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

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-) 1ß, 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.

Petasites japonicus bakkenolide B inhibits lipopolysaccharide­induced pro­inflammatory cytokines via AMPK/Nrf2 induction in microglia.

Abnormal neuroinflammatory responses have diverse roles in neuronal death, oxidative stress and neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Microglia regulate these responses via molecular signaling cascades that involve inflammatory cytokines and complement proteins. Bakkenolide B from Petasites japonicus exhibits significant anti­inflammatory and anti­allergic bioactivities. The present study investigated the anti­neuroinflammatory effects and underlying molecular mechanisms of bakkenolide B on the lipopolysaccharide (LPS)­mediated neuroinflammatory response in microglia. The results indicated that bakkenolide B pretreatment significantly reduced microglial production of interleukin (IL)­1ß, IL­6, IL­12, and tumor necrosis factor (TNF)­α. Furthermore, this effect was associated with reduced production of reactive oxygen species. The role of bakkenolide B was then evaluated in the upregulation of nuclear factor erythroid 2­related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathways. The results suggested that bakkenolide B significantly upregulated Nrf2/ARE pathway­related downstream factors, such as NADPH dehydrogenase quinone­1 (NQO­1) and heme oxygenase­1 (HO­1). Silencing of Nrf2, HO­1 and NQO­1 diminished the anti­neuroinflammatory properties of bakkenolide B. AMP­activated protein kinase (AMPK) activates the Nrf2/ARE signaling pathway, and the results of the present study demonstrated that bakkenolide B increased AMPK phosphorylation in microglia. In addition, an AMPK inhibitor abolished the bakkenolide B­induced increase in nuclear Nrf2, NQO­1 and HO­1 protein expression. Finally, an AMPK inhibitor diminished the bakkenolide B­mediated inhibition of LPS­stimulated TNF­α production. Taken together, the present results demonstrate that bakkenolide B may be an effective and therapeutically relevant AMPK/Nrf2 pathway activator for suppressing abnormal neuro-inflammation in neurodegenerative diseases.