Melatonin modulates TLR4-mediated inflammatory genes through MyD88- and TRIF-dependent signaling pathways in lipopolysaccharide-stimulated RAW264.7 cells.

Increasing evidence demonstrates that melatonin has an anti-inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll-like receptor 4 (TLR4)-mediated molecule myeloid differentiation factor 88 (MyD88)-dependent and TRIF-dependent signaling pathways in lipopolysaccharide (LPS)-stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 μg/mL) in the absence or presence of melatonin (10, 100, 1000 μm). As expected, melatonin inhibited TLR4-mediated tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and IL-10 in LPS-stimulated macrophages. In addition, melatonin significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS-stimulated macrophages. Although it had no effect on TLR4-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF-κB) in LPS-stimulated macrophages. In addition, melatonin inhibited TLR4-mediated Akt phosphorylation in LPS-stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)-regulated factor-3 (IRF3), which was involved in TLR4-mediated TRIF-dependent signaling pathway, in LPS-stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS-induced IFN-β in macrophages. In conclusion, melatonin modulates TLR4-mediated inflammatory genes through MyD88-dependent and TRIF-dependent signaling pathways.

Xia MZ ，Liang YL ，Wang H ，Chen X ，Huang YY ，Zhang ZH ，Chen YH ，Zhang C ，Zhao M ，Xu DX ，Song LH ... -  《-》

Quercetin disrupts tyrosine-phosphorylated phosphatidylinositol 3-kinase and myeloid differentiation factor-88 association, and inhibits MAPK/AP-1 and IKK/NF-κB-induced inflammatory mediators production in RAW 264.7 cells.

Quercetin is a major bioflavonoid widely present in fruits and vegetables. It exhibits anti-inflammatory, anti-tumor, antioxidant properties and reduces cardiovascular disease risks. However, the molecular mechanism of action against inflammation in RAW 264.7 cells is only partially explored. Quercetin effect on LPS-induced gene and protein expressions of inflammatory mediators and cytokines were determined. Moreover, involvement of heme-oxygenase-1, protein kinases, adaptor proteins and transcription factors in molecular mechanism of quercetin action against inflammation were examined. Quercetin inhibited LPS-induced NO, PGE₂, iNOS, COX-2, TNF-α, IL-1β, IL-6 and GM-CSF mRNA and protein expressions while it promoted HO-1 induction in a dose- and time-dependent manner. It also suppressed I-κB-phosphorylation, NF-κB translocation, AP-1 and NF-κB-DNA-binding and reporter gene transcription. Quercetin attenuated p38(MAPK) and JNK1/2 but not ERK1/2 activations and this effect was further confirmed by SB203580 and SP600125-mediated suppressions of HO-1, iNOS, and COX-2 protein expressions. Moreover, quercetin arrested Src, PI3K, PDK1 and Akt activation in a time- and dose-dependent manner, which was comparable to PP2 and LY294002 inhibition of Src, PI3K/Akt and iNOS expressions. Quercetin further arrested Src and Syk tyrosine phosphorylations and their kinase activities followed by inhibition of PI3K tyrosine phosphorylation. Moreover, quercetin disrupted LPS-induced p85 association to TLR4/MyD88 complex and it then limited activation of IRAK1, TRAF6 and TAK1 with a subsequent reduction in p38 and JNK activations, and suppression in IKKα/β-mediated I-κB phosphorylation. Quercetin limits LPS-induced inflammation via inhibition of Src- and Syk-mediated PI3K-(p85) tyrosine phosphorylation and subsequent TLR4/MyD88/PI3K complex formation that limits activation of downstream signaling pathways.

Endale M ，Park SC ，Kim S ，Kim SH ，Yang Y ，Cho JY ，Rhee MH ... -  《-》

Propofol Inhibits Lipopolysaccharide-Induced Inflammatory Responses in Spinal Astrocytes via the Toll-Like Receptor 4/MyD88-Dependent Nuclear Factor-κB, Extracellular Signal-Regulated Protein Kinases1/2, and p38 Mitogen-Activated Protein Kinase Pathways.

Zhou CH ，Zhu YZ ，Zhao PP ，Xu CM ，Zhang MX ，Huang H ，Li J ，Liu L ，Wu YQ ... -  《-》

Zedoarondiol isolated from the rhizoma of Curcuma heyneana is involved in the inhibition of iNOS, COX-2 and pro-inflammatory cytokines via the downregulation of NF-kappaB pathway in LPS-stimulated murine macrophages.

Cho W ，Nam JW ，Kang HJ ，Windono T ，Seo EK ，Lee KT ... -  《-》

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. 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.

Ju Woo H ，Jun DY ，Lee JY ，Park HS ，Woo MH ，Park SJ ，Kim SC ，Yang CH ，Kim YH ... -  《-》