Anti-inflammatory mechanisms of isoflavone metabolites in lipopolysaccharide-stimulated microglial cells

The microglial activation plays an important role in neurodegenerative diseases by producing several proinflammatory cytokines and nitric oxide ( NO). We found that three types of isoflavones and their metabolites that are transformed by the human intestinal microflora suppress lipopolysaccharide ( LPS)induced release of NO and tumor necrosis factor ( TNF)-alpha in primary cultured microglia and BV2 microglial cell lines. The inhibitory effect of the isoflavone metabolites ( aglycon form) was more potent than that of isoflavones ( glycoside form). The RNase protection assay showed that the isoflavone metabolites regulated inducible nitric oxide synthase ( iNOS) and the cytokines at either the transcriptional or post- transcriptional level. A further molecular mechanism study was performed for irisolidone, a metabolite of kakkalide, which had the most potent anti-inflammatory effect among the six isoflavones tested. Irisolidone significantly inhibited the DNA binding and transcriptional activity of nuclear factor (NF)-kappa B and activator protein-1. Moreover, it repressed the LPS-induced extracellular signalregulated kinase ( ERK) phosphorylation without affecting the activity of c- Jun N- terminal kinase or p38 mitogen- activated protein kinase. The level of NF-kappa B inhibition by irisolidone correlated with the level of iNOS, TNF-alpha, and interleukin ( IL)-1 beta suppression in LPS- stimulated microglia, whereas the level of ERK inhibition correlated with the level of TNF-alpha and IL-1 beta repression. Overall, the repression of proinflammatory cytokines and iNOS gene expression in activated microglia by isoflavones such as irisolidone might have therapeutic potential for various neurodegenerative diseases including ischemic cerebral disease.