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

Abstract

Objective

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.

Materials and Methods

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.

Results

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.

Conclusions

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.

Keywords:

• 5-Acetoxy-6,7,8,3′,4′-pentamethoxyflavone
• NF-κB/IκB
• TLR4/MyD88/MAPK
• STAT3
• microglia-mediated neuroinflammatory disorders

Authors’ contributions

MJC, HLC, CIC and CHL designed the study; JSL, HMC, WJW and CHL performed the experiments; MYL, WFC and CHL contributed to manuscript preparation. All authors have read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by grants from National Pingtung University of Science and Technology (NPUST)-Kaohsiung Medical University (KMU) Joint Research Project (#NPUST-KMU-112-P004), the KMU-KMUH Co-Project of Key Research (grant No. KMU-DK(A)112005 from Kaohsiung Medical University), Chi-Mei Medical Center Liouying Research Grant (CLFHR10832, CLFHR11026 and CLFHR11238) and Chang Gung Memorial Hospital, Kaohsiung, Taiwan (CMRPG8L1131 and CMRPG8N1271).