Received for publication July 26, 2005.
Revised August 24, 2005.
Accepted for publication August 25, 2005.

Inhibition of Inducible Nitric-Oxide Synthase Expression by (5R)-5-hydroxytriptolide (LLDT-8) in Interferon- and Bacterial Lipopolysaccharide Stimulated Macrophages

Abstract

(5R)-5-hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has anti-arthritic, hepatoprotective, and anti-allogenic transplantation rejective effects. In the present study, we report that LLDT-8 inhibited nitric oxide (NO) production and inducible nitric-oxide synthase (iNOS) expression in macrophages. LLDT-8 significantly attenuated NO production, in a dose-dependent manner, in primary peritoneal macrophages and a macrophage cell line of Raw 264.7 cells following stimulation with IFN-, LPS, and IFN- plus LPS. It also reduced the production of TNF- from LPS-stimulated Raw 264.7 cells. To further elucidate the mechanism responsible for the inhibition of NO, we examined the effect of LLDT-8 on IFN- and LPS-induced iNOS expression. Indeed, LLDT-8 prevented NO generation by inhibiting iNOS expression at mRNA level and protein level, rather than interfering its enzymatic activity. In IFN--stimulated Raw 264.7 cells, LLDT-8 suppressed the gene transcription of STAT1 and IRF-1 while displaying no apparent effect on IFN- receptor level on cell surface. Following LPS challenge, LLDT-8 further abrogated the expression of LPS receptor complex including CD14, TLR4 and MD-2, decreased the LPS-induced phosphorylation of SAPK/JNK, Erk1/2 and p38 mitogen-activated protein kinase (MAPK), retarded the degradation of IB and ameliorated the DNA binding activity of NF-B to nuclear proteins that accounts for transcriptional regulation of iNOS. Taken together, these results suggest that LLDT-8 reduces NO production and iNOS expression by inhibiting IFN--triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-B activation.

Key words: (5R)-5-hydroxytriptolide, Autoimmune disease, Immunosuppressive effect, Inducible nitric-oxide synthase, Nitric oxide, Triptolide

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