Anti-inflammatory action of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (CMEP-NQ) suppresses both the MyD88-dependent and TRIF-dependent pathways of TLR4 signaling in LPS-stimulated RAW264.7 cells

The roots of Rubia cordifolia L. have been widely used as a traditional herbal medicine in Northeast Asia for treating inflammatory diseases. To elucidate the anti-inflammatory mechanism of 2-carbomethoxy-2,3-epoxy-3- prenyl-1,4-naphthoquinone (CMEP-NQ), purified from the roots of R. cordifolia L. a...

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Published in:Journal of ethnopharmacology 2017-06, Vol.205, p.103-115
Main Authors: Ju Woo, Hyun, Jun, Do Youn, Lee, Ji Young, Park, Hae Sun, Woo, Mi Hee, Park, Sook Jahr, Kim, Sang Chan, Yang, Chae Ha, Kim, Young Ho
Format: Article
Language:English
Subjects:
2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone
Adaptor Proteins, Vesicular Transport - genetics
Adaptor Proteins, Vesicular Transport - metabolism
Animals
Anti-Inflammatory Agents - chemistry
Anti-Inflammatory Agents - pharmacology
Anti-inflammatory drug
Gene Expression Regulation - drug effects
IRF3
Lipopolysaccharides - pharmacology
LPS-treated macrophages
Mice
Molecular Structure
MyD88 and IRAK1 association
Myeloid Differentiation Factor 88 - genetics
Myeloid Differentiation Factor 88 - metabolism
Naphthoquinones - pharmacology
Plant Roots - chemistry
RAW 264.7 Cells
Rubia - chemistry
Rubia cordifolia L
Signal Transduction - drug effects
Signal Transduction - physiology
Toll-Like Receptor 4 - genetics
Toll-Like Receptor 4 - metabolism
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Summary:The roots of Rubia cordifolia L. have been widely used as a traditional herbal medicine in Northeast Asia for treating inflammatory diseases. To elucidate the anti-inflammatory mechanism of 2-carbomethoxy-2,3-epoxy-3- prenyl-1,4-naphthoquinone (CMEP-NQ), purified from the roots of R. cordifolia L. as the major anti-inflammatory component, in LPS-treated RAW264.7 murine macrophage cells. Anti-inflammatory activity of CMEP-NQ was investigated in LPS-treated RAW264.7 cells by measuring the levels of NO, PGE2, and cytokines (IL1β, IL-6, TNF-α) in the culture supernatants and the TLR4-mediated intracellular events including association of MyD88 with IRAK1, activation of IRAK1, TAK1, MAPKs, NF-κB/AP-1, and IRF3, and generation of ROS. Pretreatment of RAW264.7 cells with CMEP-NQ reduced LPS-induced production of NO and PGE2 by suppressing iNOS and COX-2 gene expression. CMEP-NQ also reduced the secretion of IL-1β, IL-6, and TNF-α by down-regulating mRNA levels. Under these conditions, TLR4-mediated MyD88-dependent events were inhibited by CMEP-NQ, including the association of MyD88 with IRAK1, phosphorylation of IRAK1, TAK1, and MAPKs (ERK, JNK and p38 MAPK), and activation of NF-κB and AP-1. As TRIF-dependent events of TLR4 signaling, phosphorylation of IRF3 and induction of iNOS protein expression were also inhibited by CMEP-NQ. However, the binding of FITC-conjugated LPS to cell surface TLR4 was not affected by CMEP-NQ. Following LPS stimulation, intracellular ROS production was first detected by DCFH-DA staining at 1h; then it continuously increased until 16h. Although CMEP-NQ failed to exhibit DPPH radical- or ABTS radical-scavenging activity in vitro, LPS-induced ROS production in RAW264.7 cells was more efficiently blocked by CMEP-NQ than by NAC. These results demonstrate that the suppressive effect of CMEP-NQ on LPS-induced inflammatory responses in RAW264.7 cells was mainly exerted via its inhibition of TLR4-mediated proximal events, such as MyD88-dependent NF-κB/AP-1 activation and ROS production, and TRIF-dependent IRF3 activation. [Display omitted]
ISSN:0378-8741
1872-7573
DOI:10.1016/j.jep.2017.04.029