The promoter of iNOS contains two consensus NF-kB binding sites, which mediate LPS-inducibility.17) The heterodimer p65/p50 is the NF-kB complex responsible for LPS-induced iNOS expression.In this study, we investigated the effect of DPT on the LPS-induced NO production and iNOS protein expression, and to clarify its mode of action in RAW264.7 macrophage cells. We found that DPT inhibited NF-kB activation through the inhibition of IkB degradation. MATERIALS AND METHODS Plant Material DPT was isolated from the dried roots ofA. sylvestris (Umbelliferae). The chemical structure of the isolated compound was established as deoxypodophyllotoxin ( Fig. 1) Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris...
We evaluated the ability of the ethylacetate fraction of marine sponge, Cliona celata (ECC), harvested from Korean seaside to regulate the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells. ECC dose-dependently inhibited both the expression of iNOS protein and mRNA, resulting in decreased production of nitric oxide (NO), with an IC(50) of 80.5 μg/mL. To investigate action mechanism by which ECC inhibits NO production and iNOS expression, we examined the activation of IκB in LPS-stimulated RAW264.7 cells. ECC clearly inhibited translocation of nuclear factor-κB (NF-κB) p65 subunits from cytosol to nucleus, which correlated with its inhibitory effects on IκB-α phosphorylation and degradation. Furthermore, ECC potently suppressed both the reporter gene expression and DNA-binding activity of NF-κB, which was associated with decreased p65 protein levels in the nucleus. Here, we show for the first time that ECC inhibits NF-κB activation through the inhibition of IκB degradation.
The Gram-negative bacteria cell wall component lipopolysaccharide (LPS) is considered as the most potent activators for macrophages. LPS-activated macrophages produces an array of proinflammatory cytokines such as tumor necrosis facto-a (TNF-a), interleukin (IL)-1b, IL-6 and other inflammatory mediators, such as nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) as well as matrix metalloproteinases (MMPs). [1][2][3][4][5] MMPs are a family of zinc requiring endopeptidases that play an important role in the turnover of extracellular matrix (ECM). Among them, MMP-2 and MMP-9 (92 kDa gelatinase) are capable of degrading basement membrane proteins. Various inflammatory cells such as macrophages, mast cells, lymphocytes, eosinophils and neutrophils produce and/or releases MMP-9 by LPS and other inflammatory mediators, such as TNF-a, IL-8, and granulocyte colony-stimulating factor. [6][7][8] Over expression or inappropriate expression of MMP-9 may contribute to the pathogenesis of conditions such as atherosclerosis, inflammation, tumor invasion and metastasis, rheumatoid arthritis, and periodontal disease. 9)It has been well established that MMP-9 induction is regulated by the transcription factor, nuclear factor-kB (NFkB).10,11) Inactive NF-kB is located in the cytoplasm by the inhibitor protein inhibitor of kB (IkB). When stimulated with any of a variety of stimuli, such as LPS, TNF-a, IL-1b and cellular stress, IkB is degraded and then NF-kB is translocated from the cytosol to the nucleus, and binds to its cognate DNA binding sites.12) LPS-stimulated macrophages activate several intracellular signaling pathways, including the NF-kB pathway and three of the mitogen-activated protein kinases (MAPKs) pathways such as extracellular signalregulated kinase (ERK) 1 and 2, c-Jun N-terminal kinase (JNK) and p38.12) Several lines of evidence have demonstrated that LPS also regulates MMP-9 expression via the MAPK signaling pathway. 13,14) Saururus chinensis (S. chinensis) has been traditionally used since long for the treatment of edema, jaundice, gonorrhea, pneumonia, and several inflammatory diseases. Previous published studies on the chemical nature of S. chinensis have reported a content of more than 20 lignans.15,16) Isolated tetrahydrofurans protect against cell adhesion, 17) inflammation, 18,19) hypercholesterolemia, 20) and sepsis. 21) Sesquinelignans such as saucerenol D inhibited the LPS-induced iNOS expression by blocking NF-kB activation.22) Previously, we had reported on isolation of compounds that protect against sepsis from this plant along with four new lignans including saucerneol G (SG) and their topoisomerase inhibitory activity.23) During the study of SG on anti-inflammatory effect using RAW264.7 cells, SG showed the MMP-9-inhibitory activity. However, the underlying mechanisms in the inhibitory activity of SG on MMP-9 induction in LPSstimulated macrophage cell line RAW264.7 cells have not been elucidated till date.Thus, in this study, we evaluated the effects of SG on LPSstimulated MMP-9 induction and co...
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