Involvement of Nuclear Factor-κB in the Inhibition of Interleukin-12 Production from Mouse Macrophages by Baicalein, a Flavonoid in Scutellaria baicalensis

Abstract

Pharmacological inhibition of interleukin-12 (IL-12) production may be a therapeutic strategy for preventing development and progression of disease in experimental models of autoimmunity. In this study we investigated the effects of baicalein, a flavonoid present in the root of Scutellaria baicalensis, on the production of IL-12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Baicalein potently inhibited the LPS-induced IL-12 production from both primary macrophages and RAW264.7 monocytic cell-line in a dose-dependent manner (the IC₅₀ values were 43.7 and 17.4 μM, respectively). The effect of baicalein on IL-12 gene promoter activation was analyzed by transfecting RAW264.7 cells with IL-12 gene promoter/luciferase constructs. The repressive effect mapped to a region in the IL-12 gene promoter containing a binding site for NF-κB. Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the NF-κB site, which significantly decreased upon addition of baicalein, indicating that baicalein inhibited IL-12 production in LPS-activated macrophages via inhibition of NF-κB binding activity.

Abbreviations

IL:interleukin

LPS:lipopolysaccharide

NK-κB:nuclear factor-κB

Th1:T helper type 1

ELISA:enzyme-linked immunosorbent assay

PCR:polymerase chain reaction

Baicalein - Scutellaria baicalensis - Labiatae - interleukin-12 - macrophage - nuclear factor-κB

References

• 1 Ma X, Trinchieri G. Regulation of interleukin-12 production in antigen-presenting cells.  Adv Immunol. 2001;  79 55-92
• 2 Kang K, Kubin M, Cooper K , Lessin S R, Trinchieri G, Rook A H. IL-12 synthesis by human Langerhans cells.  J Immunol. 1996;  156 1402-7
• 3 Gri G, Savio D, Trinchieri G, Ma X. Synergistic regulation of the human interleukin-12 p40 promoter by NF-κB and Ets transcription factors in Epstein-Barr virus-transformed B cells and macrophages.  J Biol Chem. 1998;  273 6431-8
• 4 Adorini L. Interleukin-12, a key cytokine in Th1-mediated autoimmune diseases.  Cell Mol Life Sci. 1999;  55 1610-25
• 5 Austin C A, Patel S, Ono K, Nakane H, Fisher L M. Site-specific DNA cleavage by mammalian DNA topoisomerase II induced by novel flavone and catechin derivatives.  Biochem J. 1992;  282 883-9
• 6 Wakabayashi I. Inhibitory effects of baicalein and wogonin on lipopolysaccharide-induced nitric oxide production in macrophages.  Pharmacol Toxicol. 1999;  84 288-91
• 7 Chen C H, Huang L L, Huang C C, Lin C C, Lee Y, Lu F J. Baicalein, a novel apoptotic agent for hepatoma cell lines: a potential medicine for hepatoma.  Nutr Cancer. 2000;  38 287-95
• 8 Kimata M, Inagaki N, Nagai H. Effects of luteolin and other flavonoids on IgE-mediated allergic reactions.  Planta Med. 2000;  66 25-9
• 9 Ma X, Chow J M, Gri G, Carra G, Gerosa F, Wolf S F, Dzialo R, Trinchieri G. The interleukin 12 p40 gene promoter is primed by interferon-γ in monocytic cells.  J Exp Med. 1996;  183 147-57
• 10 Kang B Y, Chung S W, Cho D, Kim T S. Involvement of p38 mitogen-activated protein kinase in the induction of interleukin-12 p40 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid.  Biochem Pharmacol. 2002;  63 1901-10
• 11 Kim T S, Kang B Y, Lee M H, Choe Y K, Hwang S Y. Inhibition of interleukin-12 production by auranofin, an anti-rheumatic gold compound, deviates CD4⁺ T cells from the Th1 to the Th2 pathway.  Br J Pharmacol. 2001;  134 571-8
• 12 Dignam J D, Lebovitz R M, Roeder R G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.  Nucleic Acids Res. 1993;  11 1475-89
• 13 DeKruyff R , Fang Y, Umetsu D T. Corticosteroids enhance the capacity of macrophages to induce Th2 cytokine synthesis in CD4⁺ lymphocytes by inhibiting IL-12 production.  J Immunol. 1998;  160 2231-7
• 14 Constantinescu C S, Goodman D B, Ventura E S. Captopril and lisinopril suppress production of interleukin-12 by human peripheral blood mononuclear cells.  Immunol Lett. 1998;  62 25-31
• 15 Moller D R, Wysocka W, Greenlee B M, Ma X, Wahl L, Flockhart D A, Trinchieri G, Karp C L. Inhibition of IL-12 production by thalidomide.  J Immunol. 1997;  159 5157-61
• 16 Panina-Bordignon P, Mazzeo D, Lucia P D, D'Ambrosio D, Lang R, Fabbri L, Self C, Sinigaglia F. β₂-Agonists prevent Th1 development by selective inhibition of interleukin 12.  J Clin Invest. 1997;  100 1513-9
• 17 D'Ambrosio D, Cippitelli M, Cocciolo M G, Mazzeo D, Di Lucia P, Lang R, Sinigaglia F, Panina-Bordignon P. Inhibition of IL-12 production by 1,25-dihydroxyvitamin D₃. Involvement of NF-κB downregulation in transcriptional repression of the p40 gene.  J Clin Invest. 1998;  101 252-62
• 18 Na S Y, Kang B Y, Chung S W, Han S J, Ma X, Trinchieri G, Im S Y, Lee J W, Kim T S. Retinoids inhibit interleukin-12 production in macrophages through physical associations of retinoid X receptor and NF-κB.  J Biol Chem. 1999;  274 7674-80
• 19 Kang B Y, Chung S W, Im S Y, Hwang S Y, Kim T S. Chloromethyl ketones inhibit interleukin-12 production in mouse macrophages stimulated with lipopolysaccharide.  Immunol Lett. 1999;  70 135-8