Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages.
(5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has antiarthritic, hepatoprotective, and antiallogenic 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 interferon (IFN)-gamma, lipopolysaccharide (LPS), and IFN-gamma plus LPS. It also reduced the production of tumor necrosis factor-alpha 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-gamma and LPS-induced iNOS expression. Indeed, LLDT-8 prevented NO generation by inhibiting iNOS expression at mRNA level and protein level, rather than by interfering its enzymatic activity. In IFN-gamma-stimulated Raw 264.7 cells, LLDT-8 suppressed the gene transcription of signal transducer and activator of transcription 1alpha and interferon regulatory factor (IRF)-1, but it displayed no apparent effect on IFN-gamma receptor level on cell surface. After LPS challenge, LLDT-8 further abrogated the expression of LPS receptor complex, including CD14, Toll-like receptor 4, and myeloid differentiation protein-2; decreased the LPS-induced phosphorylation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IkappaBalpha; and ameliorated the DNA binding activity of nuclear factor-kappaB (NF-kappaB) 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-gamma-triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-kappaB activation.
Zhou
Ru
R
Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Rd., Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China.
Zheng
Shen-Xi
SX
Tang
Wei
W
He
Pei-Lan
PL
Li
Xiao-Yu
XY
Yang
Yi-Fu
YF
Li
Yuan-Chao
YC
Geng
Jian-Guo
JG
Zuo
Jian-Ping
JP
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004224:Diterpenes / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007371:Interferon-gamma / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000096:biosynthesis*; D010766:Phosphorylation; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000096:biosynthesis; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C509074:5-hydroxytriptolide; D004224:Diterpenes; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases