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.
Triptolide is the most active component accounting for the immunosuppressive effects of the Chinese traditional herb Tripterygium wilfordii Hook. f. (TWHF) (Gu et al., 1992a,b; Chen, 2001). However, the strong toxicity of triptolide limits its application (Huynh et al., 2000). (5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide and has immunosuppressive activities similar to that of triptolide but with greatly reduced toxicities (Zhou et al., 2005). Moreover, we have found that LLDT-8 potently suppressed bovine type II collagen-induced arthritis (CIA) in DBA/1 mice and prevented graft-versus-host disease in C57BL/6 mice (Tang et al., 2005).
Nitric oxide (NO) is a short-lived free radical and signaling molecule that mediates many physiological and pathophysiological processes, including neurotransmission and inflammation (Nathan and Xie, 1994a; Garthwaite, 1995). NO is produced by nitric-oxide synthases (NOS) that catalyze the conversion of l-arginine to NO and l-citrulline (Nathan and Xie, 1994b). When properly activated, macrophages express the inducible form of NOS (iNOS) (Nathan and Xie, 1994a). There are two important binding sites that are located in the promoter region of iNOS gene and that regulate its transcription: a NF-κB binding site (site 1) and an IRF-1-binding site (site 2). Lipopolysaccharide (LPS) activates NF-κB to bind site 1, whereas IFN-γ induces IRF-1 to bind site 2. The synergistic effect between LPS and IFN-γ is the result of cooperative interaction between site 1 and site 2 (Lowenstein et al., 1993; Xie et al., 1993, 1994; Kamijo et al., 1994).
The LPS receptor complex plays a vital role in LPS signaling. The functional integrity of the LPS receptor is composed of three proteins: CD14, Toll-like receptor 4 (TLR4), and myeloid differentiation protein-2 (MD-2). LPS activates macrophages through CD14 and the TLR4-MD-2 complex on cell surface. CD14 lacks a transmembrane domain. It binds LPS but does not participate directly in signaling. TLR4 is essential for LPS signaling. MD-2, a small protein that lacks a transmembrane domain, is associated with the extracellular domain of TLR4 and is indispensable for TLR4-dependent LPS responses in vivo (Akashi et al., 2000; Watters et al., 2002; Fujihara et al., 2003). Toll-like receptors are thought to be responsible for the observed CD14-dependent, LPS-induced extracellular signal-regulated kinase (ERK) and NF-κB activation (Watters et al., 2002).
Mitogen-activated protein kinases (MAPKs) are a highly conserved family of protein serine/threonine kinases, including ERK1/2, c-Jun NH2-terminal kinase (JNK), and p38 MAPK (Widmann et al., 1999). They participate in many LPS-induced macrophage inflammatory processes. ERK1/2, SAPK/JNK, and p38 participate in TNF production at transcription, translation, or both levels, respectively, in certain human and murine macrophages (Means et al., 2000).
The activation of MAPKs can lead to the activation of NF-κB. In the cytosol, inactive NF-κB is constitutively present as homo- or heterodimers and binds to inhibitory IκB proteins. Proinflammatory cytokines or bacterial infection can induce phosphorylation, ubiquitination, and proteasome-mediated degradation of the IκB proteins, followed by translocation of NF-κB to the nucleus, binding to relevant DNA sites on the promoter region of genes, and induction of gene transcription (Tak and Firestein, 2001). LPS is known to stimulate the degradation of one of the isoforms of IκB, IκBα and to promote the activation of NF-κB DNA binding activity (Watters et al., 2002).
Despite the beneficial roles of NO in host defense against tumor cells, viral replication, and other factors (Schmidt and Walter, 1994; MacMicking et al., 1997), overproduction of NO can be harmful to the host, leading to rheumatoid arthritis (RA) (St Clair et al., 1996), experimental allergic encephalomyelitis (Cross et al., 1994), and allograft rejection (Worrall et al., 1995). The selective inhibition of iNOS can be beneficial.
Triptolide, the parent compound of LLDT-8, inhibits LPS-induced NO production and iNOS expression in murine macrophages and Raw 264.7 cells, and this effect is through abrogating JNK activation and blockading NF-κB binding to the iNOS promoter (Kim et al., 2004; Wang et al., 2004). LLDT-8 inhibited the expression of iNOS and the transcription factors STAT1α and IRF-1 in CIA (R. Zhou, Y.-F. Yang, Y.-C. Li, W. Tang, P.-L. He, X.-Y. Li, and J.-P. Zuo, manuscript in preparation). To further elucidate the molecular mechanisms involved in immunosuppressive and antiinflammatory effects of LLDT-8, in this study, we investigated the effect of LLDT-8 on NO production and iNOS expression in macrophages. Our results demonstrated that LLDT-8 reduced the IFN-γ-induced and LPS-induced iNOS expression and NO production by suppressing IRF-1 expression and MAPK phosphorylation and NF-κB activation, respectively.
Materials and Methods
Reagents. LLDT-8 (C20H24O7; mol. wt. = 376) was synthesized from triptolide that was separated from TWHF. LLDT-8 is the white amorphous powder with 99% pure by reversed-phase high-performance liquid chromatography. Stock solution of LLDT-8 was prepared in dimethyl sulfoxide (Sigma, St. Louis, MO) and diluted with culture medium. Murine recombinant IFN-γ was purchased from BD Biosciences PharMingen (San Diego, CA). LPS, cycloheximide (CHX), sulfanilamide, and N-[naphthyl]ethylene diamine dihydrochloride were purchased from Sigma.
Mice. Male C57BL/6 mice (6–8 weeks old; 20–22 g) were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, People's Republic of China). The animals were housed under specific pathogen-free conditions. All mice were allowed to acclimatize in our facility for 1 week before any experiments were started. All experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of the Shanghai Institute of Materia Medica (Shanghai, People's Republic of China).
Cell Cultures. Mouse primary peritoneal macrophages were elicited by i.p. injection of 0.5 ml of 3% Brewer's thioglycollate medium (Difco, Detroit, MI) into male C57BL/6 mice. Three days later, adherent peritoneal exudate cells were obtained by peritoneal lavage using ice-cold phosphate-buffered saline, seeded in dishes, and collected by removing the nonadherent cells after 2-h incubation at 37°C.
Raw 264.7 cells (American Type Culture Collection, Manassas, VA), a macrophage cell line, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 100 U/ml penicillin, and 10 μg/ml streptomycin.
NO Production and Quantification. Peritoneal macrophages (1 × 105/well) and Raw 264.7 cells (5 × 104/well) were cultured in triplicate in 96-well plates and stimulated with 50 U/ml IFN-γ, 100 ng/ml LPS, or both in the absence or presence of LLDT-8 (25–800 nM) for 24 h. The production of NO was determined by assaying culture supernatant for NO–2, a stable reaction product of NO. In brief, 100 μl of culture supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-[naphthyl]ethylene diamine dihydrochloride in 2.5% H3PO4) at room temperature for 10 min. Absorbance was measured at 540 nm in a microplate reader. Nitrite concentration was calculated from a NaNO2 standard curve.
TNF-α Detection. Culture supernatants from the NO production assay in Raw 264.7 cells were collected. TNF-α concentrations were determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions.
Measurement of iNOS Enzyme Activity. Raw 264.7 cells were stimulated with 50 U/ml IFN-γ, 100 ng/ml LPS, or both for 24 h. Cells were then washed to deplete the stimuli and treated with LLDT-8 in the presence of 1 μg/ml CHX for additional 24 h. The supernatants were collected and analyzed for nitrite.
Flow Cytometry Analysis. Raw 264.7 cells were stimulated with 50 U/ml IFN-γ or 100 ng/ml LPS in the absence or presence of LLDT-8 (400 and 800 nM) for 24 h. Then, 5 × 105 cells were stained with biotin-conjugated anti-mouse CD119 monoclonal antibody plus avidin-(R)-phycoerythrin or fluorescein isothiocyanate-conjugated anti-mouse CD14 monoclonal antibody (BD Biosciences PharMingen). The expressions of CD119 and CD14 were analyzed on a FAC-SCalibur (BD Biosciences, San Jose, CA).
RT-PCR Analysis. Raw 264.7 cells were pretreated with LLDT-8 (400 and 800 nM) for 2 h before stimulation with 50 U/ml IFN-γ, 100 ng/ml LPS, or both for 3 h, and lysed using TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was isolated from each cell preparation, reverse transcribed, and polymerase chain reaction amplified using specific primers (Table 1). RT-PCR products were visualized by electrophoresis through 1% agarose gels containing ethidium bromide.
Western Immunoblotting. Raw 264.7 cells were pretreated with LLDT-8 (400 and 800 nM) for 2 h before stimulation with 50 U/ml IFN-γ or 100 ng/ml LPS. Cells were lysed in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and 0.02% bromphenol blue) and boiled for 5 min at 100°C. Aliquots (∼20 μg/lane) were electrophoresed in a 10% polyacrylamide gel and transferred to nitrocellulose transfer and immobilization membranes (Whatman Schleicher & Schuell, Dassel, Germany). The membranes were treated with 10% nonfat milk for 1 h to block nonspecific binding, rinsed, and incubated with mouse antibody against GAPDH or a panel of rabbit polyclonal antibodies against iNOS (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-ERK1/2, phospho-JNK/SAPK, and phospho-p38 (Cell Signaling Technology Inc., Beverly, MA) overnight at 4°C. The membranes were then treated with a 1:2000 dilution of horseradish peroxidase-conjugated anti-rabbit IgG for 1 h. Immune complexes were detected with a chemiluminescence substrate (Pierce Chemical, Rockford, IL) and exposed to Kodak X-ray film (Eastman Kodak, Rochester, NY).
Electrophoretic Mobility Shift Assay. Raw 264.7 cells were pretreated with LLDT-8 (400 and 800 nM) for 2 h before stimulation with 100 ng/ml or 1 μg/ml LPS for 1 h. Nuclear extracts were prepared and subjected to EMSA analysis as described previously (Xia et al., 2004). In brief, the oligonucleotide corresponding to the NF-κB motifs of the iNOS promoter was synthesized: 5′-CCAACTGGGGACTCTCCCTTTGGGAACA-3′. Oligonucleotides were annealed after boiling for 5 min. It was end-labeled with [γ-32P]ATP (GE Healthcare, Little Chalfont, Buckinghamshire, UK) by T4 polynucleotide kinase. The nuclear extracts (4 μg) were incubated with binding buffer (Promega, Madison, WI) in a total volume of 9 μl of mixture for 10 min at room temperature. The 32P-labeled oligonucleotide probes were added to each of the reaction mixtures and incubated at room temperature for 20 min. Protein-DNA complexes were separated from free DNA probes through 4% nondenaturing polyacrylamide gels. Gels were dried and analyzed by autoradiography.
Statistical Analysis. Data are expressed as mean ± S.D. of indicated experiments. Student's t test was used to determine significance between two groups where appropriate. A p value <0.05 was considered significant.
Results
Inhibition of Nitric Oxide Production by LLDT-8. We have demonstrated that in vivo treatment with LLDT-8 significantly inhibited NO production in the splenocytes of CIA mice. To further investigate the effect of LLDT-8 on NO generation, murine primary peritoneal macrophages and Raw 264.7 cells were stimulated with IFN-γ, LPS, or IFN-γ plus LPS for 24 h. The nitrite production was dramatically increased over basal levels (0.1∼2.0 μM) in stimulated cells. As shown in Fig. 1, the induction in nitrite generation by these two stimuli was significantly inhibited by LLDT-8 in a dose-dependent manner. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays. There was no significant difference in viability of cells treated with LLDT-8 compared with control (data not shown).
TNF-α is an inflammatory cytokine and can act as a cosignal for NO induction. Once activated with LPS, Raw 264.7 cells produced a large amount of TNF-α over basal levels (12.5 ± 0.2 pg/ml), and LLDT-8 significantly attenuated TNF-α production, depending on the concentrations (Fig. 2).
Effect of LLDT-8 on iNOS Activity. To assess whether LLDT-8-mediated suppression of NO production was due to the suppression of iNOS catalytic activity, Raw 264.7 cells were activated with IFN-γ, LPS, or IFN-γ plus LPS for 24 h, washed, and then CHX with or without LLDT-8 was added. In these cultures, iNOS protein was already induced after 24-h stimulation, and its further induction was blocked by addition of translation inhibitor CHX. LLDT-8 failed to affect iNOS enzymatic activity in Raw 264.7 cells, because the nitrite levels remained unchanged in cells treated with or without LLDT-8 (Fig. 3).
Effects of LLDT-8 on IFN-γ-Induced iNOS Expression. To determine whether the suppressive effects of LLDT-8 on IFN-γ-activated macrophages were due to the reduced binding of IFN-γ with its receptor, we analyzed the expression of IFN-γ receptor (IFN-γR) by flow cytometry. There were no distinct differences in the receptor expression between LLDT-8-treated and untreated cells, although the level of IFN-γR seemed lower after IFN-γ stimulation (Fig. 4A). We further investigated the effect of LLDT-8 on the transcription of iNOS mRNA and its upstream regulators STAT1α and IRF-1 by RT-PCR at 3 h after IFN-γ or IFN-γ plus LPS stimulation. Irrespective of the stimulation difference, a significantly lower level of iNOS mRNA was observed in 800 nM LLDT-8-treated Raw 264.7 cells (Fig. 4B). IRF-1 was an important transcription factor for IFN-γ-induced expression of iNOS gene in murine macrophages (Kamijo et al., 1994). IFN-γ dramatically increased the transcripts of STAT1α and IRF-1, and the increase was effectively prevented by LLDT-8 (Fig. 4B). The inhibition profile of LLDT-8 was also observed in IFN-γ plus LPS stimulated cells. Furthermore, results obtained at 6, 8, and 18 h were consistent with that at 3 h (data not shown). The inhibitory effect of LLDT-8 on STAT1α, IRF-1, and iNOS expression was also tested in murine peritoneal macrophages, and similar results were obtained as in Raw 264.7 cells (data not shown). The effect of LLDT-8 on iNOS protein expression was further examined by Western immunoblot. As shown in Fig. 4C, the expression of iNOS protein was not detected in unstimulated cells, but it markedly increased 20 h after IFN-γ stimulation. Treatment with LLDT-8 (400 and 800 nM) showed a significant inhibition of iNOS protein expression.
Effects of LLDT-8 on LPS-Induced iNOS Expression. LPS binding with CD14 and forming complex with TLR4-MD-2 is the initial step of LPS signaling (Fujihara et al., 2003). To examine the effect of LLDT-8 on the initial step, Raw 264.7 cells were stimulated with LPS to analyze the surface expression of CD14 by flow cytometry and the expression of TLR4 and MD-2 by RT-PCR. Unlike the effect on IFN-γR, LLDT-8 (400 and 800 nM) significantly reduced the expression of these three components of LPS receptor complex (Fig. 5). Treatment with LPS caused a marked increase in CD14 expression (Fig. 5A). The LPS-induced CD14 expression was greatly inhibited by LLDT-8 (100, 200, 400, and 800 nM) in a dose-dependent way (data not shown). It was interesting that LPS treatment reduced TLR4 mRNA transcripts, and this was consistent with previous reports (Medvedev et al., 2000; Yeo et al., 2003). The effect of LLDT-8 on iNOS mRNA expression was examined by RT-PCR. As shown in Fig. 5B, the iNOS transcript was barely detectable in unstimulated cells, but it was dramatically increased 3 h after LPS stimulation. LLDT-8 inhibited LPS-stimulated iNOS mRNA expression. LLDT-8 also reduced iNOS protein level in LPS-stimulated Raw 264.7 cells (Fig. 5C).
Inhibition of LPS-Induced Phosphorylation of ERK1/2, SAPK/JNK, and p38 as Well as Degradation of IκB. LPS is known to activate a series of MAPKs in macrophages. The ERK1/2, SAPK/JNK, and p38 MAPK pathways are involved in LPS-induced NO production in Raw 264.7 cells (Ajizian et al., 1999). LPS strongly and transiently induced the phosphorylation of ERK1/2, SAPK/JNK, and p38, and their levels peaked at 30 min after stimulation and declined thereafter (data not shown). We examined the effect of LLDT-8 on ERK1/2, SAPK/JNK, and p38 MAPK phosphorylation in LPS-stimulated Raw 264.7 cells using Western immunoblot analysis. Treatment with LLDT-8 (400 and 800 nM) effectively decreased the phosphorylation of these three kinases, displaying a strongest inhibition on phosphorylated-SAPK/JNK (Fig. 6). Because the nuclear translocation and DNA binding of NF-κB are preceded by the degradation of IκB, we investigated the effect of LLDT-8 on the cytoplasmic levels of IκB by Western immunoblot analysis. The IκBα protein level decreased significantly 30 min after LPS stimulation. Pretreatment of cells with LLDT-8 2 h before LPS stimulation retarded IκB degradation (Fig. 6).
Inhibition of LPS-Induced NF-κB Binding Activity by LLDT-8. NF-κB is an important transcription factor in LPS-induced iNOS gene expression. Therefore, we investigated whether the inhibitory effect of LLDT-8 on iNOS expression was due to the reduction of NF-κB activity by EMSA. Treatment of Raw 264.7 cells with LPS (100 ng/ml or 1 μg/ml) caused a significant increase in the DNA binding activity of NF-κB within 60 min compared with untreated cells (Fig. 7). And the induction of NF-κB activity was markedly ameliorated by LLDT-8 at the higher concentration (800 nM). The binding specificity was confirmed by the abrogation of NF-κB binding complexes in the presence of a 50-fold excess of unlabeled probes (data not shown).
Discussion
LLDT-8 is known to have potent immunosuppressive effects in vitro and in vivo. In the present study, we demonstrated that LLDT-8 inhibits NO production and iNOS expression in macrophages and that these effects are possibly mediated through the inhibition of IRF-1 expression, MAPK phosphorylation, and NF-κB activation.
Macrophages are the major sources of iNOS-induced NO (MacMicking et al., 1997). NO in macrophages can be induced by inflammatory cytokines or bacterial products, including IFN-γ, LPS, or TNF-α (Chiou et al., 2001). IFN-γ-primed macrophages are more sensitive to other stimuli, and the synergistic effects are observed on the production of many inflammatory factors, such as iNOS (Xie et al., 1993; Kamijo et al., 1994), which was also observed in our present study. In this study, LLDT-8 significantly reduced NO production in peritoneal macrophages and Raw 264.7 cells activated by IFN-γ, LPS, or IFN-γ plus LPS. We used CHX to block the further induction of iNOS in activated Raw 264.7 cells; therefore, only the iNOS catalytic activity was involved in the NO generation. LLDT-8 failed to affect the iNOS catalytic activity, suggesting that LLDT-8 probably interfered with the iNOS induction rather than with its enzymatic activity in macrophages.
Up-regulation of iNOS expression in response to IFN-γ requires a second signal, which can be provided by LPS or endogenous TNF-α (Liu et al., 1999). LLDT-8 significantly inhibits inflammatory cytokines production, including TNF-α and interleukin-6 (Zhou et al., 2005). In this study, LLDT-8 dramatically reduced TNF-α production in LPS-stimulated Raw 264.7 cells. This reduction would abrogate the further autocrine induction of iNOS and would contribute to the effective suppression of NO production.
iNOS activity is regulated at various levels, such as transcription and translation, and transcription seems to be the primary regulatory site (Xie et al., 1993; Nathan and Xie, 1994b). IFN-γ primes macrophages through the activation of the latent transcription factor STAT1. STAT1α is the main transcription factor involved in IFN-γ-induced NO generation by macrophages with direct or indirect effects on iNOS expression. STAT1α binds to γ-interferon-activated sites located in the murine iNOS promoter to initiate transcription. And it can also increase iNOS activity by inducing gene expression of IRF-1 (Gao et al., 1997; Ohmori et al., 1997). LLDT-8 displayed no effect on IFN-γ binding. It inhibited IRF-1 mRNA transcription and its upstream regulator STAT1 in IFN-γ-stimulated Raw 264.7 cells. The inhibition of IRF-1 expression probably led to the decrease of IRF-1 binding to the promoter region of iNOS and finally reduced iNOS expression and NO production.
In the LPS signaling, LPS first forms a complex with CD14-TLR4-MD-2. This initial formation of complex is a limited step for intracellular signaling cascades. LLDT-8 potently abrogated the LPS signaling by reducing the expression of binding receptors, including CD14, TLR4, and MD-2. The formation of LPS with CD14-TLR4-MD-2 complex triggers the activation of both MAPKs and NF-κB (Beutler, 2000). The MAPKs are critical regulator for cell growth, differentiation, and control of cellular responses to cytokines and stresses, and they are involved in the signaling pathway for LPS-induced iNOS expression, in which ERK1/2, SAPK/JNK, and p38 contribute greatly (Caivano, 1998; Johnson and Lapadat, 2002). Treatment with LLDT-8 caused a significant decrease of SAPK/JNK phosphorylation, and partial reduction of ERK1/2 and p38 phosphorylation. Triptolide inhibits SAPK/JNK phosphorylation but not ERK1/2 and p38 phosphorylation (Kim et al., 2004). These results suggest that SAPK/JNK was probably the target for extracts from TWHF in the MAPK pathway.
After LPS stimulation, the NF-κB binding activity for iNOS is elevated. Because the MAPK pathway play an important role in the activation of NF-κB, LLDT-8 effectively prevented NF-κB from activation, at least partially via inhibiting phosphorylation of MAPKs and by blocking the degradation of IκBα. Moreover, the inhibition of MAPK phosphorylation and NF-κB activation by LLDT-8 might cause the reduction of TNF-α. Collectively, the inhibitory effect of LLDT-8 on NF-κB activation contributed to the suppression of NO production in LPS signaling.
In addition to the in vitro studies, we have demonstrated the inhibitory effects of LLDT-8 on CIA and graft-versus-host disease animal diseases. In CIA mice, in vivo treatment with LLDT-8 reduced iNOS gene expression and NO production, which contributed to the suppression of onset and development of arthritis. NO has been implicated in inflammation, autoimmunity, and arthritis. Excessive production of NO generated by iNOS has been described in patients with RA (Stefanovic-Racic et al., 1993), and its concentration was related to disease activity. Inhibition of NOS has beneficial effects in acute and chronic joint inflammation, because experimental arthritis was found suppressed after the nonselective inhibition of NOS (Stefanovic-Racic et al., 1994). Thus, selective inhibition of the pathologically enhanced NO synthesis may be a useful therapeutic approach in the treatment of various inflammatory diseases. LLDT-8 inhibited iNOS expression both in vitro and in vivo, suggesting it as a potential agent for the clinical treatment of patients with RA and other inflammatory diseases.
In summary, this study demonstrates that LLDT-8 significantly inhibits NO production and that this inhibitory effect occurs through reduction of iNOS at the mRNA and protein levels, rather than by interfering with its enzymatic activity in macrophages. And two signaling pathways are involved in the regulation of iNOS transcription: LLDT-8 inhibits IFN-γ-induced STAT1α and IRF-1 expression, and LLDT-8 abrogates LPS-induced cell surface binding and retards MAPKs activation and NF-κB binding to the iNOS promoter. Because NF-κB plays a critical role in the pathogenesis of many inflammatory diseases (Yamamoto and Gaynor, 2001), the mechanism involved in the suppression of NO production by LLDT-8 further suggests the therapeutic potential of LLDT-8 in inflammatory and/or autoimmune diseases.
Footnotes
-
This work was supported by Grant KSCX2-SW-202 from the Knowledge Innovation Program of Chinese Academy of Sciences.
-
doi:10.1124/jpet.105.093179.
-
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