LPS-induced Nitric Oxide Synthesis Research Articles

Anti-inflammatory effects of the Thai herbal remedy Yataprasen and biflavonoids isolated from Putranjiva roxburghii in RAW264.7 macrophages

Ethnopharmacological relevanceYataprasen is a topical Thai herbal remedy for the treatment of musculoskeletal pain and is included in Kumpe Thart Phra Narai, the first Thai textbook of traditional medicine. The herbal preparation is made from a hydroethanolic extract of a mixture of 13 medicinal plants, of which Putranjiva roxburghii Wall. leaves are the major ingredient. Aim of the studyIn this study, we investigated the underlying mechanism of action for the anti-inflammatory effects of the Yataprasen remedy, its main ingredients, and the phytochemicals isolated from P. roxburghii leaves. Materials and methodsThe anti-inflammatory effects of the Yataprasen remedy, along with its main ingredients, including the leaves of Baliospermum solanifolium (Burm.) Suresh, Melia azedarach L., P. roxburghii, Senna siamea (Lam.) Irwin & Barneby, and Tamarindus indica L. were determined by measuring prostaglandin E2 (PGE2) secretion, nitric oxide (NO) production, and the synthesis of inflammatory biomarkers in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. The active ingredients of the P. roxburghii leaves were separated by chromatography and spectroscopic measurements were used to identify their chemical structures. ResultsEthanol extracts of the Yataprasen remedy and some of its ingredients significantly suppressed LPS-induced PGE2 secretion and NO production in a dose-dependent manner. Treatment of RAW264.7 cells with ethanolic extracts of the Yataprasen remedy (50 μg/mL) significantly inhibited LPS-induced mRNA expression of TNF-α, COX-2, iNOS, and NF-κB. Among the plant ingredient extracts, P. roxburghii leaf extract exhibited the highest inhibitory effects on LPS-induced TNF-α and iNOS expression. Moreover, T. indica leaf extract showed the highest activity on the inhibition of LPS-induced COX-2 and NF-κB expression. Putraflavone, podocarpusflavone A, and amentoflavone were isolated biflavonoids from P. roxburghii leaf extract and showed the inhibitory effects on LPS-induced PGE2 secretion and NO synthesis in RAW264.7 cells. Of the isolated biflavonoids, amentoflavone exhibited the strongest anti-inflammatory activity by inhibiting the expression of TNF-α, COX-2, and iNOS. ConclusionThe results support reported the anti-inflammatory effects of the Yataprasen remedy, which are associated with the downregulation of proinflammatory mediators. P. roxburghii, along with its biflavonoids, are the impact components that contribute to the anti-inflammatory effects of the herbal remedy.

N-Acetylcysteine Suppresses Microglial Inflammation and Induces Mortality Dose-Dependently via Tumor Necrosis Factor-α Signaling.

N-acetylcysteine (NAC) is an antioxidant that prevents tumor necrosis factor (TNF)-α-induced cell death, but it also acts as a pro-oxidant, promoting reactive oxygen species independent apoptosis. Although there is plausible preclinical evidence for the use of NAC in the treatment of psychiatric disorders, deleterious side effects are still of concern. Microglia, key innate immune cells in the brain, play an important role in inflammation in psychiatric disorders. This study aimed to investigate the beneficial and deleterious effects of NAC on microglia and stress-induced behavior abnormalities in mice, and its association with microglial TNF-α and nitric oxide (NO) production. The microglial cell line MG6 was stimulated by Escherichia coli lipopolysaccharide (LPS) using NAC at varying concentrations for 24 h. NAC inhibited LPS-induced TNF-α and NO synthesis, whereas high concentrations (≥30 mM) caused MG6 mortality. Intraperitoneal injections of NAC did not ameliorate stress-induced behavioral abnormalities in mice, but high-doses induced microglial mortality. Furthermore, NAC-induced mortality was alleviated in microglial TNF-α-deficient mice and human primary M2 microglia. Our findings provide ample evidence for the use of NAC as a modulating agent of inflammation in the brain. The risk of side effects from NAC on TNF-α remains unclear and merits further mechanistic investigations.

Stimulation of murine immune response by Clerodendrum infortunatum

Background: Clerodendrum infortunatum is a medicinal plant found especially in the Sub-Himalayan regions of West Bengal. Various tribal communities of this region have been using these plants as a source of natural medicine. Objective: Medicinal values of C. infortunatum are well established, but the immunomodulatory properties have never been studied. Therefore, we wanted to investigate various immunomodulatory activities of this plant. Materials and Methods: Several parameters such as plaque-forming cell assay, hemagglutination titer, phagocytic activity of macrophages, and inhibition of lipopolysaccharide (LPS) were performed. Moreover, effects of C. infortunatum on the weight of various organs and total serum protein, albumin, and globulin levels were also determined. The phytochemical fingerprints of C. infortunatum extracts were obtained from Fourier transform infrared and gas chromatography-mass spectrometry analysis. Results: The macrophage stimulation and plaque-forming cell numbers (using sheep red blood cell as antigen) were significantly proliferated and found optimum at 100 and 250 mg/kg doses of leaf and root extracts simultaneously. The extent of stimulation in mural humoral immunity was in the order of leaf > root > stem. The anti-inflammatory activity of C. infortunatum was confirmed by the inhibition of LPS-induced nitric oxide synthesis by macrophages. Conclusion: It can be concluded that 70% hydromethanolic extract of C. infortunatum has profound immunomodulation potentials in the murine model, stimulating both humoral and innate form of the immune system. Abbreviations used: PFC: Plaque-forming cell assay; HA titer: Hemagglutination titer; LPS: Lipopolysaccharide; FTIR: Fourier transform infrared; GC-MS: Gas chromatography-mass spectrometry; SDS: Sodium dodecyl sulfate; sRBC: Sheep red blood cell; PBS: Phosphate buffer saline; PC: Phagocytic capacity; PI: Phagocytic index; NaCl: Sodium chloride; CaCl2: Calcium chloride; NBT: Nitro blue tetrazolium; DMSO: Dimethyl sulfoxide; KOH: Potassium hydroxide; H2SO4: Sulfuric acid; HOCl: Hypochlorous acid; H2O2: Hydrogen peroxide; EDTA: Ethylenediaminetetraacetic acid.

Lysophosphatidylcholine Triggers TLR2- and TLR4-Mediated Signaling Pathways but Counteracts LPS-Induced NO Synthesis in Peritoneal Macrophages by Inhibiting NF-κB Translocation and MAPK/ERK Phosphorylation

BackgroundLysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway.Methodology/Principal FindingsHEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells.Conclusions/SignificanceThe above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression.

Anti-inflammatory and immune-modulating effect of Ulmus davidiana var. japonica Nakai extract on a macrophage cell line and immune cells in the mouse small intestine

Ethnopharmacological relevanceTraditional Oriental medicine has utilized the barks of the stem and root of Ulmus davidiana var. japonica Nakai (UD) to treat inflammatory disorders. Aim of the studyThe purpose of the present study was to evaluate UD's anti-inflammatory and immune-modulating effects on a lipopolysaccharide (LPS)-stimulated macrophage cell line and small-intestinal lamina propria (LP) cells, respectively. Materials and methodsRAW 264.7 cells were stimulated with LPS in the presence of various concentrations of a UD water-soluble extract. Cell viability, nitric oxide (NO) production, and the level of inflammatory cytokines synthesis were measured. Among the mice receiving the UD water-soluble extract, changes in the LP cell populations and immunoglobulin (Ig)A production were evaluated. ResultsThe UD water-soluble extract inhibited LPS-induced NO synthesis and inflammatory cytokine production in a RAW264.7 macrophage-like cell line. Small-intestinal LP cells isolated from mice that received the UD extract displayed a decrease in the side scatter of medium-to-high cells. Those LP cells isolated from the UD-treated mice also showed a marked decrease of intracellular IgA. However, UD administration had no apparent effect on the synthesis of systemic inflammatory cytokines. ConclusionsThese results suggest that UD water-soluble extracts have anti-inflammatory properties and, as such, can be used to promote intestinal immune-homeostatic conditions.

Nitric Oxide-Mediated Maintenance of Redox Homeostasis Contributes to NPR1-Dependent Plant Innate Immunity Triggered by Lipopolysaccharides

The perception of lipopolysaccharides (LPS) by plant cells can lead to nitric oxide (NO) production and defense gene induction. However, the signaling cascades underlying these cellular responses have not yet been resolved. This work investigated the biosynthetic origin of NO and the role of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) to gain insight into the mechanism involved in LPS-induced resistance of Arabidopsis (Arabidopsis thaliana). Analysis of inhibitors and mutants showed that LPS-induced NO synthesis was mainly mediated by an arginine-utilizing source of NO generation. Furthermore, LPS-induced NO caused transcript accumulation of alternative oxidase genes and increased antioxidant enzyme activity, which enhanced antioxidant capacity and modulated redox state. We also analyzed the subcellular localization of NPR1 to identify the mechanism for protein-modulated plant innate immunity triggered by LPS. LPS-activated defense responses, including callose deposition and defense-related gene expression, were found to be regulated through an NPR1-dependent pathway. In summary, a significant NO synthesis induced by LPS contributes to the LPS-induced defense responses by up-regulation of defense genes and modulation of cellular redox state. Moreover, NPR1 plays an important role in LPS-triggered plant innate immunity.

Phospholipase D2 acts as an important regulator in LPS-induced nitric oxide synthesis in Raw 264.7 cells

The purpose of this study was to identify the role of phospholipase D2 (PLD2) in lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophage cell line, Raw 264.7 cells. When Raw 264.7 cells were stimulated with LPS, the expressions of PLDs were increased. Thus, to investigate the role of PLD in NO synthesis, we transfected PLD1, PLD2, and their dominant negative forms to Raw 264.7 cells, respectively. Interestingly, only PLD2 overexpression, but not that of PLD1, increased NO synthesis and iNOS expression. Moreover, LPS-induced NO synthesis and iNOS expression were blocked by PLD2 siRNA, suggesting that LPS upregulates NO synthesis through PLD2. Next, we investigated the S6K1-p42/44 MAPK-STAT3 signaling pathway in LPS-induced NO synthesis mechanism. Knockdown of PLD2 with siRNA also decreased phosphorylation of S6K1, p42/44 MAPK and STAT3 induced by LPS. Furthermore, we found that STAT3 bound with the iNOS promoter, and their binding was mediated by PLD2. Taken together, our results demonstrate the importance of PLD2 for LPS-induced NO synthesis in Raw 264.7 cells with involvement of the S6K1-p42/44 MAPK-STAT3 pathway.

Progesterone inhibits Toll-like receptor 4-mediated innate immune response in macrophages by suppressing NF-κB activation and enhancing SOCS1 expression

Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been implicated to have a functional role in immune response, mainly at concentrations commensurate with pregnancy. However, the underlying mechanisms remain to be fully understood. Here we present the evidences that progesterone inhibited immune response to lipopolysaccharide (LPS) and CpG oligodeoxynucleotides (CpG ODNs) through modulating Toll-like receptor (TLR) signaling. Pretreatment with progesterone can significantly inhibit TLR4 and TLR9-triggered IL-6 and nitric oxide (NO) production in macrophages. Furthermore, we found that progesterone can significantly inhibit LPS-induced nitric oxide synthesis (iNOS), TLR4 expression and nuclear factor-κB (NF-κB) activation. Consistently, as a negative feedback inhibitor, the expression of suppressor of cytokine signaling (SOCS1) protein was up-regulated by progesterone in LPS-stimulated macrophages. These results support the concept that progesterone might inhibit innate immune response by suppressing NF-κB activation and enhancement of SOCS1 expression, providing a possible mechanistic explanation for the function of progesterone in regulating innate immune responses.

Inhibitory effect of carnosine and N-acetyl carnosine on LPS-induced microglial oxidative stress and inflammation

Chronic inflammation and oxidative stress have been implicated in the pathogenesis of neurodegenerative diseases. A growing body of research focuses on the role of microglia, the primary immune cells in the brain, in modulating brain inflammation and oxidative stress. One of the most abundant antioxidants in the brain, particularly in glia, is the dipeptide carnosine, β-alanyl- l-histidine. Carnosine is believed to be involved in cellular defense such as free radical detoxification and inhibition of protein cross-linking. The more stable N-acetyl derivative of carnosine has also been identified in the brain. The aim of the present study was to examine the role of carnosine and N-acetyl carnosine in the regulation of lipopolysaccharide (LPS)-induced microglial inflammation and oxidative damage. In this study, BV2 microglial cells were stimulated with bacterial LPS, a potent inflammatory stimulus. The data shows that both carnosine and N-acetyl carnosine significantly attenuated the LPS-induced nitric oxide synthesis and the expression of inducible nitric oxide synthase by 60% and 70%, respectively. By competitive spectrophotometric measurement and electrospray mass spectrometry analysis, we demonstrated a direct interaction of N-acetyl carnosine with nitric oxide. LPS-induced TNFα secretion and carbonyl formation were also significantly attenuated by both compounds. N-acetyl carnosine was more potent than carnosine in inhibiting the release of the inflammatory and oxidative stress mediators. These observations suggest the presence of a novel regulatory pathway through which carnosine and N-acetyl carnosine inhibit the synthesis of microglial inflammatory and oxidative stress mediators, and thus may prove to play a role in brain inflammation.

Anandamide regulates lipopolysaccharide-induced nitric oxide synthesis and tissue damage in the murine uterus

In women, the association between chronic marijuana smoking and early miscarriage has long been known. Anandamide, a major endocannabinoid, mimics some of the psychotropic, hypnotic and analgesic effects of Delta(9)-tetrahydrocannabinol, the psychoactive component of marijuana. The uterus contains the highest concentrations of anandamide yet discovered in mammalian tissues and this suggests that it might play a role in reproduction. The production of small amounts of nitric oxide (NO) regulates various physiological events including implantation and myometrial relaxation, but in an inflammatory setting such as sepsis, NO has toxic effects as it is a free radical. The results presented in this study indicate that anandamide modulates NO production induced by lipopolysaccharide (LPS) in an in-vitro murine model. It was shown that LPS-induced NO synthesis and tissue damage were mediated by anandamide, as a cannabinoid receptor type I antagonist could block the effect of LPS (P < 0.001). This endotoxin inhibited anandamide uterine degradation (P < 0.05) and increased the expression of one of its synthesizing enzymes (P < 0.05). Contrary to the known anti-inflammatory and protective effects, in this model anandamide seems to act as a pro-inflammatory molecule modulating the production of NO induced by LPS. This proinflammatory effect of anandamide may be implicated in pathological reproductive events such as septic abortion.

Inhibition of lipopolysaccharide-induced nitric oxide synthesis by nicotine through S6K1-p42/44 MAPK pathway and STAT3 (Ser 727) phosphorylation in Raw 264.7 cells

Lipopolysaccharide (LPS) has been known to produce inflammatory modulators such as tumor necrosis factor α (TNF-α) or nitric oxide (NO). In this study, we examined the effects of nicotine on LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophages. LPS-induced NO synthesis and iNOS expression were significantly decreased by nicotine. To investigate the signaling mechanism of nicotine induced suppression of NO synthesis and iNOS expression induced by LPS, we focused on the possible roles of p42/44 MAPK, S6K1, and signal transducers and activators of transcription 3 (STAT3) signaling. LPS is known to activate p42/44 MAPK and S6K1, which in turn activates STAT3 to induce inflammatory regulators. Pretreatment of cells with nicotine blocked LPS-induced p42/44 MAPK and S6K1 as well as iNOS promoter activity. Furthermore, we found that LPS-induced phosphorylation of STAT3 at serine 727 is mediated by S6K1-p42/44 MAPK pathway, and this STAT3 phosphorylation was also blocked by nicotine. We also found that downregulation of STAT3 using STAT3 siRNA resulted in suppression of the NO synthesis and iNOS expression. Taken together, our results suggest that nicotine inhibits LPS-induced NO synthesis through suppression of S6K1-p42/44 MAPK pathway and phosphorylation of STAT3 in Raw 264.7 cells.

Toxoplasma gondii Prevents Neuron Degeneration by Interferon-γ-Activated Microglia in a Mechanism Involving Inhibition of Inducible Nitric Oxide Synthase and Transforming Growth Factor-β1 Production by Infected Microglia

Interferon (IFN)-gamma, the main cytokine responsible for immunological defense against Toxoplasma gondii, is essential in all infected tissues, including the central nervous system. However, IFN-gamma-activated microglia may cause tissue injury through production of toxic metabolites such as nitric oxide (NO), a potent inducer of central nervous system pathologies related to inflammatory neuronal disturbances. Despite potential NO toxicity, neurodegeneration is not commonly found during chronic T. gondii infection. In this study, we describe decreased NO production by IFN-gamma-activated microglial cells infected by T. gondii. This effect involved strong inhibition of iNOS expression in IFN-gamma-activated, infected microglia but not in uninfected neighboring cells. The inhibition of NO production and iNOS expression were parallel with recovery of neurite outgrowth when neurons were co-cultured with T. gondii-infected, IFN-gamma-activated microglia. In the presence of transforming growth factor (TGF)-beta1-neutralizing antibodies, the beneficial effect of the parasite on neurons was abrogated, and NO production reverted to levels similar to IFN-gamma-activated uninfected co-cultures. In addition, we observed Smad-2 nuclear translocation, a hallmark of TGF-beta1 downstream signaling, in infected microglial cultures, emphasizing an autocrine effect restricted to infected cells. Together, these data may explain a neuropreservation pattern observed during immunocompetent host infection that is dependent on T. gondii-triggered TGF-beta1 secretion by infected microglia.

Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production.

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO4 markedly reduced IFN-gamma + LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-gamma + LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

Inhibition of LPS-induced iNOS and NO synthesis in primary rat microglial cells

Nitric oxide (NO) has been implicated in the etiopathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), and inhibition of NO synthesis has been proposed to be a possible mechanism of action of drugs to treat MS. In the present study, we investigated the inhibitory effect on NO synthesis of various steroids, cytokines and drugs used or proposed for the treatment of MS. As a model system, we used primary rat microglial cells which produce NO synthase and subsequently release NO upon stimulation with lipopolysaccharide (LPS). Among the substances tested, the glucocorticoids prednisone, hydrocortisone, dexamethasone and progesterone as well as transforming growth factor-β (TGF-β) dose-dependently inhibited LPS-induced nitric oxide synthase (iNOS) and NO synthesis. In contrast, COP-1, the phosphodiesterase inhibitors rolipram and pentoxifylline, the cytokines interleukin-10 (IL-10) and interferon-β (IFN-β) as well as the steroids β-estradiol, testosterone, and dehydroepiandrosterone (DHEA) showed no inhibitory effect. Cholesterol slightly, but not significantly, increased LPS-induced nitric oxide synthesis. We conclude from the present study that with respect to treatment of MS, inhibition of NO synthesis may be an important mechanism of action of glucocorticoids and transforming growth factor-β, but not of other drugs used or proposed to treat MS.

Nitric oxide is necessary for CC-class chemokine expression in endotoxin-stimulated ANA-1 murine macrophages

The production of nitric oxide (NO) in response to endotoxin (LPS)-stimulation is associated with a myriad of NO-dependent regulatory functions. The study of NO-dependent genetic programs in the setting of endotoxin stimulation can be aided by determination of genes whose transcription is upregulated in the presence of NO and LPS. Using subtractive suppression hybridization analysis, we demonstrate that ANA-1 murine macrophages produce the CC class chemokines, monocyte chemoattractant protein-1 (JE/MCP-1) and macrophage inflammatory protein-related protein-1 (C10/MRP-1), in response to LPS-mediated nitric oxide (NO) production. MCP-1 and MRP-1 gene transcription and protein synthesis are upregulated in the setting of LPS-induced NO synthesis. NO alone is necessary but insufficient for induction of chemokine protein expression; an additional LPS-dependent signaling pathway is also required. This study suggests a novel mechanism by which NO induces chemokine expression and regulates the host inflammatory response to endotoxin.

Leflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes

Highly reactive gaseous free radical nitric oxide (NO), generated by astrocytes and infiltrating macrophages is implicated in inflammatory destruction of brain tissue, including that occurring in multiple sclerosis. Therefore, the influence of immunosuppressive drug leflunomide on inducible nitric oxide synthase (iNOS)-dependent NO production in rat astrocytes and macrophages was investigated. Under the same cultivating conditions, leflunomide’s active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-γ+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-γ+LPS-triggered expression of iNOS mRNA in astrocytes. In the presence of transcription inhibitor actinomycin D, A77 1726 failed to inhibit astrocyte NO production, suggesting transcriptional regulation of iNOS by leflunomide. This assumption was further supported by the ability of A77 1726 to inhibit IFN-γ+LPS-induced expression of mRNA for an important iNOS transcription factor IRF-1. PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), but not genistein, an unselective protein tyrosine kinase inhibitor, completely mimicked cell type-specific inhibition of NO synthesis by A77 1726. Therefore, previously described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for A77 1726-mediated suppression of iNOS activation in astrocytes. Accordingly to results obtained with primary astrocytes, both A77 1726 and PD98059 significantly reduced IFN-γ+LPS-induced NO synthesis in the cultures of rat astrocytoma cell line C6. The ability to suppress iNOS induction in astrocytes supports potential use of leflunomide in the treatment of multiple sclerosis and other NO-dependent inflammatory brain disorders.

Structural requirements of sesquiterpene lactones to inhibit LPS-induced nitric oxide synthesis in RAW 264.7 macrophages

Some sesquiterpene lactones were recently demonstrated to inhibit inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) synthesis. The primary objective of the present study was, therefore, to find evidence for structural requirements of sesquiterpene lactones regarding their capability to inhibit iNOS-dependent NO synthesis. Sesquiterpene lactones 1– 11 were examined for their influence on nitrite accumulation in cell culture supernatants of LPS-induced RAW 264.7 macrophages. Except the taraxinic acid β- d-glucopyranosylester 8 all compounds showed a dose-dependent inhibition of nitrite accumulation in cell culture supernatants with IC 50 values ranging from 0.5 to 36.8 μM. High activity seemed to be dependent on an α-methylene-γ-lactone functionality. Cytotoxicity and the ability to inhibit activation of transcription factor NF-κB are further biological activities of sesquiterpene lactones. The second point of interest was, therefore, whether the structural requirements of sesquiterpene lactones for these activities may differ or be the same for those needed to inhibit iNOS-dependent NO synthesis. Using concentrations of 1– 11 required to inhibit NO synthesis cell viability was determined and NF-κB binding activity was measured by gel-shift experiments. Interestingly, compounds almost equally effective in inhibiting nitrite accumulation did not show the same cytotoxic potential, and most sesquiterpene lactones inhibited nitrite accumulation at concentrations where inhibition of NF-κB activation was not significant. These results suggest that different biological activities of sesquiterpene lactones have different structural requirements.

Regulation of nitric oxide production in cultured human T84 intestinal epithelial cells by nuclear factor-kappa B-dependent induction of inducible nitric oxide synthase after exposure to bacterial endotoxin.

Intestinal epithelium is consistently in contact with lipopolysaccharide (LPS) produced by intraluminal microbes. LPS induces nitric oxide production in many rodent cells, but in human cells it is very differently regulated. To test the hypothesis that exposure to LPS up-regulates nitric oxide synthesis in human intestinal epithelium. LPS induced nitric oxide synthesis in T84 cells in a time- and dose-dependent manner whereas detectable amounts of peroxynitrite were not produced. A novel selective inducible nitric oxide synthase (iNOS) inhibitor 1400 W potently inhibited LPS-induced nitric oxide synthesis in T84 cells while dexamethasone was relatively ineffective. Nitric oxide production was sensitive to cycloheximide, indicating that it was dependent on de novo protein synthesis. Nuclear factor-kappa B (NF-kappa B) inhibitor pyrrolidinedithiocarbamate abolished iNOS and nitric oxide production. Nitric oxide synthesis was also suppressed by genistein (tyrosine kinase inhibitor) and PD 98059 (p44/42 MAP kinase inhibitor) but enhanced by SB 203580 (p38 MAP kinase inhibitor). Intestinal epithelial cells express iNOS and produce nitric oxide in a nuclear factor-kappa B-dependent manner when exposed to LPS. The process is regulated by tyrosine kinases, and p44/42 and p38 MAP kinases. Because nitric oxide acts as an antimicrobial agent and immune modulator, these findings are implicated in the regulation of gut mucosal immunity.

Down-regulation of LPS-induced nitric oxide synthesis of murine macrophages by oral administration of Sho-saiko-to

The effect of oral administration of a traditional Chinese herbal medicine, Sho-saiko-to (SST), on nitric oxide (NO) synthesis by murine macrophages was examined. SST was orally administered to mice, and NO synthesis of peritoneal exudate cells (PEC) cultured in the presence or absence of lipopolysaccharide (LPS) was measured. NO synthesis of PEC in response to LPS was reduced by oral administration of SST, whereas alkaline phosphatase (ALP) activity expressed by splenic B cells in response to LPS was enhanced. This finding suggests that augmented ALP activity possibly resulted from a decrease in macrophage functions, including NO synthesis. These activities were observed when SST or a low molecular weight fraction of SST was orally administered to mice, suggesting that down-regulation of macrophage functions depend on low molecular weight substances in SST.

Effects of radiodetoxified endotoxin on nitric oxide production in J774 macrophages and in endotoxin shock

Radiodetoxified lipopolysaccharide (RD-LPS) is a60Co-gamma-irradiated LPS with a modified structure, which decreases its toxic effects. To obtain a better understanding of the mechanism of the reduced toxicity of RD-LPS, here we studied the effect of RD-LPS on the regulation of nitric oxide (NO) production in vitro and in vivo. In control cells, stimulation by native LPS (10 μg/ml) induced the expression of the inducible NO synthase (iNOS) and production of NO, as measured by increase in the concentration of nitrite, breakdown product of NO. Pre-exposure of the cells for 24 h to a subthreshold concentration of RD-LPS (10 ng/ml) induced a complete desensitization to the LPS-induced NO production in comparison to control cells (P &lt; 0.01). On the contrary, pre-exposure of the cells with native LPS (10 ng/ml) did not reduce LPS-induced NO synthesis. RD-LPS induced a smaller production of tumor necrosis factor (TNF) than native LPS, but did not induce a desensitization against subsequent LPS-induced TNF synthesis. ln in vivo studies, pretreatment of rats with repeated doses of sublethal RD-LPS (1 mg/kg/day i.p. for 4 days) inhibited increase of plasma nitrate/nitrite levels, NO production in peritoneal macrophages ex vivo and induction of lung iNOS activity, in response to a high-dose LPS challenge (15 mg/kg i.p.) given at the fifth day. Pretreatment with repeated sublethal doses of the native LPS (1 mg/kg/day i.p.) did not affect NO production in rats subjected to endotoxic shock. The results demonstrate that RD-LPS induces tolerance to the stimulatory effect of LPS on NO synthesis. Suppression of iNOS induction was only observed with RD-LPS, but not with native LPS in the models used herein. It remains to be further investigated whether suppression of iNOS induction by RD-LPS contributes to the protective effects of this compound in shock and inflammation.