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The Regulation of Bis-N-norgliovictin on TLR4 Signaling Pathway

Author: SongYuXian
Tutor: HouYaYi
School: Nanjing University
Course: Pharmacology
Keywords: bis-N-norgliovictin Toll like receptor4 inflammation MyD88 TRIF
CLC: R392.1
Type: Master's thesis
Year: 2011
Downloads: 31
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Abstract

Toll-like receptors (TLRs) play pivotal roles in mammalian immune system for their detection of microorganisms, initiation and modulation of innate and adaptive immune responses. TLR4 is significant among TLRs. Since dysregulation of TLR4-mediated immune responses has been reported to be closely linked to many stubborn diseases (such as cancer, viral infection, autoimmune illness, etc), strict and fine modulation of TLR4 signaling by small molecules may have therapeutic potentials against such diseases.Lipopolysaccharide (LPS) is the most typical ligand of TLR4, which can activate TLR4 signaling pathway and produce multiple inflammatory mediators. To discover new small molecule inhibitors of TLR4 signaling, srceening among a series of novel small-molecule compounds using mouse macrophage cell line RAW264.7 stimulated with LPS was carried out. Intriguingly, several small molecules were identified to have suppressive effects on TNF-a and MCP-1 production in RAW264.7 cells induced by LPS. Bis-N-norgliovictin, a diketopiperazine from marine fungus, is one of the best candidates. Further investigation was supposed to carry out to elucidate the anti-inflammatory activity and its molecular mechanisms.First of all, we used CCK-8 assay to evaluate cell viability and found that bis-N-norgliovictin didn’t affect cell viability in RAW264.7 cells. This suggests that the inhibitory effect of bis-N-norgliovictin on TNF-a and MCP-1 production was not mediated by the cytotoxicity. Subsequently, we identified that bis-N-norgliovictin inhibited LPS-induced TNF-α, IL-6, MCP-1 and IFN-βproduction and their mRNA expression in RAW264.7 cells. What’s more, the anti-inflammatory effects were also shown in mouse peritoneal macrophages and in vivo. It was noteworthy that bis-N-norgliovictin didn’t affect LPS recognition and internalization by mouse peritoneal macrophages.It is generally believed that TLR4 induces two signaling pathways including the MyD88-dependent and TRIF-dependent pathways. Activated MyD88-dependent pathway induces the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB), leading to the release of pro-inflammatory cytokines (such as TNF-αand IL-6). The TRIF- dependent signaling pathway activates the downstream of TRIF kinases apart from MAPKs and NF-κB, leading to the phosphorylation and activation of interferon regulatory factor3 (IRF3) and the consequent expression of typeⅠinterferons and interferon inducible genes (such as IFN-βand IP-10). We have demonstrated that bis-N-norgliovictin inhibited both MyD88-dependent and TRIF-dependent inflammatory mediators, which led us to assumption that it regulated both of these pathways. So we investigate the mechanism of bis-N-norgliovictin in these two pathways.Results of western blot showed that bis-N-norgliovictin (2μg/ml) inhibited LPS-induced phosphorylation and degradation of IκBαin RAW264.7 cells. What’s more, we observed down-regulation of LPS-induced NF-κB translocation after bis-N-norgliovictin administration. Meanwhile, the phosphorylations of JNK and p38 were obviously attenuated, but not ERK. We also found by gene report assay that it depressed the AP-1 activity. As to the TRIF- dependent IRF3 activation, bis-N-norgliovictin could inhibit its phosphorylation.In summary, we have discovered bis-N-norgliovictin among scores of fungi products that can effectively inhibit TLR4 mediated inflammation pathway in macrophages and in vivo. The inhibitory activity of bis-N-norgliovictin is attributed to the down-regulation of both MyD88 and TRIF-dependent pathways. All these results suggest that bis-N-norgliovictin is a new small-molecule inhibitor of TLR4, and might be a useful therapeutic candidate for the treatment of inflammatory diseases related to TLR4.

The Regulation of Bis-N-norgliovictin on TLR4 Signaling Pathway

Abstract

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