The Naturally Occurring Flavolignan, Deoxypodophyllotoxin, Inhibits Lipopolysaccharide-Induced iNOS Expression through the NF-κB Activation in RAW264.7 Macrophage Cells

Abstract

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...

IVSE, isolated from Inula japonica,suppresses LPS-induced NO production via NF-κB and MAPK inactivation in RAW264.7 cells

“…Thus, downregulating NO production offers a therapeutic strategy in the management of inflammation. In order to find new anti-inflammatory agents, we established a bio-assay method [14] to search for compounds that block NO production. By using such a bio-assay, we recently isolated several compounds including SE, IBSE, and IVSE from I. japonica at non-cytotoxic concentrations.…”

NF-κB and Therapeutic Approach

“…NF-κB expression (Dat et al, 2008) Deguelin (N) R1, R2= H NF-κB expression (Dat et al, 2008) Licochalcone A (N) NF-κB transactivation & p65 phosphorylation (Furusawa et al, 2009) Deoxypodophyllotoxin (N) DNA binding (Jin et al, 2008) a4-OMGA (N) DNA-binding (Lee et al, 2006) Diarctigenin (N) DNA binding (Kim et al, 2008b) …”

“…Deoxypodophyllotoxin, a naturally occurring flavolignan from Anthriscus sylvestris, potently suppressed both reporter gene activity and DNA binding activity of NF-κB (Jin et al, 2008). Diarctigenin, an inhibitor of nitric oxide (NO) production in macrophages from the seeds of Arctium lappa, inhibited the transcriptional activity and DNA binding ability of NF-κB in zymosan-activated macrophages but did not affect the degradation and phosphorylation of I-κB proteins (Kim et al, 2008b).…”

Prediction of Deoxypodophyllotoxin Disposition in Mouse, Rat, Monkey, and Dog by Physiologically Based Pharmacokinetic Model and the Extrapolation to Human

“…Accumulating evidences have demonstrated that DPT possesses a variety of pharmacological activities, such as anti-tumor, anti-viral, anti-inflammatory, and anti-platelet aggregation effects (Gordaliza et al, 1994; Chen et al, 2000; Jin et al, 2008), among which anti-tumor effect is the most attractive. Its analogs etoposide and teniposide have already been widely used for the treatment of lung cancer, leukemia and lymphoma (Smith et al, 1994; Baldwin and Osheroff, 2005; Wu et al, 2014).…”