Neuroinflammation and oxidative stress play key roles in the progression of neurodegenerative diseases. Thus, the use of potent anti-inflammatory/antioxidant agents has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we investigated the anti-inflammatory and antioxidant effects of nootkatone (NKT), a sesquiterpenoid compound isolated from grapefruit, in in vitro and in vivo models of neuroinflammation. In lipopolysaccharide (LPS)-stimulated BV2 microglial cells, NKT inhibited the expression of iNOS, COX-2, and pro-inflammatory cytokines, and increased the expression of the anti-inflammatory cytokine, IL-10. In addition, NKT inhibited reactive oxygen species (ROS) production and upregulated the expression of antioxidant enzymes, such as NQO1 and HO-1. Molecular mechanistic studies showed that NKT inhibited Akt, p38 MAPK, and NF-κB activities, while increasing AMPK, PKA/CREB, and Nrf2/ARE signaling in LPS-stimulated BV2 cells. Since NKT dramatically increased NQO1 expression, we investigated the role of this enzyme using pharmacological inhibition or knockdown experiments. Treatment of BV2 cells with the NQO1-specific inhibitor, dicoumarol, or with NQO1 siRNA significantly blocked NKT-mediated inhibition of NO, ROS, TNF-α, IL-1β, and upregulation of IL-10. Furthermore, NQO1 inhibition reversed the effects of NKT on pro- and anti-inflammatory signaling molecules. Intriguingly, we found that the AMPK inhibitor, compound C, mimicked the effects of dicoumarol, suggesting the presence of a crosstalk between NQO1 and AMPK. Finally, we demonstrated that NKT inhibited microglial activation, lipid peroxidation, and the expression of pro-inflammatory markers in the brains of LPS-injected mice, which was also reversed by dicoumarol. These data collectively suggest that NQO1 plays a critical role in mediating the anti-inflammatory and antioxidant effects of NKT in LPS-induced neuroinflammation by modulating AMPK and its downstream signaling pathways.
