Differential inhibitory effects of inotilone on inflammatory mediators, inducible nitric oxide synthase and cyclooxygenase-2, in LPS-stimulated murine macrophage

The inhibitory effects of inotilone and methylinotilone on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS were investigated. The results show that both hydroxyl groups on the benzene ring of the inotilone molecule are required for better anti-inflammatory effect. Western blotting and RT-PCR analyses demonstrated that inotilone blocked protein and mRNA expression of iNOS but not COX-2. Instead, inotilone inhibited prostaglandin E₂ production through decreasing the enzyme activity of COX-2. The repression of iNOS but not COX-2 expression may come from the differential effect of inotilone on nuclear factor-κB (NFκB) and CCAAT/enhancer-binding protein beta Treatment with inotilone resulted in the reduction of LPS-induced nuclear translocation of NFκB subunit and the NFκB-dependent transcriptional activity by blocking phosphorylation of inhibitor κB(IκB)α and p65 and subsequent degradation of inhibitor κBα. Inotilone also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Our results suggest that inotilone may have potential to be developed into an effective anti-inflammatory agent.