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

Publication date: Available online 30 April 2017
Source:Journal of Ethnopharmacology
Author(s): Hyun Ju Woo, Do Youn Jun, Ji Young Lee, Hae Sun Park, Mi Hee Woo, Sook Jahr Park, Sang Chan Kim, Chae Ha Yang, Young Ho Kim
Ethnopharmacological relevanceThe roots of Rubia cordifolia L. have been widely used as a traditional herbal medicine in Northeast Asia for treatment of inflammatory diseases.Aim of the studyTo 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.Materials and methodsAnti-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.ResultsPretreatment 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 influenced by CMEP-NQ. Following LPS stimulation, intracellular ROS production was first detected by DCFH-DA staining at 1h; thereafter, 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.ConclusionsThese results demonstrate that the suppressive effect of CMEP-NQ on LPS-induced inflammatory responses in RAW264.7 cells was mainly exerted via its inhibitory function toward TLR4-mediated proximal events, such as MyD88-dependent NF-κB/AP-1 activation and ROS production, and TRIF-dependent IRF3 activation.

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