Induced Nitrite Production Research Articles

Synthesis and Biological Evaluations of Betulinic Acid Derivatives With Inhibitory Activity on Hyaluronidase and Anti-Inflammatory Effects Against Hyaluronic Acid Fragment Induced Inflammation.

We previously reported that the structural modifications of pentacyclic triterpenoids including oleanolic acid resulted in enhanced hyaluronidase inhibitory activity but whether this applies to other pentacyclic triterpenoids such as betulinic acid (BA) is unknown. Herein, we synthesized BA derivatives with an α,β-unsaturated ketene moiety and evaluated for their: 1) hyaluronidase inhibitory activity and, 2) anti-inflammatory effects against lipopolysaccharides (LPS) induced inflammation. Compared to BA, the BA derivatives exerted improved anti-hyaluronidase activity (26.3%–72.8% vs. 22.6%) and anti-inflammatory effects by reducing nitrite production in BV2 cells (3.9%–46.8% vs. 3.4%) and RAW264.7 cells (22.7%–49.2% vs. 20.4%). BA derivatives inhibited LPS-induced production of pro-inflammatory cytokines in THP-1 cells (15.2%–22.4%). BA derivatives also exerted promising anti-inflammatory effects against hyaluronic acid fragment induced nitrite production (8.6%–35.6%) in THP-1 cells. BA derivatives showed augmented anti-hyaluronidase and anti-inflammatory effects but further biological evaluations using in vivo models are warranted to confirm their efficacy.

Eicosapentaenoic acid inhibits lipopolysaccharide (LPS)-induced nitrite production and cytokine release from J774 macrophages

Abstract Background Eicosapentaenoic acid (EPA), an omega-3 (ω-3) fatty acid, reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT) but the mechanism is not fully understood. Activated macrophages, characterized by cytokine release and increased inducible nitric oxide synthase (iNOS) activity, contribute to atherosclerosis. As both a substrate for and potential inhibitor of cyclooxygenase (COX), EPA may reduce iNOS activity. Purpose The purpose of this study was to evaluate the dose-dependent effects of EPA on nitrite and cytokine release from lipopolysaccharide (LPS)-activated macrophages. Methods Murine J774 macrophages were pretreated with vehicle or EPA at 10, 20 and 40 μM for 2 h, then challenged with LPS at 1.0 μg/ml. After 24 hr, iNOS activity was measured by nitrite production using the Griess assay. EPA was compared to the COX inhibitor diclofenac at 1.0 μg/ml. Levels of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) in cell supernatant were measured by immunochemistry using colchicine as a positive control. Results Activated macrophages caused a >4-fold increase in nitrite production (p<0.001) that was reduced by EPA in a dose-dependent manner. EPA decreased nitrite levels by 40, 62 and 77% at 10, 20 and 40 μM, respectively (p<0.01). Diclofenac separately reduced nitrite levels by 40% (p<0.01). EPA also reduced expression of IL-1β and TNF-α by 40% and 31%, respectively (p<0.01), in a manner similar to equimolar colchicine (10 μM). The reductions in IL-1β and TNF-α with EPA were dose-dependent. Conclusions EPA reduced macrophage activation as evidenced by decreased nitrite production and cytokine release similar to other anti-inflammatory agents. These findings indicate a novel effect of EPA on mechanisms of inflammation associated with vascular disease. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Carajurin: a anthocyanidin from Arrabidaea chica as a potential biological marker of antileishmanial activity

Leishmaniasis is a group of neglected tropical diseases whose treatment with antimonials bears limitations and has changed little in over 80 years. Medicinal plants have been evaluated as a therapeutic alternative for leishmaniasis. Arrabidaea chica is popularly used as a wound healing and antiparasitic agent, especially as leishmanicidal agent. This study examined the leishmanicidal activity of a crude extract (ACCE), an anthocyanidin-rich fraction (ACAF), and three isolated anthocyanidins from A. chica: carajurin, 3′-hydroxy-carajurone, and carajurone. We evaluated the antileishmanial activity against promastigote and intracellular amastigote forms of Leishmania amazonensis and determined cytotoxicity in BALB/c peritoneal macrophages, as well as nitrite quantification, using the Griess method. Molecular docking was carried out to evaluate interactions of carajurin at the nitric oxide synthase enzyme. All compounds were active against promastigotes after 72 h, with IC50 values of 101.5 ± 0.06 μg/mL for ACCE and 4.976 ± 1.09 μg/mL for ACAF. Anthocyanidins carajurin, 3′-hydroxy-carajurone, and carajurone had IC50 values of 3.66 ± 1.16, 22.70 ± 1.20, and 28.28 ± 0.07 μg/mL, respectively. The cytotoxicity assay after 72 h showed results ranging from 9.640 to 66.74 µg/mL for anthocyanidins. ACAF and carajurin showed selectivity against intracellular amastigote forms (SI> 10), with low cytotoxicity within 24 h, a statistically significant reduction in all infection parameters, and induced nitrite production. Molecular docking studies were developed to understand a possible mechanism of activation of the nitric oxide synthase enzyme, which leads to an increase in the production of nitric oxide observed in the other experiments reported. These results encourage us to suggest carajurin as a biological marker of A. chica.

Role of Coffee Caffeine and Chlorogenic Acids Adsorption to Polysaccharides with Impact on Brew Immunomodulation Effects.

Coffee brews have High Molecular Weight (HMW) compounds with described immunostimulatory activity, namely polysaccharides and melanoidins. Melanoidins are formed during roasting and are modified during brews technological processing. In addition, brews have Low Molecular Weight (LMW) compounds, namely free chlorogenic acids and caffeine, with well-known anti-inflammatory properties. However, this study shows that both espresso and instant coffee brews did not present immunostimulatory neither anti-inflammatory in vitro activities. It is possible that the simultaneous existence of compounds with antagonistic effects can mitigate their individual effects. To test this hypothesis, an ultrafiltration separation process was applied, studying the behavior of coffee brews’ HMW on retention of LMW compounds. Several ultrafiltration sequential cycles were required to separate retentates from LMW compounds, suggesting their retention. This effect was higher in instant coffee, attributed to its initial higher carbohydrate content when compared to espresso. Separation of HMW and LMW compounds boosted their immunostimulatory (6.2–7.8 µM nitrites) and anti-inflammatory (LPS induced nitrite production decrease by 36–31%) in vitro activities, respectively. As coffee anti-inflammatory compounds are expected to be first absorbed during digestion, a potential in vivo fractionation of LMW and HMW compounds can promote health relevant effects after coffee intake.

Green Pepper (Piper nigrum L.) Extract Suppresses Oxidative Stress and LPS-Induced Inflammation via Regulation of JNK Signaling Pathways

In this study, we compared the physicochemical properties and the antioxidant capacities of black and green pepper. Green pepper from India (GPI) and Sri Lanka (GPS) had higher Hunter L* and b* values and lower a* values than black pepper from India (BPI) and Sri Lanka (BPS). The contents of chlorophyll a and b, flavonoids, and phenolic compounds in GPI and GPS were higher than those in BPI and BPS. The peppercorns showed the following decreasing order of 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging capacities: GPI > GPS > BPI > BPS. High-performance liquid chromatography showed that the highest piperine content was present in GPI (8613.27 ± 45.86 mg/100 g). We further investigated the anti-inflammatory capacity of the green pepper. GPS and GPI significantly suppressed lipopolysaccharide (LPS)-induced nitrite production and inducible nitric oxide synthase expression without being cytotoxic to RAW 264.7 cells. GPS and GPI also suppressed the LPS-induced phosphorylation of mitogen-activated protein kinases, but not p65. GPS had a higher inhibitory effect on LPS-induced c-Jun phosphorylation and translocation from the cytosol to the nucleus than GPI. Thus, the findings of our study suggest that green pepper has the potential to be an effective nutraceutical against oxidative and inflammatory stress.

Transcriptome analysis of alcohol-treated microglia reveals downregulation of beta amyloid phagocytosis

BackgroundMicroglial activation contributes to the neuropathology associated with chronic alcohol exposure and withdrawal, including the expression of inflammatory and anti-inflammatory genes. In the current study, we examined the transcriptome of primary rat microglial cells following incubation with alcohol alone, or alcohol together with a robust inflammatory stimulus.MethodsPrimary microglia were prepared from mixed rat glial cultures. Cells were incubated with 75 mM ethanol alone or with proinflammatory cytokines (“TII”: IL1β, IFNγ, and TNFα). Isolated mRNA was used for RNAseq analysis and qPCR. Effects of alcohol on phagocytosis were determined by uptake of oligomeric amyloid beta.ResultsAlcohol induced nitrite production in control cells and increased nitrite production in cells co-treated with TII. RNAseq analysis of microglia exposed for 24 h to alcohol identified 312 differentially expressed mRNAs (“Alc-DEs”), with changes confirmed by qPCR analysis. Gene ontology analysis identified phagosome as one of the highest-ranking KEGG pathways including transcripts regulating phagocytosis. Alcohol also increased several complement-related mRNAs that have roles in phagocytosis, including C1qa, b, and c; C3; and C3aR1. RNAseq analysis identified over 3000 differentially expressed mRNAs in microglia following overnight incubation with TII; and comparison to the group of Alc-DEs revealed 87 mRNAs modulated by alcohol but not by TII, including C1qa, b, and c. Consistent with observed changes in phagocytosis-related mRNAs, the uptake of amyloid beta1–42, by primary microglia, was reduced by alcohol.ConclusionsOur results define alterations that occur to microglial gene expression following alcohol exposure and suggest that alcohol effects on phagocytosis could contribute to the development of Alzheimer’s disease.

Liver X receptor and STAT1 cooperate downstream of Gas6/Mer to induce anti-inflammatory arginase 2 expression in macrophages.

Mer signaling increases the transcriptional activity of liver X receptor (LXR) to promote the resolution of acute sterile inflammation. Here, we aimed to understand the pathway downstream of Mer signaling after growth arrest-specific protein 6 (Gas6) treatment that leads to LXR expression and transcriptional activity in mouse bone-marrow derived macrophages (BMDM). Gas6-induced increases in LXRα and LXRβ and expression of their target genes were inhibited in BMDM from STAT1−/− mice or by the STAT1-specific inhibitor fludarabine. Gas6-induced STAT1 phosphorylation, LXR activation, and LXR target gene expression were inhibited in BMDM from Mer−/− mice or by inhibition of PI3K or Akt. Gas6-induced Akt phosphorylation was inhibited in BMDM from STAT1−/− mice or in the presence of fludarabine. Gas6-induced LXR activity was enhanced through an interaction between LXRα and STAT1 on the DNA promoter of Arg2. Additionally, we found that Gas6 inhibited lipopolysaccharide (LPS)-induced nitrite production in a STAT1 and LXR pathway-dependent manner in BMDM. Additionally, Mer-neutralizing antibody reduced LXR and Arg2 expression in lung tissue and enhanced NO production in bronchoalveolar lavage fluid in LPS-induced acute lung injury. Our data suggest the possibility that the Gas6-Mer-PI3K/Akt-STAT1-LXR-Arg2 pathway plays an essential role for resolving inflammatory response in acute lung injury.

Brazilin Ameliorates High Glucose-Induced Vascular Inflammation via Inhibiting ROS and CAMs Production in Human Umbilical Vein Endothelial Cells

Vascular inflammatory process has been suggested to play a key role in the initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Recent studies have shown that brazilin exhibits antihepatotoxic, antiplatelet, cancer preventive, or anti-inflammatory properties. Thus, we investigated whether brazilin suppresses vascular inflammatory process induced by high glucose (HG) in cultured human umbilical vein endothelial cells (HUVEC). HG induced nitrite production, lipid peroxidation, and intracellular reactive oxygen species formation in HUVEC cells, which was reversed by brazilin. Western blot analysis revealed that brazilin markedly inhibited HG-induced phosphorylation of endothelial nitric oxide synthase. Besides, we investigated the effects of brazilin on the MAPK signal transduction pathway because MAPK families are associated with vascular inflammation under stress. Brazilin blocked HG-induced phosphorylation of extracellular signal-regulated kinase and transcription factor NF-κB. Furthermore, brazilin concentration-dependently attenuated cell adhesion molecules (ICAM-1 and VCAM-1) expression induced by various concentrations of HG in HUVEC. Taken together, the present data suggested that brazilin could suppress high glucose-induced vascular inflammatory process, which may be closely related with the inhibition of oxidative stress, CAMs expression, and NF-κB activation in HUVEC. Our findings may highlight a new therapeutic intervention for the prevention of vascular diseases.

ATF6 upregulates XBP1S and inhibits ER stress-mediated apoptosis in osteoarthritis cartilage

As we previously reported, transcription factor XBP1S enhances BMP2-induced chondrocyte differentiation and acts as a positive mediator of chondrocyte hypertrophy. The purpose of this study was to determine (1) whether XBP1S influences ER stress-mediated apoptosis in osteoarthritis (OA); (2) whether ATF6 regulates IRE1/XBP1 signal pathway in OA cartilage; (3) what are the associated molecules affecting apoptosis in osteoarthritis and the molecular events underlying this process. Herein, we examined and found that ER stress-associated molecules were activated in OA patients, specifically XBP1S splice and expression were increased markedly by TNF-α and IL-1β treatments. Transcription factor ATF6 can specifically bind to the promoter of XBP1 gene and enhance the expression of XBP1S spliced by IRE1α in osteoarthritis cartilage. Furthermore, siXBP1S can enhance ER stress-mediated apoptosis and main matrix degradation in osteoarthritis. Whereas AdXBP1S can inhibit ER stress-mediated apoptosis and TNFα induced nitrite production in OA cartilage. In a word, our observations demonstrate the importance of XBP1S in osteoarthritis. ATF6 and IRE1α can regulate endogenous XBP1S gene expression synergistically in OA cartilage. More significantly, XBP1S was a negative regulator of apoptosis in osteoarthritis by affecting caspase 3, caspase 9, caspase 12, p-JNK1, and CHOP.

Carvacrol attenuates mechanical hypernociception and inflammatory response

Carvacrol is a phenolic monoterpene present in the essential oil of the family Lamiaceae, as in the genera Origanum and Thymus. We previously reported that carvacrol is effective as an analgesic compound in various nociceptive models, probably by inhibition of peripheral mediators that could be related with its strong antioxidant effect observed in vitro. In this study, the anti-hypernociceptive activity of carvacrol was tested in mice through models of mechanical hypernociception induced by carrageenan, and the involvement of important mediators of its signaling cascade, as tumor necrosis factor-alpha (TNF-α), prostaglandin E(2) (PGE(2)), and dopamine, were assessed. We also investigated the anti-inflammatory effect of carvacrol on the model of carrageenan-induced pleurisy and mouse paw edema, and the lipopolysaccharide (LPS)-induced nitrite production in murine macrophages was observed. Systemic pretreatment with carvacrol (50 or 100mg/kg; i.p.) inhibited the development of mechanical hypernociception and edema induced by carrageenan and TNF-α; however, no effect was observed on hypernociception induced by PGE(2) and dopamine. Besides this, carvacrol significantly decreased TNF-α levels in pleural lavage and suppressed the recruitment of leukocytes without altering the morphological profile of these cells. Carvacrol (1, 10, and 100μg/mL) also significantly reduced (p < 0.001) the LPS-induced nitrite production in vitro and did not produce citotoxicity in the murine peritoneal macrophages in vitro. The spontaneous locomotor activity of mice was not affected by carvacrol. This study adds information about the beneficial effects of carvacrol on mechanical hypernociception and inflammation. It also indicates that this monoterpene might be potentially interesting in the development of novel tools for management and/or treatment of painful conditions, including those related to inflammatory and prooxidant states.

Naringenin more effectively inhibits inducible nitric oxide synthase and cyclooxygenase-2 expression in macrophages than in microglia

Macrophages and microglia are thought to account for initial disease progression in acute myocardial infarction and acute ischemic stroke. Before our study, the inhibitory effects of naringenin, a flavonoid, on lipopolysaccharide (LPS)-induced inflammation in macrophages and microglia have not been fully reported and compared. We hypothesized that naringenin can effectively inhibit LPS-induced inflammation of macrophages and microglia at different concentrations, the range of which is broader, with the lowest concentration more easily achieved in macrophages. In this study, we compared the anti-inflammatory effects of naringenin on LPS-stimulated RAW 274.6 macrophages and BV2 microglia and the suppression effects of naringenin and vitamin C (a well-known anti-inflammatory agent) on LPS-induced nitrite production. The results show that macrophages could maintain cell viability at higher naringenin concentrations and were more easily activated by LPS in comparison to microglia (200 vs 100 μmol/L; 0.1 vs 1 μg/mL). Under LPS (1 μg/mL) stimulation in both cell types, naringenin (up to 200 μmol/L in macrophages and 100 μmol/L in microglia) inhibited nitrite production and inducible nitric oxide synthase and cyclooxygenase-2 expression in a dose-dependent manner. The range of naringenin concentrations for inhibition was broader, and the lowest concentration was more easily achieved in macrophages; the lowest effective concentrations of naringenin to achieve constant suppression effect were 50 μmol/L in macrophages and 100 μmol/L in microglia, respectively. Vitamin C (100 μmol/L), compared with naringenin (100 μmol/L), had less and no suppression effect on LPS (1μg/mL)-induced nitrite production in macrophages and microglia, respectively. In conclusion, naringenin more effectively inhibits the LPS-induced inflammatory status, including nitrite production and inducible nitric oxide synthase and cyclooxygenase-2 expression, in macrophages than in microglia. The findings of the present study suggest that consumption of naringenin-containing flavonoids might be beneficial to the cardiovascular and cerebrovascular inflammatory process.

Activation of microglial cells via protease-activated receptor 2 mediates neuronal cell death in cultured rat primary neuron

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2–PKC–MAPK pathway-induced microglial activation was NF-κB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-α and IL-1β. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X L, and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N G -nitro- l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.

Responses to Amyloids of Microbial and Host Origin Are Mediated through Toll-like Receptor 2

Curli fibrils are proteinaceous bacterial structures formed by amyloid fibrils composed of the major curli subunit CsgA. Like beta-amyloid 1-42, which is associated with brain inflammation and Alzheimer's disease, curli fibrils have been implicated in the induction of host inflammatory responses. However, the underlying mechanisms of amyloid-induced inflammation are not fully understood. In a mouse sepsis model, we show that curli fibrils contributed to Nos2 expression, a hallmark of inflammation, by stimulating Toll-like receptor (TLR) 2. The TLR2 agonist activity was reduced by an amyloidogenicity-lowering amino acid substitution (N122A) in CsgA. Amyloid-forming synthetic peptides corresponding to beta-amyloid 1-42 or CsgA 111-151 stimulated Nos2 production in macrophages and microglia cells through a TLR2-dependent mechanism. This activity was abrogated when an N122A substitution was introduced into the synthetic CsgA peptide. The induction of TLR2-mediated responses by bacterial and eukaryotic amyloids may explain the inflammation associated with amyloids and the resulting pathologies.

Inhibitory Effects of Ginsenoside Rb1,Rg3, and Panax ginseng Head Butanol Fraction on Inflammatory Mediators from LPS-Stimulated RAW 264.7 Cells

Panax ginseng C.A. Mayer (Araliaceae, P. ginseng) has been used for the enhancement of vascular and immune functions in Korea and Japan for a long time. Ginsenoside <TEX>$Rb_1$</TEX> and <TEX>$Rg_3$</TEX> isolated from P. ginseng head-part butanolic extract (PGHB) were investigated for anti-inflammatory activity. Ginsenosides and PGHB did not affect the cell viability within <TEX>$0\;-\;100\;{\mu}g/ml$</TEX> concentration to RAW 264.7 murine macrophage cells. Ginsenosides and PGHB inhibited partly lipopolysaccharide (LPS)-induced nitrite production in a dose-dependent manner. The ginsenosides and PGHB showed partially chemical nitric oxide (NO) quenching (maximum 40%) in the cell-free system. Also, ginsenoside <TEX>$Rb_1$</TEX> and <TEX>$Rg_3$</TEX> inhibited markedly approximately 74 and 54% of inducible nitric oxide synthase (iNOS) mRNA transcription from LPS-induced RAW 264.7 cells. Taken together, the inhibitory effect of ginsenosides and PGHB on NO production did not occur as a result of cell viability, but was caused by both the chemical NO quenching and the regulation of iNOS. Additionally, the ginsenoside <TEX>$Rb_1$</TEX> and PGHB inhibited prostaglandin <TEX>$E_2$</TEX> (<TEX>$PGE_2$</TEX>) synthesis in a concentration-dependent manner, showed approximately 70-98% inhibition at <TEX>$100\;{\mu}g/ml$</TEX> concentration. And the treatment with ginsenosides and PGHB attenuated partially LPS-upregulated cyclooxygenase-2 (COX-2) gene transcription. Ginsenoside <TEX>$Rg_3$</TEX> suppressed LPS-stimulated interleukin-6 (IL-6) level to the basal in RAW 264.7 cells. From these results, ginsenoside <TEX>$Rb_1,\;Rg_3$</TEX>, and PGHB may be useful for the relief and retardation of immunological inflammatory responses and its action may occur through the reduction of inflammatory mediators, including NO, <TEX>$PGE_2$</TEX>, and IL-6 production.

Multidrug resistance related protein (ABCC1) and its role on nitrite production by the murine macrophage cell line RAW 264.7

Multidrug resistance related protein 1 (MRP1/ABCC1) is an ABC transporter protein related to the extrusion of reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH-conjugates, as well as leukotriene C 4 and cyclopentane prostaglandins. Inhibition of ABCC1 activity impairs lymphocyte activation. The present work studied ABCC1 expression and activity on a murine macrophage cell line, RAW 267.4 and the effects of ABCC1 classical inhibitors, as well as GSH metabolism modulators, on LPS induced activation. Approximately, 75% of resting cells were positive for ABCC1 and the classical ABCC1 reversors (indomethacin, 0.1–2 mM; probenecid, 0.1–10 mM and MK571, 0.01–1 mM) were able to enhance intracellular CFDA accumulation in a concentration-dependent manner, suggesting ABCC1 inhibition. After LPS (100 ng/ml) activation 50% of the population was positive for ABCC1, and this protein was still active. In LPS-activated cells, ABCC1 activity was also impaired by BSO (1 mM), an inhibitor of GSH synthesis. Conversely, GSH (5 mM) reversed the BSO effect. ABCC1 inhibition by indomethacin, probenecid or MK571 decreased LPS induced nitrite production in a concentration-dependent manner, the same result was observed with BSO and again GSH reversed its effect. The ABCC1 reversors were also able to inhibit iNOS expression. In conclusion, LPS modulated the expression and activity of ABCC1 transporters in RAW macrophages and inhibitors of these transporters were capable of inhibiting nitrite production suggesting a role for ABCC1 transporters in the inflammatory process.

Synthesis and evaluation of some lipidic aminoalcohols and diamines as immunomodulators

Lymphoproliferation inhibition and cytotoxicity of a number of lipidic aminoacids, aminoalcohols and diamines were evaluated as a preliminary screening to select potential immunomodulators. The four most potent/less toxic compounds were submitted to delayed hypersensibility (DTH) assays to define the best to be evaluated further Graft-vs-Host, NO production and other immunoevaluation (CD4 +, CD45, CD8, CD11b, I-Ek, and NK cells) assays, to establish their immunomodulation potential for being further considered as auxiliary agents for vaccination against some parasitic infections. Compounds 5d, 6d, 6f, 7a, and 9a, fairly inhibited the lymphoproliferation (71.6–79.5%, at 3.2–2.4 nM), while the aminoalcohol derivative 6f and the diamine 7a gave the most promising results in the DTH assays. Diamine derivative 8b induced nitrite production on normal macrophages, whereas compounds 6f and 7a induced nitrite production on LPS pre-stimulated macrophages. These two last compounds have been selected to follow in vivo vaccination assays.

Laxifolone A Suppresses LPS/IFN-γ-Induced NO Synthesis by Attenuating NF-κB Translocation: Role of NF-κB p105 Level

Laxifolone A is a triterpene isolated from Euonymus laxiflorus Champ. Exposure of RAW264.7 macrophages to laxifolone A concentration-dependently suppressed lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced nitrite production (IC50 = 0.37 +/- 0.05 microM), inducible NO synthase (iNOS) protein, and iNOS mRNA expression. Translocation of nuclear factor-kappaB (NF-kappaB) into the nucleus with subsequent activation of iNOS gene transcription is essential in NO signaling. Western blot analysis indicated that the cytosolic NF-kappaB/p65 was obviously decreased after LPS/IFN-gamma stimulation for 30 min and this phenomenon could be reversed by laxifolone A. Similarly, a time-related NF-kappaB/p65 nuclear translocation induced by LPS/IFN-gamma was diminished in the presence of laxifolone A. However, laxifolone A failed to interfere in LPS/IFN-gamma-evoked IkappaB degradation. Our results also showed that LPS/IFN-gamma-stimulation resulted in the degradation of NF-kappaB p105, the NF-kappaB precursor, and laxifolone A treatment significantly counteracted this effect. Furthermore, laxifolone A itself was able to enhance NF-kappaB p105 protein expression. In summary, these results suggest that inhibition of NF-kappaB p105 degradation in cytoplasm may participate in the abrogation of LPS/IFN-gamma-induced NF-kappaB translocation and subsequent NO synthesis by laxifolone A.

Enteropathogenic Escherichia coli Outer Membrane Proteins Induce iNOS by Activation of NF-κB and MAP Kinases

Enteropathogenic Escherichia coli (EPEC) infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. Nitric oxide (NO) is an important modulator of intestinal inflammatory response. The aim of the present study was to investigate whether EPEC outer membrane proteins (OMPs) up regulate epithelial cell expression of inducible nitric oxide synthase (iNOS) and to examine the role of NF-kappaB and MAP kinases (MAPK) on nitrite production. iNOS mRNA expression was assessed by RT-PCR. Nitrite levels were measured by Griess reaction. NF-kappaB activation by OMPs was evaluated by EMSA and immunoblotting was done to detect MAPK activation. EPEC OMP up regulated iNOS, induced nitrite production and NF-kappaB and MAPK were activated in caco-2 cells. The nitrite levels decreased when NF-kappaB and MAPK inhibitors were used. Thus, EPEC OMPs induce iNOS expression and NO production through activation of NF-kappaB and MAPK.

Induction of Nitric Oxide Production by the Cytostatic Macrolide Apicularen A [2,4-Heptadienamide,N-[(1E)-3-[(3S,5R,7R,9S)-3,4,5,6,7,8,9,10-octahydro-7,14 Dihydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)] and Possible Role of Nitric Oxide in Apicularen A-Induced Apoptosis in RAW 264.7 Cells

We previously reported that apicularen A [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-3,4,5,6,7,8,9,10-octahydro-7,14 dihydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)], a highly cytostatic macrolide isolated from the myxobacterial genus Chondromyces, induces apoptosis in the mouse leukemic monocyte cell line RAW 264.7. To analyze the action mechanism of apicularen A for the induction of apoptosis, effects of apicularen A on nitric oxide (NO) production in RAW 264.7 cells were examined. It was demonstrated that apicularen A at 10 and 100 nM induced nitrite production, whereas apicularen B [2,4-heptadienamide, N-[(1E)-3-[(3S,5R,7R,9S)-7-[[2-(acetylamino)-2-deoxy-beta-d-glucopyranosyl]oxy]-3,4,5,6,7,8,9,10-octahydro-14-hydroxy-1-oxo-5,9-epoxy-1H-2-benzoxacyclododecin-3-yl]-1 propenyl]-, (2Z,4Z)-(9CI)], an N-acetyl-glucosamine glycoside of apicularen A, had no effect at 100 nM. The apicularen A-induced nitrite production was accompanied by an increase in the level of inducible nitric-oxide synthase (iNOS) and its mRNA and was suppressed by the NOS inhibitor N(G)-monomethyl-l-arginine acetate (l-NMMA). In addition, apicularen A activated nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and decreased the level of IkappaB-alpha and increased that of phosphorylated c-Jun N-terminal kinase (JNK). Furthermore, the apicularen A-induced nitrite production was suppressed by the NF-kappaB inhibitor Bay 11-7082 [(E)-3-(4-methylphenylsulfonyl)-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one]. These findings suggested that apicularen A activates NF-kappaB and AP-1, thus triggering the expression of iNOS mRNA and iNOS protein and induces NO production. Finally, apicularen A decreased cell growth and survival and cell viability and disrupted the mitochondrial membrane potential. The addition of l-NMMA partially recovered the apicularen A-induced decrease in cell growth and survival and cell viability and the disruption of mitochondrial membrane potential. These findings suggested that NO produced by apicularen A treatment participate partially in the apicularen A-induced apoptosis in RAW 264.7 cells.

C-Reactive Protein Directly Inhibits Nitric Oxide Production by Cytokine-stimulated Vascular Smooth Muscle Cells

C-reactive protein (CRP) is an important risk factor for atherosclerosis and coronary artery disease. The authors investigated the effects of CRP on nitric oxide (NO) synthesis in vascular smooth muscle cells (VSMCs). Production of nitrite, a stable metabolite of NO, was measured, as was the expression of inducible NO synthase (iNOS) messenger RNA and protein in cultured rat VSMCs. Incubation of cultures with interleukin (IL)-1beta (10 ng/mL) caused a significant increase in nitrite production. C-reactive protein significantly decreased the IL-1beta-induced nitrite production by VSMCs in a dose-dependent manner (10-100 microg/mL). Incubation with IL-1beta induced expression of iNOS mRNA and protein in VSMCs, whereas CRP showed a suppressive effect on their expression. Addition of IL-1beta activated nuclear factor-kappaB (NFkappaB) in VSMCs, whereas CRP did not affect IL-1beta-induced NFkappaB activation. In conclusion, CRP acts on VSMCs and inhibits NO synthesis, which might contribute to the development of atherosclerosis and vascular events.