Nodakenin Suppresses Lipopolysaccharide-Induced Inflammatory Responses in Macrophage Cells by Inhibiting Tumor Necrosis Factor Receptor-Associated Factor 6 and Nuclear Factor-κB Pathways and Protects Mice from Lethal Endotoxin Shock

Nodakenin, a coumarin isolated from the roots of Angelicae gigas, has been reported to possess neuroprotective, antiaggregatory, antibacterial, and memory-enhancing effects. In the present study, we investigated the anti-inflammatory effects of nodakenin by examining its in vitro inhibitory effects on inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and mouse peritoneal macrophages and its in vivo effects on LPS-induced septic shock in mice. Our results indicate that nodakenin concentration-dependently inhibits iNOS and COX-2 at the protein, mRNA, and promoter binding levels, and these inhibitions cause attendant decreases in the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2). Furthermore, we found that nodakenin inhibits the production and mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-1 beta induced by LPS. Molecular data revealed that nodakenin suppressed the transcriptional activity and translocation of nuclear factor-kappa B (NF-kappa B) by inhibiting inhibitory kappa B-alpha degradation and I kappa B kinase-alpha/beta phosphorylation. In addition, nodakenin was found to significantly inhibit the LPS-induced binding of transforming growth factor-beta-activated kinase 1 to tumor necrosis factor receptor-associated factor 6 (TRAF6) by reducing TRAF6 ubiquitination. Pretreatment with nodakenin reduced the serum levels of NO, PGE(2), and proinflammatory cytokines and increased the survival rate of mice with LPS-induced endotoxemia. Taken together, our data suggest that nodakenin down-regulates the expression of the proinflammatory iNOS, COX-2, TNF-alpha, IL-6, and IL-1 beta genes in macrophages by interfering with the activation of TRAF6, thus preventing NF-kappa B activation.