Vol. 302, Issue 1, 138-144, July 2002

Inhibition of Inducible Nitric-Oxide Synthase Expression by Silymarin in Lipopolysaccharide-Stimulated Macrophages

Jong S. Kang, Young J. Jeon, Hwan M. Kim, Seung H. Han and Kyu-Hwan Yang

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, Korea (J.S.K., K.-H.Y.); Department of Pharmacology, Chosun University School of Medicine, Kwangju, Korea (Y.J.J.); Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea (H.M.K.); and International Vaccine Institute, Seoul, Korea (S.H.H.)

Silymarin, a polyphenolic flavonoid antioxidant, is known to have anti-inflammatory, hepatoprotective, and anticarcinogenic effects. In the present study, we report the inhibitory effect of silymarin on nitric oxide production and inducible nitric-oxide synthase (iNOS) gene expression in macrophages. In vivo administration of silymarin attenuated nitric oxide production by peritoneal macrophages in lipopolysaccharide (LPS)-treated mice. Silymarin also dose dependently suppressed the LPS-induced production of nitric oxide in isolated mouse peritoneal macrophages and RAW 264.7, a murine macrophage-like cell line. Moreover, iNOS mRNA and its protein expression were completely abrogated by silymarin in LPS-stimulated RAW 264.7 cells. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by silymarin, we examined the effect of silymarin on LPS-induced nuclear factor-B (NF-B)/Rel activation, which regulates various genes involved in immune and inflammatory response. In RAW 264.7 cells, the LPS-induced DNA binding activity of NF-B/Rel was significantly inhibited by silymarin, and this effect was mediated through the inhibition of the degradation of inhibitory factor-B. Silymarin also inhibited tumor necrosis factor--induced NF-B/Rel activation, whereas okadaic acid-induced NF-B/Rel activation was not affected. NF-B/Rel-dependent reporter gene expression was also suppressed by silymarin in LPS-stimulated RAW 264.7 cells. Further study showed that silymarin suppressed the production of reactive oxygen species generated by H₂O₂ in RAW 264.7 cells. Collectively, these results suggest that silymarin inhibits nitric oxide production and iNOS gene expression by inhibiting NF-B/Rel activation. Furthermore, the radical-scavenging activity of silymarin may explain its inhibitory effect on NF-B/Rel activation.