Urolithin A attenuates pro-inflammatory mediator production by suppressing PI3-K/Akt/NF-κB and JNK/AP-1 signaling pathways in lipopolysaccharide-stimulated RAW264 macrophages: Possible involvement of NADPH oxidase-derived reactive oxygen species.

Urolithin A, a gut microbial metabolite of ellagic acid, is reported to exert anti-inflammatory effects in vitro and in vivo. However, complete mechanisms underlying the regulation of inflammatory responses by urolithin A remain unclear. This study aimed to evaluate the anti-inflammatory potential of urolithin A and its underlying mechanisms in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. Urolithin A significantly attenuated the pro-inflammatory mediator production in LPS-stimulated RAW264 and mouse peritoneal macrophages. This compound significantly suppressed the LPS-elicited nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activation. The phosphorylation of Akt and c-Jun N-terminal kinase (JNK) was also inhibited by the treatment with urolithin A. Through experiments using kinase inhibitors, urolithin A abolished the LPS-induced phosphatidylinositol 3-kinase (PI3-K)/Akt/NF-κB and JNK/AP-1 signaling pathways, resulting in suppression of pro-inflammatory mediator production. Furthermore, treatment with this compound significantly reduced the intracellular accumulation of reactive oxygen species, which are known to act as secondary messengers in the activation of redox-sensitive transcription factors NF-κB and AP-1. Urolithin A treatment also diminished the LPS-evoked activation of NADPH oxidase (NOX), which is the main source of reactive oxygen species in activated macrophages. The inhibition of this activity by urolithin A led to the prevention of LPS-elicited NF-κB and AP-1 activation as well as Akt and JNK phosphorylation, resulting in the reduction of pro-inflammatory mediator production. Collectively, these results indicate that urolithin A treatment attenuates pro-inflammatory mediator production by suppressing NOX-derived reactive oxygen species-mediated PI3-K/Akt/NF-κB and JNK/AP-1 signaling pathways in LPS-stimulated macrophages.