Nobiletin derivative, 5-acetoxy-6,7,8,3',4'-pentamethoxyflavone, inhibits neuroinflammation through the inhibition of TLR4/MyD88/MAPK signaling pathways and STAT3 in microglia.

Microglia in the central nervous system regulate neuroinflammation that leads to a wide range of neuropathological alterations. The present study investigated the anti-neuroinflammatory properties of nobiletin (Nob) derivative, 5-acetoxy-6,7,8,3′,4′-pentamethoxyflavone (5-Ac-Nob), in lipopolysaccharide (LPS)-activated BV2 microglia. By using the MTT assay, Griess method, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), we determined the cell viability, the levels of nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory factors (interleukin 1 beta; IL-1β, interleukin 6; IL-6, tumor necrosis factor alpha; TNF-α and prostaglandin E2; PGE2) in LPS-stimulated BV2 microglia. Toll-like receptor 4 (TLR4)-mediated myeloid differentiation primary response gene 88 (MyD88)/nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK) signaling pathway and signal transducer and activator of transcription 3 (STAT3) were measured by western blotting. Analysis of NO generation and mRNA of pro-inflammatory cytokines was confirmed in the zebrafish model. 5-Ac-Nob reduced cell death, the levels of NO, ROS, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and pro-inflammatory factors in LPS-activated BV-2 microglial cells. TLR4-mediated MyD88/NF-κB and MAPK pathway (p38, ERK and JNK) after exposure to 5-Ac-Nob was also suppressed. Moreover, 5-Ac-Nob inhibited phosphorylated STAT3 proteins expression in LPS-induced BV-2 microglial cells. Furthermore, we confirmed that 5-Ac-Nob decreased LPS-induced NO generation and mRNA of pro-inflammatory cytokines in the zebrafish model. Our findings suggest that 5-Ac-Nob represses neuroinflammatory responses by inhibiting TLR4-mediated signaling pathway and STAT3. As a result of these findings, 5-Ac-Nob has potential as an anti-inflammatory agent against microglia-mediated neuroinflammatory disorders. [ABSTRACT FROM AUTHOR]