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

1 Andalusol, ent-6 alpha,8 alpha,18-trihydroxy-13(16),14-labdadiene, is a naturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo models 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 the synthesis of nitrite caused by LPS (1 mu g ml(-1)) in concentration and time-dependent manners. The maximal inhibition was observed when andalusol was added 30 min before LPS stimulation and decreased progressively as the interval between andalusol and LPS challenge increased up to 14 h. 3 Incubation of J774 cells with LPS resulted in the expression of NOS-2 protein (130 kDa) as identified by Western blot analysis. The levels of this enzyme 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 compound also inhibited the degradation of I kappa B alpha favouring the retention of the inactive NF-kappa B complexes in the cytosol. 5 Related compounds to andalusol but lacking the polyol groups were less effective inhibiting NOS-2 expression in LPS-activated macrophages. The present findings provide a mechanism by which the anti-inflammatory properties of this diterpene could be mediated.