AMPK/Nrf2 signaling is involved in the anti-neuroinflammatory action of Petatewalide B from Petasites japonicus against lipopolysaccharides in microglia

Abstract

Objectives: Abnormal microglia secrete neuroinflammatory factors that play a pivotal role in neurodegenerative-disorder development. Thus, regulating abnormal microglia-activation could be a promising therapeutic strategy. The purposes of this study included investigating the effect of Petatewalide B on lipopolysaccharide- (LPS-) stimulated microglia and exploring the role of the AMPK/Nrf2- (adenosine monophosphate-activated protein kinase/nuclear factor erythroid 2-related factor 2) signaling pathway in the anti-neuroinflammatory function of Petatewalide B.

Methods: We divided the microglia into four groups: a control group, a Petatewalide B-treated group, an LPS-treated group, and an LPS and Petatewalide B-treated group. The four groups of microglia were experimented with, using the NO, ELISA, and promoter assays, and western blotting was conducted to determine LPS-stimulated neuroinflammatory responses.

Results: We found that pretreatment with Petatewalide B strongly alleviates interleukin- (IL-) 1β, IL-6, and tumor-necrosis-factor-α (TNF-α) production, and suppresses iNOS and nitric oxide (NO) overexpression in LPS-stimulated microglia. The AMPK/Nrf2-signaling pathway is important for inducing anti-neuroinflammatory responses. Mechanistic studies report that Petatewalide B increases nuclear-Nrf2 translocation, and heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) expression in a dose-dependent manner. Furthermore, Petatewalide B significantly up-regulates HO-1 and NQO1 by specifically improving antioxidant-response-elements-transcription activity. We then investigated whether Nrf2/HO-1/NQO1 contribute to the anti-neuroinflammatory properties of Petatewalide B. Nrf2, HO-1, and NQO1 small-integrating-ribonucleic-acids (siRNAs) significantly blocked Petatewalide B-attenuated iNOS-promoter-activity in LPS-stimulated microglia. Furthermore, Petatewalide B also up-regulated AMPK-phosphorylation in a dose-dependent manner. We next evaluated whether blocking AMPK-phosphorylation using an inhibitor (compound C) would critically affect anti-neuroinflammatory responses. We found that the AMPK-phosphorylation is associated with nuclear-Nrf2 translocation and elevated HO-1 and NQO1 expression levels. Our data also showed that AMPK-inhibitor pretreatment significantly reverses Petatewalide B-attenuated iNOS-promoter-activity in LPS-stimulated microglia.

Conclusions: Our findings provide the possible mechanism of the anti-neuroinflammatory properties of Petatewalide B that result from beneficial responses in the AMPK/Nrf2-signaling pathway.

Keywords:

• Petatewalide B
• AMPK
• Nrf2
• anti-neuroinflammation
• microglia

Disclosure statement

The authors have no financial conflict of interest.

Additional information

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education [NRF-2015R1D1A1A01059450 and NRF-2016R1D1A3B03934083].