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J Ethnopharmacol. 2017 Jun 9;205:103-115. doi: 10.1016/j.jep.2017.04.029. Epub 2017 Apr 30.

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.

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Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea.
Institute of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, South Korea.
College of Pharmacology, Daegu Catholic University, Gyeongsan 712-702, South Korea.
College of Oriental Medicine, Daegu Hanny University, Daegu 706-060, South Korea.
Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, South Korea. Electronic address:



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.


2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone; ABTS (PubChem CID:35687); Anti-inflammatory drug; DCFH-DA (PubChem CID:104913); DPPH (PubChem CID:74358); IRF3; LPS (PubChem CID:11970143); LPS-treated macrophages; MyD88 and IRAK1 association; NAC (PubChem CID:12035); Rubia cordifolia L; ethyl acetate (PubChem CID:8857); methanol (PubChem CID:887); methylene chloride (PubChem CID:6344); n-butanol (PubChem CID:263)

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