Nitric oxide and tumor necrosis factor-alpha production by oleanolic acid via nuclear factor-kappaB activation in macrophages.

Oleanolic acid (OA), a pentacyclic triterpene acid, is reported to have antitumor activities; however, the mechanism underlying its antitumorigenic effects is poorly understood. To further determine the mechanism of OA, we investigated the effects of OA on the release of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) and on the level of inducible nitric oxide synthase (iNOS) and TNF-alpha gene expression in mouse macrophages. We found that OA elicited a dose-dependent increase in NO and TNF-alpha production. Reverse transcription-polymerase chain reaction showed that the increased NO and TNF-alpha secretion were due to an increase in iNOS mRNA and TNF-alpha mRNA, respectively. Since iNOS and TNF-alpha transcription have recently been shown to be under the control of the NF-kappaB transcription factor, the effects of OA on NF-kappaB activation were examined using a transient transfection assay and an electrophoretic mobility shift assay (EMSA). Transient expression assays with NF-kappaB binding sites linked to the luciferase gene revealed that the increased levels of iNOS mRNA and TNF-alpha mRNA induced by OA were mediated by the NF-kappaB transcription factor complex. Using DNA fragments containing the NF-kappaB binding sequence, OA was shown to activate the protein/DNA binding of NF-kappaB to its cognate site as measured by EMSA. These results demonstrate that OA stimulates NO and TNF-alpha release and is able to upregulate iNOS and TNF-alpha expression through NF-kappaB transactivation, which may be the mechanism whereby OA elicits its biological effects.