Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced signaling pathways involved in the release of tumor necrosis factor-alpha

Administration of bacterial lipopolysaccharide (LPS) to laboratory animals and cultured macrophages induces tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine. Pretreatment with Ginkgo biloba extract (EGb 76 1) inhibited the in vivo production of TNF-alpha (measured by ELISA) after challenge with LPS. To begin to understand the mechanism of this inhibition , we evaluated the in vitro effects of EGb 761 and its flavonoid component, quercetin, on LPS-treated RAW 264.7 macrophages. Pretreatment with EGb 761 or quercetin concentration-dependently inhibited TNF-a release, as measure d by the L929 fibroblast assay. Northern blotting demonstrated that quercet in inhibited LPS-induced TNF-alpha mRNA, but did not alter its half-life. A ctivation of mitogen-activated protein kinases (MAPKs) and the redox-sensit ive transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1), are key events in the signal transduction pathways mediat ing TNF-alpha induction. Phosphorylation of extracellular signal-related ki nases 1 and 2 (ERK 1/2), p38 MAPK, and Jun N-terminal kinase/stress-activat ed protein kinase. (JNK/SAPK), members of the MAPK family, was analyzed by western blotting. Our results suggest that quercetin is unique in its abili ty to inhibit TNF-alpha transcription by inhibiting the phosphorylation and activation of JNK/SAPK and, therefore, suppressing AP-1-DNA binding [asses sed by electrophoretic mobility shift analysis (EMSA)]. Results from wester n analysis, EMSA, and transient transfections suggest that EGb 761 diminish es LPS-induced NT-KB but has no effect on LPS-induced TNF-alpha transcripti on. Both EGb 761 and quercetin inhibited ERK1/2 phosphorylation and p38 MAP K activity, which are important in the post-transcriptional regulation of T NF-alpha mRNA. (C) 2001 Elsevier Science Inc. All rights reserved.