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

Crotafuran B, a natural pterocarpanoid isolated from Crotalaria pallida, inhibited the lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (IC50 16.4 ± 0.7 μM) and inducible nitric oxide synthase (iNOS) protein and mRNA expression (IC50 11.5 ± 0.6 μM and 11.8 ± 2.2 μM, 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 μM) in piNOS-LUC-transfected cells. Crotafuran B treatment inhibited the p65 nuclear translocation and the nuclear factor-κB (NF-κB) DNA binding activity in LPS-activated macrophages. Crotafuran B also reduced the NF-κB transcriptional activity in pNF-κB-LUC-transfected cells. Crotafuran B had no effect on the LPS-induced phosphorylation of inhibitory κBα (IκBα), but enhanced the cellular level of IκBα that rebounded to the basal levels and increased the IκBα 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-κB activation through the increase in IκBα synthesis.