Inhibition of inducible nitric-oxide synthase expression by silymarin in lipopolysaccharide-stimulated macrophages

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-κB (NF-κB)/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-κB/Rel was significantly inhibited by silymarin, and this effect was mediated through the inhibition of the degradation of inhibitory factor-κB. Silymarin also inhibited tumor necrosis factor-α-induced NF κB/Rel activation, whereas okadaic acid-induced NF-κB/Rel activation was not affected. NF-κB/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-κB/Rel activation. Furthermore, the radical-scavenging activity of silymarin may explain its inhibitory effect on NF-κB/Rel activation.