Induced Nitric Oxide Production Research Articles

Activation of macrophages by natural TLR4 ligand isolated from Paecilomyces cicadae (P4496)

Abstract Many polysaccharides isolated from mushroom are considered to be biological response modifiers and have been shown to enhance various immune responses. Paecilomyces cicadae have been reported to have immunomodulatory properties. In this study, we investigated the membrane receptor and intracellular signaling responsible for the activation of macrophage by polysaccharide (PCP) isolated from P. cicadae. PCP induced the production of nitric oxide (NO) and the mRNA expression of IL-1β, IL-6, and TNF-α in RAW 264.7 cells. To investigate the membrane receptor involved in the activation of NO production, we examined the effect of PCP on primary macrophages isolated from C3H/HeN mice having normal-TLR4 and C3H/HeJ mice having mutant-TLR4. PCP induced NO production and cytokine gene expression in macrophages from C3H/HeN, but not from tlr4-mutated C3H/HeJ mice, which suggests that TLR4 is the membrane receptor for PCP. As a mechanism of action, PCP induced the phosphorylation of ERK, JNK, and p38, and the nuclear translocation of NF-κB p50/p65, which are the main signaling molecules down-stream from TLR4. In addition, p38 and NF-κB inhibitor attenuated PCP-mediated induction of NO production by macrophages. These results indicate that PCP activates macrophages through the TLR4 signaling pathway

Mechanisms of shear stress mediated nitric oxide production by inner medullary collecting duct cells

We previously reported that increased shear stress (SS) stimulates nitric oxide (NO) production in mouse inner medullary collecting duct‐3 (mIMCD3) cells. Recent work in mpk‐cortical collecting duct cells determined that SS‐induced endothelin‐1 (ET1) expression was mediated via activation of the epithelial sodium channel (ENaC). We have shown that ET1 via ETB receptor stimulation of NOS1 mediates NO production in mIMCD3 cells. Thus, we hypothesized that SS‐induced NO production in mIMCD3 cells is dependent on an ENaC‐ET1‐ETB‐NOS1 mechanism. mIMCD3 cells were grown to confluency on glass slides, subjected to SS (30 dynes/cm2 for 1 h), and nitrite production measured by HPLC and calculated as fold change from static conditions. Cells were pretreated with ENaC inhibitors, 1 μM amiloride or 0.2 μM benzamil, ETB antagonist 1000 nM BQ‐788, or 100 nM 1400W (NOS2‐selective inhibitor) or 1 μM VNIO (NOS1‐selective inhibitor), and nitrite production determined. Both ENaC inhibitors significantly blunted the SS‐induced increase in nitrite (control = 1.8 ± 0.3 vs 1.3 ± 0.2 and 0.9 ± 0.3 respectively N = 4, P < 0.05). BQ‐788 attenuated the SS mediated increase in NO (control = 3.9 ± 1.0 vs 1.7 ±0.1, N =5, P < 0.05). NOS2 inhibition had no significant effect on SS‐induced nitrite production (p>;0.05), while NOS1 inhibition lead to an ~50% reduction in SS‐mediated nitrite (control = 2.8 ± 0.5 vs 1.5 ± 0.4 N= 5–10, P < 0.05). In conclusion, mIMCD3 SS induced NO production is mediated via activation of ENaC, ETB receptor, and NOS1. Future studies will determine whether ENaC via ET1/ETB/NOS1 directly regulates mIMCD3 NO production. Funding support from AHA to KAH and NIH to JSP.

β‐arrestin‐mediated signal transduction participates in laminar shear stress‐induced production of nitric oxide in endothelial cells

Endothelial cells are capable of converting mechanical stimuli into intracellular signals generating vasoactive factors such as nitric oxide (NO). A great body of evidence suggests that β‐arrestins play a role not only on GPCR desensibilization but also in mechanotransduction. The aim of this study was to test the hypothesis that laminar shear stress (SS)‐induced NO production by endothelial cells requires the activation of the β‐arrestin signaling pathway. Towards this end, human saphenous vein endothelial cells (SVEC) were transfected with siRNA against the isoforms 1 and 2 of β‐arrestins and subsequently subjected to SS (15 dynes/cm2) for 10 minutes. We found that the SS‐induced production of nitrite in the cell culture medium was significantly decreased in SVEC silenced for β‐arrestin 1 and 2 compared to control cells (326±44 vs. 166±17%; P < 0.001, n=5). Furthermore, silencing of β‐arrestin 1 and 2 significantly attenuated the phosphorylation levels of AKT at the serine residue 473 (99±22 vs. 293±42% in control; P <0.001, n=5) and led to a non‐significant trend towards an increase of ERK phosphorylation (157±71 vs.107±5% in control; P= 0.3670, n=5). Altogether, we provide evidence that β‐arrestins play a role on laminar SS‐induced generation of NO associated with AKT activation in endothelial cells.Supported by FAPESP, CAPES and CNPq.

ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

Anti-inflammatory properties of potato glycoalkaloids in stimulated Jurkat and Raw 264.7 mouse macrophages

AimsThe potato glycoalkaloids, α-chaconine, α-solanine and solanidine, along with potato peel extracts were investigated for potential anti-inflammatory effects in vitro. Their potential to reduce two biomarkers of inflammation, cytokine and nitric oxide (NO) productions, were assessed in the stimulated Jurkat and macrophage models, respectively. Main methodsCytokine and nitric oxide productions were stimulated in Jurkat and Raw 264.7 macrophages with Concanavalin A (Con A; 25μg/ml) and lipopolysaccaride (LPS; 1μg/ml), respectively. Selective concentrations of glycoalkaloids and potato peel extracts were added simultaneously with Con A or LPS for 24h to investigate their potential to reduce inflammatory activity. Key findingsα-Chaconine and solanidine significantly reduced interleukin-2 (IL-2) and interleukin-8 (IL-8) productions in Con A-induced Jurkat cells. The potato peel extracts did not influence cytokine production. In LPS-stimulated Raw macrophages, α-solanine, solanidine and two potato peel extracts significantly reduced induced NO production. SignificanceOur findings suggest that sub-cytotoxic concentrations of potato glycoalkaloids and potato peel extracts possess anti-inflammatory effects in vitro and with further investigation may be useful in the prevention of anti-inflammatory diseases.

Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation

BackgroundDemyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage.Methods/Principal FindingsTo monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage.ConclusionThe model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.

Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts

We previously demonstrated that 11,12 and 14,15-epoxeicosatrienoic acids (EETs) produce cardioprotection against ischemia-reperfusion injury in dogs and rats. Several signaling mechanisms have been implicated in the cardioprotective actions of the EETs; however, their mechanisms remain largely elusive. Since nitric oxide (NO) plays a significant role in cardioprotection and EETs have been demonstrated to induce NO production in various tissues, we hypothesized that NO is involved in mediating the EET actions in cardioprotection. To test this hypothesis, we used an in vivo rat model of infarction in which intact rat hearts were subjected to 30-min occlusion of the left coronary artery and 2-hr reperfusion. 11,12-EET or 14,15-EET (2.5mg/kg) administered 10min prior to the occlusion reduced infarct size, expressed as a percentage of the AAR (IS/AAR), from 63.9±0.8% (control) to 45.3±1.2% and 45.5±1.7%, respectively. A nonselective nitric oxide synthase (NOS) inhibitor, L-NAME (1.0mg/kg) or a selective endothelial NOS inhibitor, L-NIO (0.30mg/kg) alone did not affect IS/AAR but they completely abolished the cardioprotective effects of the EETs. On the other hand, a selective neuronal NOS inhibitor, nNOS I (0.03mg/kg) and a selective inducible NOS inhibitor, 1400W (0.10mg/kg) did not affect IS/AAR or block the cardioprotective effects of the EETs. Administration of 11,12-EET (2.5mg/kg) to the rats also transiently increased the plasma NO concentration. 14,15-EET (10μM) induced the phosphorylation of eNOS (Ser1177) as well as a transient increase of NO production in rat cardiomyoblast cell line (H9c2 cells). When 11,12-EET or 14,15-EET was administered at 5min prior to reperfusion, infarct size was also reduced to 42.8±2.2% and 42.6±1.9%, respectively. Interestingly, L-NAME (1.0mg/kg) and a mitochondrial KATP channel blocker, 5-HD (10mg/kg) did not abolish while a sarcolemmal KATP channel blocker, HMR 1098 (6.0mg/kg) and a mitochondrial permeability transition pore (MPTP) opener, atractyloside (5.0mg/kg) completely abolished the cardioprotection produced by the EETs. 14,15-EET (1.5mg/kg) with an inhibitor of MPTP opening, cyclosporin A (CsA, 1.0mg/kg) produced a greater reduction of infarct size than their individual administration. Conversely, an EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 2.5mg/kg) completely abolished the cardioprotective effects of CsA, suggesting a role of MPTP in mediating the EET actions. Taken together, these results suggest that the cardioprotective effects of the EETs in an acute ischemia-reperfusion model are mediated by distinct mediators depending on the time of EET administration. The cardioprotective effects of EETs administered prior to ischemia were regulated by the activation of eNOS and increased NO production, while sarcKATP channels and MPTP were involved in the beneficial effects of the EETs when administered just prior to reperfusion.

Quercetin negatively regulates TLR4 signaling induced by lipopolysaccharide through Tollip expression

Polyphenolic compounds have been regarded as one of the most promising dietary agents for the prevention and treatment of inflammation-related chronic diseases; however, the anti-inflammatory activities of flavonoids, such as quercetin, are not completely characterized, and many features remain to be elucidated. In this study, we showed the molecular basis for the downregulation of TLR4 signal transduction by quercetin. Quercetin markedly elevated the expression of the Toll-interacting protein, a negative regulator of TLR signaling. Lipopolysaccharide-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by quercetin, and this action was prevented by Toll-interacting protein silencing. In addition, quercetin-treated macrophages inhibited lipopolysaccharide-induced activation of mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase, and the translocation of nuclear factor-κB and p65 through Toll-interacting protein. Treatment with quercetin resulted in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels as well as inducible nitric oxide synthase-mediated nitric oxide production induced by lipopolysaccharide. Taken together, these findings represent new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.

Bradykinin decreases nitric oxide release from microglia via inhibition of cyclic adenosine monophosphate signaling

Bradykinin (BK) is a major potent inflammatory mediator outside the central nervous system. In Alzheimer's disease, BK release and BK receptor expression in brain tissues are upregulated relatively early during the course of the disease. Hence, BK was believed to promote neuroinflammation. However, BK was recently reported to possess anti-inflammatory and neuroprotective roles. Exposure of BV2 microglial cell line to BK lead to a decrease in NO release from unstimulated cells as well as a dose-dependent attenuation, mediated by both B1 and B2 receptors, in lipopolysaccharide (LPS)-induced NO production. In this study we examined whether cyclic adenosine monophosphate (cAMP) signaling is involved in BK-mediated effect in microglial nitric oxide (NO) production. A protein kinase A (PKA) inhibitor mimicked the effects of BK, while cAMP elevating agents antagonized BK-mediated NO decrease. Moreover, BK inhibited the activation of cAMP responsive element binding protein (CREB). In addition, BK protected microglial cells from death triggered by combinations of LPS and each of the cAMP elevating agents. Finally, the addition of Gαi protein inhibitor abrogated the effects of BK on NO release, and the expression of Gαi protein in the plasma membrane was induced by BK. These results suggest that BK-mediated reduction in microglial NO production depends on coupling to Gi protein and also involves inhibition of cAMP-PKA-CREB signaling.

1α,25-Dihydroxycholecalciferol (Vitamin D3) Induces NO-Dependent Endothelial Cell Proliferation and Migration in a Three-Dimensional Matrix

Background/Aims: The 1α,25-dihydroxycholecalciferol (Vit. D) induces eNOS dependent nitric oxide (NO) production in human umbilical vein endothelial cells (HUVEC). To our knowledge, there are no reports directly relating Vit. D induced NO production to proliferation and/or migration in endothelial cells (EC). The aim of this study was to evaluate whether Vit. D addition to porcine EC could affect their proliferation and/or migration in a three-dimensional matrix via NO production. Materials and Methods: Porcine aortic endothelial cells (PAE) were used to evaluate Vit. D effects on cell proliferation and migration in a three-dimensional matrix. Results: Vit. D induced NO production in PAE cells. Moreover, it induced a significant increase in cellular proliferation and migration in a three-dimensional matrix. These effects were NO dependent, as inhibiting eNOS activity by L-NAME PAE migration was abrogated. This effect was strictly related to MMP-2 expression and apparently dependent on Vit. D and NO production. Conclusions: Vit. D can promote both endothelial cells proliferation and migration in a three-dimensional matrix via NO-dependent mechanisms. These findings cast new light on the role of Vit. D in the angiogenic process, suggesting new applications for Vit. D in such fields as tissue repair and wound healing.

Bacterial Clearance in Septic Mice Is Modulated by MCP-1/CCL2 and Nitric Oxide

Bacterial clearance is one of the most important beneficial consequences of the innate immune response. Chemokines are important mediators controlling leukocyte trafficking and activation, whereas reactive oxygen and nitrogen species are effectors in bacterial killing. In the present work, we used in vivo and in vitro models of infections to study the role of monocyte chemoattractant protein 1 (MCP-1)/CCL2 and nitric oxide (NO) in the bacterial clearance in sepsis. Our results show that MCP-1/CCL2 and NO levels are increased in the peritoneal cavity of mice 6 h after sepsis induced by cecal ligation and puncture. Pretreatment with anti-MCP-1/CCL2 monoclonal antibodies increased the number of colony-forming units (CFUs) recovered in the peritoneal lavage fluid. Moreover, CFU counts were increased in the peritoneal fluid of CCR2 mice subjected to cecal ligation and puncture. In vitro stimulation of peritoneal macrophages with recombinant MCP-1/CCL2 reduced CFU counts in the supernatant after challenge with Escherichia coli. Conversely, treatment with anti-MCP-1/CCL2 increased CFU counts under the same experimental condition. Stimulation of cultured macrophages with MCP-1/CCL2 and interferon had a synergistic effect on NO production. Macrophages from CCL2 mice showed a consistent decrease in NO production when compared with wild-type controls after stimulation with LPS + interferon. Finally, we showed incubation of macrophages with E. coli, and the ERK inhibitor U0126 increased CFU numbers and decreased intracellular levels of NO. In conclusion, we demonstrated for the first time that MCP-1/CCL2 has a crucial role in the clearance of bacteria by mechanisms involving increased expression of inducible NO synthase and production of NO by ERK signaling pathways.

Compared effects of inhibition and exogenous administration of hydrogen sulphide in ischaemia-reperfusion injury

IntroductionHaemorrhagic shock is associated with an inflammatory response consecutive to ischaemia-reperfusion (I/R) that leads to cardiovascular failure and organ injury. The role of and the timing of administration of hydrogen sulphide (H2S) remain uncertain. Vascular effects of H2S are mainly mediated through K+ATP-channel activation. Herein, we compared the effects of D,L-propargylglycine (PAG), an inhibitor of H2S production, as well as sodium hydrosulphide (NaHS), an H2S donor, on haemodynamics, vascular reactivity and cellular pathways in a rat model of I/R. We also compared the haemodynamic effects of NaHS administered before and 10 minutes after reperfusion.MethodsMechanically ventilated and instrumented rats were bled during 60 minutes in order to maintain mean arterial pressure at 40 ± 2 mmHg. Ten minutes prior to retransfusion, rats randomly received either an intravenous bolus of NaHS (0.2 mg/kg) or vehicle (0.9% NaCl) or PAG (50 mg/kg). PNU, a pore-forming receptor inhibitor of K+ATP channels, was used to assess the role of K+ATP channels.ResultsShock and I/R induced a decrease in mean arterial pressure, lactic acidosis and ex vivo vascular hyporeactivity, which were attenuated by NaHS administered before reperfusion and PNU but not by PAG and NaHS administered 10 minutes after reperfusion. NaHS also prevented aortic inducible nitric oxide synthase expression and nitric oxide production while increasing Akt and endothelial nitric oxide synthase phosphorylation. NaHS reduced JNK activity and p-P38/P38 activation, suggesting a decrease in endothelial cell activation without variation in ERK phosphorylation. PNU + NaHS increased mean arterial pressure when compared with NaHS or PNU alone, suggesting a dual effect of NaHS on vascular reactivity.ConclusionNaHS when given before reperfusion protects against the effects of haemorrhage-induced I/R by acting primarily through a decrease in both proinflammatory cytokines and inducible nitric oxide synthase expression and an upregulation of the Akt/endothelial nitric oxide synthase pathway.Keywords: hydrogen sulphide, inflammation mediators, therapeutic use, shock, hemorrhagic/drug therapy, haemodynamics/drug effects

Inhibitory Effect of the Culture Broth Extract of Aspergillus tamarii on Nitric Oxide Production and Its Antioxidative Activity

Many studies have explored suppression of aflatoxin produced by Aspergillus Genus. On the other hand, this study examined the inhibitory effect of the culture broth extract (CE) of A. tamarii obtained from dead silkworm on nitric oxide (NO) production and its antioxidative activity. The culture broth was extracted with EtOAc, dried, and then used in this experiment. As a result, CE did not show cytotoxicity on RAW 264.7 cells at any concentration. Moreover, CE suppressed lipopolysaccharide (LPS)-induced NO production of RAW 264.7 cells in a dose-dependent manner. The total phenol content according to the Folin-Dennis method, the antioxidative activity by DPPH, and the nitrate radical scavenging capacity of CE were increased in a dose-dependent manner. Thus, many of the phenolic compounds were considered to represent the antioxidative activity.

The procyanidin trimer C1 inhibits LPS-induced MAPK and NF-κB signaling through TLR4 in macrophages

Natural products and dietary components rich in polyphenols have been shown to reduce inflammation; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. This research was carried out to clarify the potential role of procyanidin trimer C1 in the anti-inflammatory effect of polyphenols. Procyanidin C1 inhibited inducible nitric oxide synthase-mediated nitric oxide production and the release of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-α) in lipopolysaccharide (LPS)-induced macrophages. Treatment with procyanidin C1 resulted in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels, as well as the expression of cell surface molecules (CD80, CD86, and MHC class II), which was induced by LPS. Furthermore, our data demonstrated that the anti-inflammatory effect of procyanidin C1 occurs through inhibition of mitogen-activated protein kinase (p38 and c-Jun N-terminal kinase) and nuclear factor-κB signaling pathways. These 2 factors play a major role in controlling inflammation, through toll-like receptor 4, suggesting that procyanidin C1 plays a potent role in promoting anti-inflammatory activity in macrophages. These results represent a novel and effective therapeutic intervention for the treatment of inflammatory disease.

Generation of avirulent Leishmania parasites and induction of nitric oxide production in macrophages by using polyacrylic acid

Polyacrylic acid (PAA) is one of the anionic synthetic polyelectrolytes and is used in various immunological and pharmaceutical applications. PAA has been used as adjuvant in veterinary vaccines, in particular. However, to our knowledge, there are no studies that document immunostimulant properties of PAA in Leishmania infection. The main aim of this study was to investigate the interaction of Leishmania parasites with PAA: the possible effects on the infectivity of Leishmania promastigotes; and, induction of nitric oxide (NO) production in macrophages in vitro. The cytotoxicity of PAA on both macrophages and Leishmania infantum promastigotes were determined by MTT assay. NO production in the macrophage culture supernatant was measured by the Griess method. A significant, dose-dependent and time-dependent decrease in the infection index was observed after PAA exposure. The value of this decrease was found to be between 93% and 100% for all concentration and time points. PAA (molecular weight (MW) 30, 100 kDa at 1mg/1h)-exposed parasites stimulate NO production significantly at 48 h post-infection (PI), when compared to the control. This study demonstrates that Leishmania parasites lost their virulence upon interaction with PAA, and this interaction induced NO production in infected macrophages. These results may have important implications in the development of anti-leishmanial vaccines and amelioration of immune response.

Effects of Curcumin on the Proliferation and Mineralization of Human Osteoblast-Like Cells: Implications of Nitric Oxide

Curcumin (diferuloylmethane) is found in the rhizomes of the turmeric plant (Curcuma longa L.) and has been used for centuries as a dietary spice and as a traditional Indian medicine used to treat different conditions. At the cellular level, curcumin modulates important molecular targets: transcription factors, enzymes, cell cycle proteins, cytokines, receptors and cell surface adhesion molecules. Because many of the curcumin targets mentioned above participate in the regulation of bone remodeling, curcumin may affect the skeletal system. Nitric oxide (NO) is a gaseous molecule generated from l-arginine during the catalization of nitric oxide synthase (NOS), and it plays crucial roles in catalization and in the nervous, cardiovascular and immune systems. Human osteoblasts have been shown to express NOS isoforms, and the exact mechanism(s) by which NO regulates bone formation remain unclear. Curcumin has been widely described to inhibit inducible nitric oxide synthase expression and nitric oxide production, at least in part via direct interference in NF-κB activation. In the present study, after exposure of human osteoblast-like cells (MG-63), we have observed that curcumin abrogated inducible NOS expression and decreased NO levels, inhibiting also cell prolifieration. This effect was prevented by the NO donor sodium nitroprusside. Under osteogenic conditions, curcumin also decreased the level of mineralization. Our results indicate that NO plays a role in the osteoblastic profile of MG-63 cells.

The nitric oxide donor sodium nitroprusside regulates polyamine and proline metabolism in leaves of Medicago truncatula plants

Nitric oxide (NO), polyamines, and proline have all been suggested to play key roles in a wide spectrum of physiological processes and abiotic stress responses. Although exogenous application of polyamines has been shown to induce NO production, the effect of NO on polyamine biosynthesis has not yet been elucidated. Several reports exist that demonstrate the protective action of sodium nitroprusside (SNP), a widely used NO donor, which acts as a signal molecule in plants responsible for the regulation of the expression of many defense-related enzymes. This study attempted to provide a novel insight into the effects of application of low (100μΜ) and high (2.5mM) concentrations of SNP on the biosynthesis of two major abiotic stress response-related metabolites, polyamines and proline, in mature (40 day) and senescing (65 day) Medicago truncatula plants. Physiological data showed that long-term (24h), higher SNP concentration resulted in decreased photosynthetic rate and stomatal conductance followed by intracellular putrescine and proline accumulation, as a result of an increase in biosynthetic arginine decarboxylase (ADC) and Δ11Abbreviations used: ROS, reactive oxygen species; RNS, reactive nitrogen species; SNP, sodium nitroprusside; PA, polyamine; ODC, ornithine decarboxylase; ADC, arginine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase; SAMDC, S-adenosylmethionine decarboxylase; DAO, diamine oxidase; PAO, polyamine oxidase; Put, putrescine; Spd, spermidine; Spm, spermine; P5CS, Δ1-pyrroline-5-carboxylate synthetase; P5CR, pyrroline-5-carboxylate reductase. -pyrroline-5-carboxylate synthetase (P5CS) enzymatic activity, respectively. Further analysis of polyamine oxidase (PAO)/diamine oxidase (DAO) polyamine catabolic enzymes indicated that DAO enzymatic activity increased significantly in correlation with putrescine accumulation, whereas PAO activity, involved in spermidine/spermine degradation, increased slightly. Moreover, transcriptional analysis of polyamine and proline metabolism genes (P5CS, P5CR, ADC, SPMS, SPDS, SAMDC, PAO, DAO) further supported the obtained data and revealed a complex SNP concentration-, time-, and developmental stage-dependent mechanism controlling endogenous proline and polyamine metabolite production. This is the first report to provide a global analysis leading to a better understanding of the role of the widely used NO donor SNP in the regulation of key stress-related metabolic pathways.

Daintain/AIF-1 (Allograft Inflammatory Factor-1) accelerates type 1 diabetes in NOD mice

A large body of experimental evidence suggests that cytokines trigger pancreatic β-cell death in type 1 diabetes mellitus. Daintain/AIF-1 (Allograft Inflammatory Factor-1), a specific marker for activated macrophages, is accumulated in the pancreatic islets of pre-diabetic BB rats. In the present study, we demonstrate that daintain/AIF-1 is released into blood and the levels of daintain/AIF-1 in the blood of type 1 diabetes-prone non-obese diabetic (NOD) mice suffering from insulitis are significantly higher than that in healthy NOD mice. When injected intravenously into NOD mice, daintain/AIF-1 stimulates white blood cell proliferation, increases the concentrations of blood glucose, impairs insulin expression, up-regulates nitric oxide (NO) production in pancreases and accelerates diabetes in NOD mice, while the antibody against daintain/AIF-1 delays or prevents insulitis in NOD mice. These results imply daintain/AIF-1 triggers type 1 diabetes probably via arousing immune cells activation and induction of NO production in pancreas of NOD mice.

Bluetongue virus – 23 stimulates inducible nitric oxide synthase expression and nitric oxide production in mononuclear cells of blood and/or regional lymphoid organs

Mononuclear leukocytes of peripheral blood mononuclear cells (PBMCs) and regional lymphoid organs (RLOs) play a critical role in primary BTV replication and subsequent viral dissemination to distant systemic organs. The lesions in animals develop primarily as a result of vascular insult, presumably induced by the activity of viral and/or proinflammatory vasoactive mediators. Hence, the current study was designed in sheep to investigate the responses of potent vasoactivators, inducible nitric oxide synthase (iNOS) and/or nitric oxide (NO) in mononuclear leukocytes of PBMCs and RLOs. The results show that BTV infection of sheep led to enhanced transcription of iNOS in PBMCs and in particular RLOs. The BTV RNAs and/or antigens were readily demonstrable in these mononuclear leukocytes, suggesting the possible role of BTV in iNOS induction. Moreover, upon in vitro infection of PBMCs with BTV-23, iNOS was up-regulated in time-dependent fashion and correlated with increased NO production. The results from these in vivo and in vitro studies thus suggest iNOS and NO produced by mononuclear leukocytes may potentially contribute to vascular-related pathology of BT.

Hyperbaric oxygen treatment prevents nitric oxide‐induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70

Heat shock proteins (HSPs), inflammatory cytokines, nitric oxide (NO), and localized hypoxia-induced apoptosis are thought to be correlated to the degree of cartilage injury. We investigated the effect of hyperbaric oxygen (HBO) on (1) interleukin-1β (IL-1β)-induced NO production and apoptosis of rabbit chondrocytes and (2) healing of articular cartilage defects. For the in vitro study, RT-PCR and Western blotting were performed to detect mRNA and protein expressions of HSP70, inducible NO synthase (iNOS), and caspase 3 in IL-1β-treated chondrocytes. To clarify that the HSP70 was necessary for anti-iNOS and anti-apoptotic activity by HBO, we treated the cells with an HSP70 inhibitor, KNK437. For the in vivo study, cartilage defects were created in rabbits. The HBO group was exposed to 100% oxygen at 2.5 ATA for 1.5 h a day for 10 weeks. The control group was exposed to normal air. After sacrifice, specimen sections were sent for examination using a scoring system. Immunohistochemical analyses were performed to detect the expressions of iNOS, HSP70, and caspase 3. Our results suggested that HBO upregulated the mRNA and protein expressions of HSP70 and suppressed those of iNOS and caspase 3 in chondrocytes. KNK437 inhibited the HBO-induced downregulation of iNOS and casapase 3 activities. The histological scores showed that HBO markedly enhanced cartilage repair. Immunohistostaining showed that HBO enhanced HSP70 expression and suppressed iNOS and caspase 3 expressions in chondrocytes. Accordingly, HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70.