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

Objectives: Abnormal microglia secrete neuroinflammatory factors that play a pivotal role in neurode-generative-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-) 1 beta, IL-6, and tumor-necrosis-factor-alpha (TNF-alpha) production, and suppresses iNOS and nitric oxide (NO) overexpres-sion in LPS-stimulated microglia. The AMPK/Nrf2-signaling pathway is important for inducing anti-neu-roinflammatory 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.