Abstract
(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)-γ, lipopolysaccharide (LPS), and IFN-γ plus LPS. It also reduced the production of tumor necrosis factor-α 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-γ 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-γ-stimulated Raw 264.7 cells, LLDT-8 suppressed the gene transcription of signal transducer and activator of transcription 1α and interferon regulatory factor (IRF)-1, but it displayed no apparent effect on IFN-γ 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 NH2-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IκBα; and ameliorated the DNA binding activity of nuclear factor-κB (NF-κB) 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-γ-triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-κB activation.
Footnotes
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This work was supported by Grant KSCX2-SW-202 from the Knowledge Innovation Program of Chinese Academy of Sciences.
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doi:10.1124/jpet.105.093179.
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ABBREVIATIONS: TWHF, Tripterygium wilfordii Hook. f.; LLDT-8, (5R)-5-hydroxytriptolide; CIA, collagen-induced arthritis; NOS, nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; NF-κB, nuclear factor-κB; IRF, interferon regulatory factor; LPS, lipopolysaccharide; CHX, cycloheximide; ERK, extracellular signal-regulated kinase; IFN, interferon; TLR4, Toll-like receptor 4; MD-2, myeloid differentiation protein-2; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; p38, p38 mitogen-activated protein kinase; SAPK, stress-activated protein kinase; TNF, tumor necrosis factor; IκB, inhibitory factor-κB; RA, rheumatoid arthritis; IFN-γR, interferon-γ receptor; STAT, signal transducer and activator of transcription; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
- Received July 23, 2005.
- Accepted August 25, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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