Inhibition of NOS-2 expression in macrophages through the inactivation of NF-kappa B by andalusol

1 Andalusol, ent-6 alpha,8 alpha,18-trihydroxy-13(16),14-labdadiene, is a n aturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo mode ls of paw and ear inflammation (Navarro et al., 1997: Z. Naturforsch., 52, 844-849). The pharmacological effects of this diterpene have been analysed on the activation of the macrophage cell line J774 with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). 2 Incubation of J774 macrophages with andalusol (0.1-100 mu M) inhibited th e synthesis of nitrite caused by LPS (1 mu g ml(-1)) in concentration and t ime-dependent manners. The maximal inhibition was observed when andalusol w as added 30 min before LPS stimulation and decreased progressively as the i nterval between andalusol and LPS challenge increased up to 14 h. 3 Incubation of J774 cells with LPS resulted in the expression of NOS-2 pro tein (130 kDa) as identified by Western blot analysis. The levels of this e nzyme decreased significantly in the presence of andalusol (IC50=10.5 mu M) , suggesting that this diterpene inhibited NOS-2 expression. 4 Andalusol inhibited nuclear factor KB activation, a transcription factor necessary for NOS-2 expression in response to LPS and IFN-gamma. This compo und also inhibited the degradation of I kappa B alpha favouring the retenti on of the inactive NF-kappa B complexes in the cytosol. 5 Related compounds to andalusol but lacking the polyol groups were less ef fective inhibiting NOS-2 expression in LPS-activated macrophages. The prese nt findings provide a mechanism by which the anti-inflammatory properties o f this diterpene could be mediated.