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Inhibition of lipopolysaccharide-stimulated NO production by crotafuran B in RAW 264.7 macrophages involves the blockade of NF-kappaB activation through the increase in IkappaBalpha synthesis.

Crotafuran B, a natural pterocarpanoid isolated from Crotalaria pallida, inhibited the lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (IC50 16.4+/-0.7 microM) and inducible nitric oxide synthase (iNOS) protein and mRNA expression (IC50 11.5+/-0.6 microM and 11.8+/-2.2 microM, respectively), but not via its cytotoxicity or the inhibition of iNOS enzyme activity, in RAW 264.7 macrophages. Crotafuran B also reduced the iNOS promoter activity (IC50 13.4+/-0.1 microM) in piNOS-LUC-transfected cells. Crotafuran B treatment inhibited the p65 nuclear translocation and the nuclear factor-kappaB (NF-kappaB) DNA binding activity in LPS-activated macrophages. Crotafuran B also reduced the NF-kappaB transcriptional activity in pNF-kappaB-LUC-transfected cells. Crotafuran B had no effect on the LPS-induced phosphorylation of inhibitory kappaBalpha (IkappaBalpha), but enhanced the cellular level of IkappaBalpha that rebounded to the basal levels and increased the IkappaBalpha mRNA expression. These results indicate that the crotafuran B inhibition of NO production involves a decrease in the iNOS gene expression via the inhibition of NF-kappaB activation through the increase in IkappaBalpha synthesis.