Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatory cytokine production in macrophages

Flavonoids are naturally occurring polyphenolic compounds with a wide distr ibution throughout the plant kingdom. In the present study, we compared the ability of several flavonoids to modulate the production of proinflammator y molecules from lipopolysaccharide (LPS)-stimulated macrophages and invest igated their mechanism(s) of action. Pretreatment of RAW 264.7 with luteoli n, luteolin-7-glucoside, quercetin, and the isoflavonoid genistein inhibite d both the LPS-stimulated TNF-alpha and interleukin-6 release, whereas erio dictyol and hesperetin only inhibited TNF-alpha release. From the compounds tested luteolin and quercetin were the most potent in inhibiting cytokine production with an IC50 of less than 1 and 5 muM for TNF-alpha release, res pectively. To determine the mechanisms by which flavonoids inhibit LPS sign aling, we used luteolin and determined its ability to interfere with total protein tyrosine phosphorylation as well as Akt phosphorylation and nuclear factor-kappaB activation. Pretreatment of the cells with luteolin attenuat ed LPS-induced tyrosine phosphorylation of many discrete proteins. Moreover , luteolin inhibited LPS-induced phosphorylation of Akt. Treatment of macro phages with LPS resulted in increased I kappaB-alpha phosphorylation and re duced the levels of I kappaB-alpha. Pretreatment of cells with luteolin abo lished the effects of LPS on I kappaB-alpha. To determine the functional re levance of the phosphorylation events observed with I kappaB-alpha, macroph ages were transfected either with a control vector or a vector coding for t he luciferase reporter gene under the control of kappaB cis-acting elements . Incubation of transfected RAW 264.7 cells with LPS increased luciferase a ctivity in a luteolin-sensitive manner. We conclude that luteolin inhibits protein tyrosine phosphorylation, nuclear factor-kappaB-mediated gene expre ssion and proinflammatory cytokine production in murine macrophages.