NF-kappaB signaling pathway, not IFN-beta/STAT1, is responsible for the selenium suppression of LPS-induced nitric oxide production

Upon stimulation of macrophages with lipopolysaccharide (LPS), Toll-like receptor 4 recognizes LPS, leading to expression of inducible nitric oxide synthase (iNOS), via MyD88/NF-kappaB and TRIF/IFN-beta/STAT pathways. Although selenium (Se) was reported to inhibit nitric oxide (NO) production, it is unclear which signaling pathway is inhibited by Se. Here, we investigated how Se inhibits NO production in LPS-stimulated RAW 264.7 cells. When the cells were pretreated with Se for 1 h followed by LPS treatment, iNOS mRNA expression and subsequent NO production declined significantly in a dose-dependent manner. Se inhibited IkappaBalpha degradation in the cytosol and NF-kappaB binding to its recognition site in the nucleus of the LPS-stimulated cells. Meanwhile, Se did not inhibit IFN-beta mRNA induction or STAT1 phosphorylation in the LPS-stimulated cells. These results suggest that Se down-regulates iNOS gene expression and NO production in the LPS-stimulated macrophages through inhibition of the NF-kappaB activation pathway but not the IFN-beta/STAT1 signaling pathway.     

Inhibition of inducible nitric oxide synthase by beta-lapachone in rat alveolar macrophages and aorta

Beta-lapachone, a plant product, has been shown to be a novel inhibitor of DNA topoisomerase. In this study, we performed experiments to examine the effects of beta-lapachone on lipopolysaccharide (LPS)-induced inducible nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages and aortic rings. In alveolar macrophages, incubation with LPS (10 microg ml(-1)) for various time intervals resulted in a significant increase in nitrite production and iNOS protein synthesis, that was inhibited by coincubation with beta-lapachone (1-4.5 microM) without any cytotoxic effects. However, addition of beta-lapachone after induction of NO synthase by LPS failed to affect the nitrite production. Treatment with LPS (10 microg ml(-1)) for 6 h resulted in significant expression of mRNA for iNOS which was significantly inhibited in the presence of beta-lapachone (3 microM) in alveolar macrophages. In endothelium-intact rings of thoracic aorta, beta-lapachone (1 and 3 microM) markedly inhibited the hypocontractility to phenylephrine in aortic rings treated with LPS (10 microg ml(-1)) for 4 h. When beta-lapachone was added 3 h after LPS into the medium, the contractions evoked by phenylephrine were not significantly different in the presence or absence of beta-lapachone. Treatment with LPS (10 microg ml(-1)) for 4 h resulted in a significant increase in iNOS protein synthesis which was inhibited in the presence of beta-lapachone (3 microM), but did not affect the constitutive (endothelial and neuronal) NOS forms in aortic rings. These results indicate that beta-lapachone is capable of inhibiting expression and function of iNOS in rat alveolar macrophages and aortic rings. It is considered that beta-lapachone can be developed as a potential anti-inflammatory agent in the future.     

Inhibitory effect of trimidox on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages

We examined the effect of trimidox (3,4,5-trihydroxybenzamidoxime) on the production of nitric oxide (NO) by lipopolysaccharide (LPS) in mouse RAW 264.7 macrophages. Trimidox (50 - 300 microM) concentration-dependently inhibited NO production by LPS (0.01, 0.1, or 1 microg/ml) after incubation for 24 h. LPS-induced expression of inducible NO synthase (iNOS) and degradation of IkappaBalpha were prevented by trimidox. The protective effect against NO production by LPS was not only observed in prior incubation but also later incubation with trimidox until iNOS was activated by LPS. These results suggest that trimidox has a predominantly protective effect against LPS-induced production of NO via iNOS expression.     

Enhancement of nitric oxide synthase induction in alveolar macrophages by in vivo administration of docetaxel

Effects of docetaxel, a taxane-derivative anti-cancer drug, on lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were investigated in alveolar macrophages isolated from rats. LPS-induced NO production and inducible NO synthase (iNOS) expression were significantly enhanced in the macrophages isolated from rats injected intraperitoneally with docetaxel (4 mg/kg body weight per day for 5 consecutive days) compared with those in macrophages from control rats administrated a vehicle. In vivo administration of docetaxel augmented LPS-induced p38 activation but not extracellular signal-related kinase (ERK) activation in isolated macrophages. On the other hand, in vitro treatment of macrophages with docetaxel (5 and 10 μg/ml) inhibited LPS-induced NO production and iNOS expression. Levels of lactate dehydrogenase released from macrophages were neither affected by in vitro treatment with docetaxel (up to 10 μg/ml) nor by its in vivo administration. These results suggest that docetaxel has an enhancing action in vivo on LPS-induced iNOS expression and subsequent NO production through stimulation of p38 activity in alveolar macrophages.     

CO from enhanced HO activity or from CORM-2 inhibits both O2- and NO production and downregulates HO-1 expression in LPS-stimulated macrophages

Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O(2)(-)) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50microM [Ru(CO)(3)Cl(2)](2), a CO-releasing molecule (CORM-2), was abolished and the production of O(2)(-) by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50microM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O(2)(-) and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.     

Inhibitory effects of ivermectin on nitric oxide and prostaglandin E2 production in LPS-stimulated RAW 264.7 macrophages

We have previously shown that ivermectin inhibits LPS-induced production of inflammatory cytokines. In the present study, we investigated the effect of ivermectin on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages. Ivermectin inhibited LPS-induced NO and PGE₂ production. Consistent with these observations, the protein and mRNA expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes were inhibited by ivermectin in a concentration-dependent manner. Furthermore, the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW 264.7 cells was suppressed by ivermectin in a dose-dependent manner. These results suggest that ivermectin suppresses NO and PGE₂ production, as well as iNOS and COX-2 expression, by inhibiting phosphorylation of mitogen-activated protein kinases (MAPK) (p38, ERK1/2, and JNK) in LPS-stimulated RAW 264.7 cells.     

Increased induction of inducible nitric oxide synthase expression in aortae of type 2 diabetes rats

The aim of this study was to determine whether the pathway of inducible NO synthase (iNOS) in blood vessels is changed by type 2 diabetes. Lipopolysaccharide (LPS)-induced nitric oxide (NO) production and expression of iNOS and effects of LPS on phenylephrine-induced contractile force were compared in aortae isolated from Goto-Kakizaki (G-K) diabetes rats and aortae isolated from control Wistar rats. Both LPS-stimulated nitrite generation and iNOS expression levels were significantly higher in aortae from G-K rats than in those from control rats. Phenylephrine-induced contractile force in the presence of LPS was significantly lower in aortae from G-K rats than in those from control rats, while contractile force in the absence of LPS was comparable in the diabetic and control groups. On the other hand, incubation of aortae in high glucose-containing medium did not affect the LPS-stimulated nitrite accumulation and iNOS expression and the phenylephrine-induced contractile force, regardless of the presence of LPS. These results suggest that LPS-induced NO production through the iNOS pathway is increased and subsequent attenuation of contractile force by excess NO is enhanced in arteries of rats with type 2 diabetes.     

Inhibition of lipopolysaccharide-stimulated NO production by a novel synthetic compound CYL-4d in RAW 264.7 macrophages involving the blockade of MEK4/JNK/AP-1 pathway

In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, NF-kappaB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-kappa B alpha (I kappa B alpha) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH(2)-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.     

YL-I-108, a synthetic chalcone derivative,inhibits lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 murine macrophages: Involvement of heme oxygenase-1 induction and blockade of activator protein-1

Chalcones, a group of phenolic compounds, exhibit potent anti-inflammatory properties. In the present study, we synthesized chalcone derivative, YL-I-108 ((E)-1-(2-methoxy-4,6-bis(methoxymethoxy)phenyl)-3-(3-nitrophenyl)prop-2-en-1-one), and examined its effect on the production of pro-inflammatory mediators. Treatment of RAW 264.7 macrophages with YL-I-108 potently inhibited nitrite production stimulated by LPS. YL-I-108 treatment also markedly inhibited expressions of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α). Treatment of cells with YL-I-108 significantly inhibited LPS-stimulated activator protein-1 (AP-1)-dependent reporter gene expression, whereas nuclear factor-κB (NF-κB) activity was not affected, indicating that down-regulation of iNOS expression by YL-I-108 is attributed by blockade of AP-1. In addition, YL-I-108 treatment led to an increase in heme oxygenase-1 (HO-1) mRNA and protein expression, accompanied with the increased expression of nuclear factor-erythroid 2-related factor 2 (Nrf2). Treatment with SnPP, a selective HO-1 inhibitor, reversed YL-I-108-mediated suppression of nitrite production, suggesting that HO-1 induction is implicated in the suppression of NO production by YL-I-108. In contrast, SnPP treatment did not reverse YL-I-108-mediated suppression of AP-1 activation, suggesting that AP-1 inhibition by YL-I-108 is independent of HO-1 induction. Together, these results indicate that YL-I-108 suppresses NO production in LPS-stimulated macrophages via simultaneous induction of HO-1 expression and blockade of AP-1 activation.     

Inhibitory effects of aclarubicin on nitric oxide production in aortic smooth muscle cells and macrophages

The effects of aclaruhicin (ACR), an anthracycline antibiotic, on inducible nitric oxide (NO) synthesis was investigated in rat aortic smooth muscle cells (RASMCs) and RAW macrophages. ACR at concentrations as low as 0.1 microM significantly inhibited NO production induced by interleukin-1beta in RASMCs. About 5- to 10-fold higher concentrations of ACR were required for inhibition of interferon-gamma and lipopolipopolysaccharide-induced NO production in RAW cells. When ACR was subsequently administered to inducible NO synthase (iNOS) induction, the NO production was barely suppressed in RASMCs. Moreover, ACR (up to 10 microM) lacked direct inhibitory effects on iNOS activity in homogenates of these cells. ACR (0.1 microM) inhibited the expression of iNOS protein and mRNA in RASMCs without concomitant cytotoxic effects. ACR (>0.5 microM)-induced inhibition of NO production in RAW cells was associated with substantial cytotoxic effects as shown by measurement of lactate dehydrogenase release. These results suggest that ACR is a potent inhibitor of iNOS induction in vascular smooth muscle, but inhibits iNOS induction in macrophage only at high cytotoxic     

Serum albumin induces iNOS expression and NO production in RAW 267.4 macrophages

1. We investigated the effects of serum albumin on inducible nitric oxide synthase (iNOS) expression in RAW 267.4 macrophages. Crude fraction-V type albumin as well as bovine serum albumin filtrated for endotoxin induced concentration-dependent iNOS expression in macrophages. Accordingly, NO production (estimated by supernatant nitrite) was markedly (up to 10-fold) increased in the presence of albumin. 2. Albumin-induced expression of iNOS protein was inhibited by cycloheximide and NO production was abolished after incubation of the cells with an iNOS inhibitor, N(G)-monomethyl-l-arginine (LNMMA). 3. An inhibitor of the NF-kappaB pathway, pyrrolidine dithiocarbamate (PDTC), as well as inhibitors of JAK2/STAT and ERK, AG490 and U0126, respectively, significantly reduced albumin-induced iNOS expression and NO production, while an inhibitor of the p38 pathway, SB203580, did not significantly affect NO production induced by albumin. 4. Both types of serum albumin were contaminated with traces of endotoxin. The endotoxin levels were found not to be sufficient for the observed induction of nitrite production in RAW 267.4 cells. In addition, the albumin-stimulated induction of iNOS was not reduced by preincubation of albumin-containing media with polymyxin B, a LPS inhibitor. 5. Polymerised albumin fractions were detected in the commercially available albumin tested in this study. A monomeric albumin-rich fraction, separated by ultrafiltration, showed a potent inducing effect on iNOS expression and NO production, while a polymer-rich fraction showed a smaller effect. 6. Advanced glycation endproducts (AGE) of albumin were not formed by interaction with glucose in incubation medium, as AGE was not increased even after long-time (4 weeks) incubation in albumin-containing media [3.2-4.4 microg ml(-1) (basal) vs 4.8-5.6 microg ml(-1) (in glucose-containing media)]. However, the duration of albumin exposure to glucose influenced the basal stimulatory properties of albumin. 7. Our results suggest that serum albumin fractions, as gained by cold alcoholic extraction, may include determinants that stimulate or further enhance stimulation of RAW 267.4 cells and are different from endotoxin, polymeric albumin and AGE.     

Inhibitory mechanisms of YC-1 and PMC in the induction of iNOS expression by lipoteichoic acid in RAW 264.7 macrophages

In the present study, the signal pathways involved in NO formation and iNOS expression in RAW 264.7 macrophages stimulated by LTA were investigated. We also compared the relative inhibitory activities and mechanisms of PMC, a novel potent antioxidant of alpha-tocopherol derivatives, with those of YC-1, an sGC activator, on the induction of iNOS expression by LTA in cultured macrophages in vitro and LTA-induced hypotension in vivo. LTA induced concentration (0.1-50 microg/mL)- and time (4-24 hr)-dependent increases in nitrite (an indicator of NO biosynthesis) in macrophages. Both PMC (50 microM) and YC-1 (10 microM) inhibited NO production, iNOS protein, mRNA expression, and IkappaBalpha degradation upon stimulation by LTA (20 microg/mL) in macrophages. On the other hand, PMC (50 microM) almost completely suppressed JNK/SAPK activation, whereas YC-1 (10 microM) only partially inhibited its activation in LTA-stimulated macrophages. Moreover, PMC (10 mg/kg, i.v.) and YC-1 (5 mg/kg, i.v.) significantly inhibited the fall in MAP stimulated by LTA (10 mg/kg, i.v.) in rats. In conclusion, we demonstrate that YC-1 shows more-potent activity than PMC at abrogating the expression of iNOS in macrophages in vitro and reversing delayed hypotension in rats with endotoxic shock stimulated by LTA. The inhibitory mechanisms of PMC may be due to its antioxidative properties, with a resulting influence on JNK/SAPK and NF-kappaB activations. YC-1 may be mediated by increasing cyclic GMP, followed by, at least partly, inhibition of JNK/SAPK and NF-kappaB activations, thereby leading to inhibition of iNOS expression.     

Inhibitory effects of nardostachin on nitric oxide, prostaglandin E2, and tumor necrosis factor-alpha production in lipopolysaccharide activated macrophages

Nardostachin, which is an iridoid isolated from Patrinia saniculaefolia, was examined by assessing its effect on the production of tumor necrosis factor-alpha (TNF-alpha) and expression of 2 enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated Raw264.7 macrophages. This compound consistently inhibited the production of nitric oxide (NO) and TNF-alpha production in a dose-dependent manner, with respective IC(50) values of 12.3 and 16.2 microM. The decrease in quantity of NO products was accompanied by a decrease in the iNOS protein level, as assessed by Western blotting probed with specific anti-iNOS antibodies. In addition, this compound also reduced the COX-2 protein expression level and the attendant PGE(2) production in LPS-stimulated macrophages. These results suggest that nardostachin may be useful for inhibiting the production of inflammatory mediators such as TNF-alpha, NO and PGE(2) in inflammatory diseases.     

8-Prenylkaempferol suppresses inducible nitric oxide synthase expression through interfering with JNK-mediated AP-1 pathway in murine macrophages

8-Prenylkaempferol is a prenylflavonoid isolated from the roots of Sophora flavescens, a Chinese herb with anti-inflammatory properties. However whether 8-prenylkaempferol itself displayed an anti-inflammatory activity remained unclear. In this study, we evaluated the effect of 8-prenylkaempferol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. 8-Prenylkaempferol inhibited significantly LPS-induced NO production through suppressing inducible NO synthase (iNOS) expression at both protein and mRNA levels but failed to affect sodium nitroprusside-triggered NO production, iNOS enzyme activity, and cell viability. Further investigation of the mechanisms revealed that 8-prenylkaempferol inhibited LPS-induced c-Jun phosphorylation (a major component of activator protein-1, AP-1), but did not attenuate IkB-alpha degradation nor NF-kappaB nuclear translocation. Cellular signaling analysis using mitogen-activating protein kinase (MAPK) inhibitors including 2'-amino-3'-methoxyflavone (PD98059, MEK1/2 inhibitor), 4-[5-(4-fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580, p38 kinase inhibitor) and anthra[1-9-cd]pyrazol-6(2H)-one (SP600125, c-Jun N-terminal kinase inhibitor) demonstrated that extracellular signal-regulated kinase1/2 (ERK1/2), p38 and JNK all participated in LPS-stimulated iNOS expression and NO production, but 8-prenylkaempferol interfered selectively with JNK phosphorylation. On the other hand, LPS-induced c-Jun phosphorylation was attenuated in the presence of SP600125. We suggested that interfering with JNK-mediated c-Jun phosphorylation and thus blocking AP-1 activation might contribute to the suppression effects of 8-prenylkaempferol on iNOS. These findings provided the first molecular basis that 8-prenylkaempferol is an effective agent for attenuating pro-inflammatory NO induction.     

Potent inhibition of lipopolysaccharide-inducible nitric oxide synthase expression by dibenzylbutyrolactone lignans through inhibition of I-kappaBalpha phosphorylation and of p65 nuclear translocation in macrophages.

AIMS: Arctigenin and demethyltraxillagenin, dibenzylbutyrolactone lignans, are phenylpropanoid metabolites with antioxidant and anti-inflammatory activities. The effects of arctigenin and demethyltraxillagenin on the nuclear factor-kappaB (NF-kappaB)-mediated inducible nitric oxide synthase (iNOS, EC1.14.13.39) gene expression were studied in Raw264.7 cells. METHODS: Activation of NF-kappaB was determined by gel mobility shift assay, immunocytochemistry and immunoblot analysis of I-kappaBalpha. Expression of the iNOS gene was assessed by Northern and Western blot analyses. NO production was monitored by chemiluminescent detection using a nitric oxide analyzer. RESULTS: Arctigenin (1 microM) inhibited lipopolysaccharide (LPS)-inducible nuclear NF-kappaB activation and nuclear translocation of p65, which was accompanied by inhibition of I-kappaBalpha phosphorylation, whereas demethyltraxillagenin was less active. LPS-inducible increase in the iNOS mRNA was 80-90% inhibited by 0.01-1 microM arctigenin, whereas similar extents of inhibition were noted by 50-100 microM demethyltraxillagenin. Immunoblot analysis revealed that arctigenin potently inhibited the induction of iNOS by LPS (IC50 < 0.01 microM). The IC50 value of demethyltraxillagenin was approximately 50 microM. Production of nitrite and nitrate by LPS in culture medium was also comparably suppressed by the lignans. CONCLUSION: These results demonstrated that arctigenin potently inhibited LPS-inducible iNOS expression in murine macrophages through suppression of I-kappaBalpha phosphorylation and nuclear translocation of p65. Potent inhibition of LPS-inducible NO production in macrophages may constitute anti-inflammatory effects of the dibenzylbutyrolactone lignans.