iNOS mRNA Synthesis Research Articles

Essential amino acid tryptophan inhibits induction of inducible nitric oxide synthase gene expression in interleukin-1β stimulated hepatocytes

Background/objective: Tryptophan exerts protective effects against a variety of organ inflammation and injury, including liver. However, there are few scientific reports about the mechanisms involved in the action. Pro-inflammatory cytokine interleukin (IL)-1β stimulates the induction of inducible nitric oxide synthase (iNOS) expression and NO production in cultured hepatocytes (“in vitro liver injury model”), and the prevention of iNOS expression and NO production is considered to be an indicator of liver protection. This study aimed to examine whether tryptophan influences the induction of iNOS gene expression and the mechanisms.Methods: Tryptophan was added into primary cultures of rat hepatocytes stimulated by IL-1β. The iNOS induction, NO production and its signaling pathway were analyzed.Results: IL-1β induced iNOS gene expression, which was followed by iNOS expression and NO production. Tryptophan inhibited the expression of iNOS mRNA and protein, and decreased the production of NO. Transfection experiments with iNOS promoter-luciferase constructs revealed that tryptophan reduced the activities of iNOS mRNA synthesis and its stability. Tryptophan blocked two essential signaling pathways, the activation of nuclear factor (NF)-κB and upregulation of type I IL-1receptor (IL-1RI).Conclusions: Results indicate that tryptophan can prevent the NO production by the inhibition of iNOS gene expression, in part through NF-κB activation and IL-1RI upregulation, in inflamed hepatocytes. Tryptophan may be a potential therapeutic treatment in injured organs, including liver.Key words: tryptophan, inducible nitric oxide synthase, nitric oxide, cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor

Zanamivir Diminishes Lung Damage in Influenza A Virus-infected Mice by Inhibiting Nitric Oxide Production.

Severe pulmonary influenza A virus (IAV) infection causes lung inflammation and expression of inducible nitric oxide synthase (iNOS), leading to overproduction of nitric oxide (NO). We studied whether zanamivir reduces pulmonary inflammation through inhibition of NO production in mice. We treated IAV-infected mice daily with intranasal zanamivir. Controls were infected and either placebo-treated or untreated, or not infected and placebo-treated. Mice were weighed daily. After euthanasia on day 3, lungs were excised and bronchoalveolar lavage was performed and fluid nitrite concentration was determined. Lungs were analyzed microscopically. iNOS and IAV RNA levels in lungs were assessed using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Mice undergoing zanamivir treatment had less weight loss, viral replication, and lung damage, as well as significant reductions of local NO and iNOS mRNA synthesis (p<0.05). Zanamivir is associated with an anti-inflammatory effect mediated through inhibition of NO production in IAV-infected mice.

L-Carnitine has a liver-protective effect through inhibition of inducible nitric oxide synthase induction in primary cultured rat hepatocytes

Background: L-Carnitine has protective effects on various injured organs. However, it has not been reported whether L-carnitine influences the induction of inducible nitric oxide synthase (iNOS) expression during inflammation. Nitric oxide (NO) produced by iNOS is an inflammatory indicator in organs which become inflamed, including the liver.Objective: This study aimed to examine whether L-carnitine influences the induction of iNOS gene expression in inflammatory cytokine-stimulated hepatocytes and the mechanisms involved in the action. Methods: L-Carnitine was added into the primary cultures of rat hepatocytes stimulated by interleukin-1β (an in vitro liver injury model). The production of NO and induction of iNOS and its signaling pathway were analyzed.Results: Transfection experiments with iNOS promoter-luciferase constructs revealed how L-carnitine inhibited iNOS mRNA synthesis activity and reduced its stability. In support of this observation, L-carnitine reduced iNOS mRNA and iNOS protein expression levels, resulting in reduced NO production. L-Carnitine blocked two essential pathways for iNOS induction: IκB kinase (IκB degradation/NF-κB activation) and phosphatidylinositol 3-kinase/Akt (type I IL-1 receptor upregulation).Conclusions: L-Carnitine inhibited the induction of inflammatory mediator iNOS, partially through inhibition of NF-κB activation, which demonstrated L-carnitine has protective effects in an in vitro liver injury model. L-Carnitine may have therapeutic potential for organ injuries, including the liver.Keywords: L-carnitine, hepatic encephalopathy, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor

Immunomodulatory role of outer membrane vesicles of Shigella in mouse model

In our previous studies, we discussed the protective efficacy of the two types of vaccine formulation namely SOMVs (single-serotype outer membrane vesicles) and MOMVs (multi-serotype outer membrane vesicles). Here, we compared the immunogenic roles of these two types of formulations and also studied general immunomodulation by Shigella OMVs in adult BALB/c mice. The production of various pro-inflammatory (TNF-α, IL-1β, IL-6, IL-12, IL-18, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokine profile were assessed by in vivo, ex vivo and in vitro studies. MOMVs treated mice showed significantly enhanced cytokine production compared to SOMVs treated mice. MOMVs treatment has also upregulated iNOS mRNA synthesis in macrophages. Overall the OMVs of Shigella were found to show a mixed Th1/Th2 response and maintain the balance between pro-inflammation and anti-inflammation in mice. This will be crucial in the development of the next generation OMVs based vaccine against shigellosis.

Sivelestat Suppresses iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes

Recent evidence has indicated that sivelestat, a neutrophil elastase inhibitor, has liver-protective effects in a variety of liver injuries. Proinflammatory cytokines including interleukin (IL)-1β stimulate the induction of inducible nitric oxide synthase (iNOS) gene expression, leading to excess production of NO and resulting in liver damage. We hypothesized that inhibition of iNOS induction underlies the protective effects of sivelestat on the liver. The objective of this study was to investigate whether sivelestat directly influences iNOS induction in cultured hepatocytes, which is used as a simple in vitro injury model, and to determine the mechanism involved. Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of sivelestat. The induction of iNOS and its signaling pathway were analyzed. Sivelestat inhibited the induction of iNOS mRNA and its protein, followed by decreased production of NO. Transfection and iNOS gene antisense-transcript experiments revealed that sivelestat reduced the levels of iNOS mRNA at both the promoter activation and mRNA stabilization steps. However, sivelestat had no effects on the degradation of IκB and nuclear translocation of NF-κB subunit p65, although it moderately blocked the activation of NF-κB. In contrast, sivelestat blocked the upregulation of IL-1 receptor I through the inactivation of phosphatidylinositol 3-kinase/Akt. Delayed sivelestat addition experiments demonstrated that the destabilization of the iNOS mRNA contributed more significantly to the inhibitory effects of sivelestat than the reduction in iNOS mRNA synthesis. Sivelestat may provide useful therapeutic effects through the suppression of iNOS induction involved in liver injury.

Nitric Oxide Causes Anoikis through Attenuation of E-Cadherin and Activation of Caspase-3 in Human Gastric Carcinoma AZ-521 Cells Infected with Mycoplasma hyorhinis

Mycoplasma hyorhinis (M. hyorhinis) infection leads cultured cells to various biological alterations in cell metabolism including apoptosis. Apoptosis induced by M. hyorhinis has mainly been considered to be due to mycoplasmal endonucleases. We previously reported that apoptosis in a human carcinoma cell line AZ-521 infected with M. hyorhinis was enhanced by addition of L-ascorbic acid to cell cultures. Since both L-ascorbic acid addition and M. hyorhinis infection activated cellular iNOS, we examined the hypothesis that nitric oxide (NO) exerts an apoptotic effect on M. hyorhinis-infected cells and down-regulates E-cadherin. In this study, we showed that M. hyorhinis infection activates iNOS mRNA synthesis, NO production, and caspase-3 activity and attenuates E-cadherin mRNA synthesis by quantitative real-time PCR, Griess assay and fluorescence caspase-3 detection. L-NAME decreased the numbers of apoptotic cells through inhibition caspase-3 activity. Our results indicate that NO causes anoikis throughout attenuation of E-cadherin and activation of caspase-3 in human gastric carcinoma cell line AZ-521 cells infected with M. hyorhinis.

Characterisation of the anti-inflammatory potential of Vitex trifolia L. (Labiatae), a multipurpose plant of the Pacific traditional medicine

Aim of the study Vitex trifolia L. (Labiatae) is a plant commonly employed against Ciguatera Fish Poisoning (CFP) in the Pacific region. Here, the anti-inflammatory potential of an aqueous extract of Vitex trifolia leaves was evaluated by monitoring its effects on the modulation of cytokines, the mediators of inflammation, as well as on the expression profiles of inducible nitric oxide synthase (iNOS) which produces the free radical nitric oxide (NO). Materials and methods We prepared an aqueous extract from Vitex trifolia leaves and evaluated its anti-inflammatory potency by monitoring its effect on the lipopolysaccharide (LPS)-induced cytokines and iNOS mRNA over-production in RAW 264.7 macrophages using quantitative Polymerase Chain Reaction (qPCR) and Enzyme-Linked Immunosorbent Assay (ELISA) methods. Results Aqueous extract of Vitex trifolia leaves showed significant dose- and time-dependent inhibitory activity on interleukin (IL)-1β, IL-6 and iNOS mRNA synthesis, but slight effect on tumor necrosis factor (TNF)-α, all of which are involved in the inflammatory response. Moreover, the plant extract seemed to induce the LPS-dependant IL-10 anti-inflammatory cytokine. These results were further confirmed by ELISA using specific antibodies to mouse IL-6, IL-10 and TNF-α. Conclusion The anti-inflammatory effects of Vitex trifolia could validate its utilization as a traditional remedy against CFP and emphasises its potential therapeutic value against other inflammatory diseases. Therefore, this plant is a promising candidate for further screening of its active compounds through activity-guided fractionation.

Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin

To investigate the possible involvement of the nitric oxide radical (NO) in ciguatera fish poisoning (CFP), the in vitro effects of the main Pacific ciguatoxin (P-CTX-1B) and bacterial lipopolysaccharide (LPS) were comparatively studied on neuroblastoma Neuro-2a and on macrophage RAW 264.7 cell lines. NO accumulation was quantified by measuring nitrite levels in cellular supernatant using Griess reagent while the up-regulation of inducible nitric oxide synthase (iNOS) at the mRNA level was quantified via Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). P-CTX-1B caused a concentration- and time-dependent induction of iNOS in RAW 264.7 cells but not in Neuro-2a cells. NO production was evidenced by increased nitrite levels in the 10 μM range after 48 h of RAW 264.7 cells exposure to LPS and P-CTX-1B (0.05 μg/ml and 6 nM, respectively). The expression of iNOS mRNA peaked at 8 h for LPS then gradually decreased to low level at 48 h. In contrast, a sustained level was recorded with P-CTX-1B in the 8–48 h time interval. The addition of N ω-nitro- l-arginine methyl ester ( l-NAME), a stereoselective NOS inhibitor, strongly diminished NO formation but had no effect on iNOS mRNA synthesis. The implication of NO in CFP paves the way for new therapies for both western and traditional medicines.

Sustained Nitric Oxide Synthesis Contributes to Immunopathology in Ongoing Myocarditis Attributable to Interleukin-10 Disorders

Ongoing coxsackievirus B3 (CVB3) myocarditis is characterized by persistence of viral RNA and chronic inflammation primarily mediated by macrophages and T cells. Activated macrophages produce anti-viral effector molecules comprising reactive nitrogen intermediates; however, reactive nitrogen intermediates also contribute to host tissue damage. Controlled activation of macrophages depends on interferon (IFN)-gamma and interleukin (IL)-10. To evaluate mechanisms involved in CVB3-induced pathogenesis of myocarditis, we determined the relationship of inducible nitric-oxide synthase (iNOS) mRNA expression with IFN-gamma and IL-10 secretion during CVB3 infection in different mouse strains. We found in susceptible A.BY/SnJ mice that develop ongoing myocarditis, a low and delayed IFN-gamma secretion and highly diminished IL-10 production compared with resistant C57BL/6 mice. Consequently, iNOS mRNA synthesis was delayed but clearly prolonged in susceptible mice. IL-10 gene-deficient mice confirmed the regulatory role of IL-10 in the outcome of CVB3 myocarditis. These mice did not establish a persistent cardiac infection and revealed IFN-gamma secretion kinetics similar to resistant mice but showed a slightly elongated cardiac iNOS mRNA expression resulting in extended myocarditis. We conclude that coordinated secretion of IFN-gamma and IL-10 is crucial for the effective resolution of CVB3 myocarditis. Moreover, lack of regulatory IL-10 leads to uncontrolled iNOS mRNA production, thus contributing to ongoing myocardial injury.

Inhibitory activity of nitric oxide production in RAW 264.7 cells of daldinals A–C from the fungus Daldinia childiae and other metabolites isolated from inedible mushrooms

Three benzophenone derivatives, daldinals A–C, from the Japanese fungus Daldinia childiae were evaluated for their inhibitory activity of nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in RAW 264.7 cells. They strongly suppressed the LPS-induced production of NO with IC50 values of 15.2, 4.6 and 6.4 μM, respectively. To clarify the mechanism involved, total RNA extraction, followed by reverse-transcribed, polymerase chain reaction (PCR) for inducible nitric oxide synthase (iNOS) mRNA and finally electrophoresis on agarose gel were performed. These experimental results suggest that the inhibition of the LPS-induced NO production of daldinal B is due to the inhibition of iNOS mRNA synthesis. Further, their biological activities were compared with those of other metabolites obtained during our studies on biologically active substances from inedible fungi in recent years.

Green tea polyphenol epigallocatechin-3-gallate inhibits the expression of nitric oxide synthase and generation of nitric oxide induced by ultraviolet B in HaCaT cells

Nitic oxide (NO) has been implicated in the pathogenesis of various inflammatory diseases, including sunburn and pigmentation induced by ultraviolet irradiation. Epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can protect skin from ultraviolet-induced damage. The purpose of this study was to investigate the protective mechanisms of EGCG on inducible nitric oxide synthase (iNOS) expression and NO generation by ultraviolet B (UVB) irradiation in HaCaT cells. HaCaT cells were irradiated with UVB 30 mJ/cm 2 and pretreated with EGCG at varying concentrations. The iNOS mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and NO production was quantified by spectrophotometric method. The expression of NF-kappaB P65 was measured by immunofluorescence cytochemistry staining. The expression of iNOS mRNA and generation of NO in HaCaT cells were increased by UVB irradiation. EGCG down regulated the UVB-induced iNOS mRNA synthesis and NO generation in a dose dependent manner. The UVB-induced ctivation and translocation of NF-kappaB were also down regulated by EGCG treatment in HaCaT cells (P < 0.01). Green tea derived-EGCG can inhibit and down regulate the UVB-induced activation and translocation of NF-kappaB, expression of iNOS mRNA and generation of NO respectively, indicating EGCG may play a protective role from UVB-induced skin damage.

Acute alterations of endothelin-1 and iNOS expression and control of the brain microcirculation after head trauma

The biosynthetic equilibrium between endothelin-1 (ET-1, a vasoconstricting agent) and nitric oxide (NO, a gas with vasodilating effects) is thought to play a role in the autoregulation of microvessel contractility and maintenance of adequate perfusion after traumatic brain injury. ET-1 is a constitutively expressed peptide, while the gene that encodes for the inducible nitric oxide synthase (iNOS, an enzyme responsible for the synthesis of excessive and toxic amounts of NO) is solely activated after brain injury. We employed the Marmarou acceleration impact model of brain injury (400 g from 2 m) to study the effect of closed head trauma on the rat brain microcirculation. Following head trauma we analyzed changes of cerebral cortex perfusion using laser Doppler flowmetry and ultrastructural alterations of endothelial cells. We temporally correlated these changes with the expression of ET-1 (immunocytochemistry) and iNOS ( in situ hybridization) to assess the role of these vasoactive agents in vascular contractility and cortical perfusion. Cortical perfusion was reduced by ~ 50% during the second hour as compared to values during preceding time points after TBI, reached a peak minutes before 3 h, and subsequently showed a trend towards normalization. A significant reduction in the lumen of microvessels and severe distortion of their shape were observed after the fourth hour post-trauma. At the same time period ET-1 expression in endothelial cells was stronger than in microvessels of control animals. ET-1 expression was further increased at 24 h after TBI. iNOS mRNA synthesis was strongly upregulated in the same cells at 4 h but was undetectable at 24 h post trauma. Our combined functional, cellular and molecular approach supports the notion that ET-1 and iNOS are expressed differentially in time within individual endothelial cells of cortical microvessels for the control of cortical blood flow following closed head trauma. This differential expression further indicates a reciprocal interaction in the synthesis of these two molecules which may underlie the control of microvascular autoregulation. [Neurol Res 2001; 23: 139-143]

Inhibition of inducible nitric oxide synthesis by oxidized lipoprotein(a) in a murine macrophage cell line

Increased plasma levels of human lipoprotein(a) (Lp(a)) are highly correlated with the development of atherosclerotic lesions. During our study, we investigated the effects of native and hypochlorite oxidized lipoprotein(a) (ox-Lp(a)) on nitric oxide production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide/interferon stimulated mouse macrophages (J774A.1). Ox-Lp(a) (0–2 μg/ml) induces a dose dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a dose dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of ox-Lp(a). Ox-Lp(a) decreases iNOS mRNA synthesis as shown by reverse transcription-polymerase chain reaction. Ox-Lp(a) induced iNOS inhibition might contribute to the development of atherosclerotic lesions by reducing the anti-atherogenic effects of nitric oxide.

Low Environmental pH Is Responsible for the Induction of Nitric-oxide Synthase in Macrophages: EVIDENCE FOR INVOLVEMENT OF NUCLEAR FACTOR-κB ACTIVATION

Stimulation of macrophages with endotoxin and/or cytokines is responsible for the expression of the inducible isoform of nitric oxide synthase (iNOS). Because macrophages are exposed to low pH within the microenvironment of inflammatory lesions, the potential role of acidic pH as an additional regulator of iNOS was investigated. Substitution of the culture medium of rat peritoneal macrophages at pH 7.4 with medium at pH 7.0 up-regulated iNOS activity, as reflected by a 2.5-fold increase in nitrite accumulation. The increase in iNOS activity was associated with a similar increase in iNOS mRNA expression that reflected an increase in iNOS mRNA synthesis rather than stability. Low environmental pH-induced iNOS gene transcription involved the activation of nuclear factor-kappaB (NF-kappaB) transcription factor since exposure of macrophages to low environmental pH both increased NF-kappaB binding activity in the nucleus and enhanced NF-kappaB-driven reporter gene expression. In addition, treatment of macrophages with pyrrolidine dithiocarbamate or n-acetyl-leucinyl-leucinyl-norleucinal, two drugs preventing NF-kappaB translocation to the nucleus, canceled low pH-induced nitrite accumulation. The overall mechanism required the synthesis of tumor necrosis factor alpha (TNFalpha). Indeed, 1) elevated TNFalpha bioactivity was observed in the medium of macrophages exposed to pH 7.0, and 2) incubation of macrophages with a neutralizing anti-TNFalpha antibody impaired both NF-kappaB activation and nitrite accumulation in response to acid challenge. In summary, exposure of macrophages to acidic microenvironment in inflammatory lesions leads to the up-regulation of iNOS activity through the activation of NF-kappaB.

Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat.

The role of nitric oxide in obliterative bronchiolitis development, i.e., chronic rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in the intragraft inducible nitric oxide synthase (iNOS) mRNA and mononuclear inflammatory cell iNOS immunoreactivity was demonstrated during progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared to syngeneic grafts. In nontreated allografts, however, intragraft nitric oxide production was decreased, most likely because of loss of iNOS epithelial expression. Treatment with aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase, was associated with enhanced proliferation of alpha-smooth muscle actin immunoreactive cells and the intensity of obliterative bronchiolitis early after transplantation. Aminoguanidine treatment did not affect iNOS mRNA synthesis or intragraft nitric oxide production, but decreased iNOS immunoreactivity in smooth muscle cells. Treatment with L-arginine, a precursor of nitric oxide, significantly reduced obliterative changes. L-arginine supplementation enhanced intragraft iNOS mRNA synthesis and iNOS immunoreactivity in capillary endothelial and smooth muscle cells as well as intragraft nitric oxide production. Immunohistochemical analysis of allografts showed that neither iNOS inhibition nor supplementation of the nitric oxide pathway affected the number of graft-infiltrating CD4+ and CD8+ T cells, ED1+ and ED3+ macrophages, immune activation with expression of IL-2R or MHC class II, or production of macrophage or Th1 cytokines. In contrast, L-arginine treatment was associated with increased staining for Th2 cytokines IL-4 and IL-10. In conclusion, this study demonstrates that nitric oxide has a protective role in obliterative bronchiolitis development in this model, and suggests that nitric oxide either directly or indirectly inhibits smooth muscle cell proliferation and modulates immune response towards Th2 cytokines.

Hyperbaric oxygenation reduces the cytostatic activity and transcription of nitric oxide synthetase gene of mouse peritoneal macrophages

The effects of hyperbaric oxygenation (HBO) and hyperbaric air (HBA) on the cytostatic activity, peroxynitrite synthesis and transcription of the inducible nitric oxide synthetase (iNOS) gene of murine peritoneal macrophages were studied in vitro. Exposure of mice to HBO or HBA significantly reduced the cytostatic activity, peroxynitrite synthesis and transcription of iNOS mRNA of their macrophages stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). These results indicate that HBO and HBA treatments of mice reduce the cytostatic activity of peritoneal macrophages by reducing iNOS mRNA synthesis.