Inducible Nitric Oxide Synthase Enzyme Research Articles

A new era of diagnostic modalities for type 1 leprosy reactions: Promise for the future.

Type 1 reactions (T1R) are known to occur in upto 30 per cent of patients in the borderline spectrum of leprosy1. It indicates an unstable immunity in a patient. The reactions can be either upgrading, towards the tuberculoid pole or downgrading, towards the lepromatous pole. These may be purely cutaneous, neural or a combination of both2. In early stages the diagnosis may be missed and hence, initiation of treatment delayed. This may have detrimental effects including permanent neurological sequlae. Though a thorough clinico-histological evaluation still remains the gold standard for diagnosis, various diagnostic assays are being used in research. These include cytokine mRNA levels, e.g. interferon-γ (IFN-γ), interleukin (IL)-1β, IL-2, IL-12, tumour necrosis factor α (TNFα) and enzyme-inducible nitric oxide synthase (iNOS)3. Previous studies indicate that potential biomarkers of T1R include CXCL10 and IL6 whereas IL7, platelet derived growth factor-BB (PDGF-BB) and IL6, may be markers of type 2 reaction (TR2)4. Cytokine assays have been the focus of research in the recent past. The gene expression of CXCL10 and its receptor, CXCR3 has been the focus of several recent studies. CXCL10 is a chemokine induced primarily by IFN-γ, produced by macrophages, T cells, and keratinocytes. It causes recruitment of CXCR3+ cells into the tissue. CXCR3+ cells also release Th1 cytokines leading to further upregulation of CXCR3 ligands leading to T1R response and granuloma formation5. The expression of these cytokines is higher in type 1 reaction, in comparison to non-reactional skin biopsies throughout the whole spectrum of leprosy. However, serological assays at baseline and immediately before T1R have not been predictive4. It has been seen that patients with high cytokine levels have a poor recovery from nerve function impairment during an acute episode of TIR6. They also have a high risk of reactivation of symptoms during treatment, and a high risk of recurrence of T1R within two months of completing the steroid regimen. Manandhar et al7 found that the levels of IFN-γ and TNF-α fell during treatment with steroids. However, the levels of TNFα increased as the steroid dose was reduced. The levels of IL-10 increased throughout the duration of treatment for T1R7. It would be worthwhile to study the utility of aforementioned markers to objectively document response to treatment. Though the study by Sharma and colleagues in this issue8 supports these observations, unfortunately, no assay has yet been described which can predict accurately the development of T1R. TNFα and CXCR3 have shown some promise and should be researched more extensively. It would also be worth studying the utility of newer markers to further subclassify the reaction into ‘ upgrading ‘ and ‘downgrading’ reactions and to predict neural involvement and severity. This might give us better insight into the pathomechanisms of reactions in leprosy. The use of cytokine assays is currently restricted to research, and as of today, we are relying on a thorough clinical assessment and histology if available, is still the most cost-effective, rapid and reliable way to diagnose T1R.

Melatonin treatment improves aging-related conditions in the liver of Apo E-KO mice

Aging is a complex, dynamic, and multifaceted process that is currently still poorly understood [1]. Hypercholesterolemia increases and exacerbates stress signals contributing to aging-related disorders in different organs such as liver [2]. Aging may contribute to the loss of cells in vital structures or organs through several mechanisms. Sirtuin1 (SIRT1) is a member of the sirtuin family of protein deacetylases involved in life span extension; however, its involvement in the aging is not yet completely defined. Recently, it was shown that melatonin, a pleiotropic molecule, activates SIRT1 and modulates oxidative stress-induced senescence and pro-survival pathways [3]. We treated 6and 15-week-old apolipoprotein E-deficient mice (ApoE -/-) with melatonin to evaluate its anti-aging effects. Morphological changes and expression of SIRT1, inducible nitric oxide synthase (iNOS) and antioxidant enzymes (SOD, CAT) were evaluated in the liver of these mice. We observed that SIRT1 expression and antioxidants enzyme activities were decreased in the liver of ApoE deficient mice between 6 and 15 weeks. On the other hand, hypercholesterolemia induced an elevated expression of iNOS in the liver of ApoE -/animals. The treatment with melatonin improves the morphological impairment of the liver, reduced the oxidative stress and inhibited liver aging processes most likely via SIRT1 regulation.

Microglia and motor neurons during disease progression in the SOD1G93A mouse model of amyotrophic lateral sclerosis: changes in arginase1 and inducible nitric oxide synthase.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the motor system. Although the etiology of the disease is not fully understood, microglial activation and neuroinflammation are thought to play a role in disease progression.MethodsWe examined the immunohistochemical expression of two markers of microglial phenotype, the arginine-metabolizing enzymes inducible nitric oxide synthase (iNOS) and arginase1 (Arg1), in the spinal cord of a mouse model carrying an ALS-linked mutant human superoxide dismutase transgene (SOD1G93A) and in non-transgenic wild-type (WT) mice. Immunolabeling for iNOS and Arg1 was evaluated throughout disease progression (6 to 25 weeks), and correlated with body weight, stride pattern, wire hang duration and ubiquitin pathology. For microglia and motor neuron counts at each time point, SOD1G93A and WT animals were compared using an independent samples t-test. A Welch t-test correction was applied if Levene’s test showed that the variance in WT and SOD1G93A measurements was substantially different.ResultsDisease onset, measured as the earliest change in functional parameters compared to non-transgenic WT mice, occurred at 14 weeks of age in SOD1G93A mice. The ventral horn of the SOD1G93A spinal cord contained more microglia than WT from 14 weeks onwards. In SOD1G93A mice, Arg1-positive and iNOS-positive microglia increased 18-fold and 7-fold, respectively, between 10 and 25 weeks of age (endpoint) in the lumbar spinal cord, while no increase was observed in WT mice. An increasing trend of Arg1- and iNOS-expressing microglia was observed in the cervical spinal cords of SOD1G93A mice. Additionally, Arg1-negative motor neurons appeared to selectively decline in the spinal cord of SOD1G93A mice, suggesting that Arg1 may have a neuroprotective function.ConclusionsThis study suggests that the increase in spinal cord microglia occurs around and after disease onset and is preceded by cellular pathology. The results show that Arg1 and iNOS, thought to have opposing inflammatory properties, are upregulated in microglia during disease progression and that Arg1 in motor neurons may confer protection from disease processes. Further understanding of the neuroinflammatory response, and the Arg1/iNOS balance in motor neurons, may provide suitable therapeutic targets for ALS.

The role of sex hormones in induced-systemic inflammation in female albino rats

Estrogen (E(2)) and progesterone (P) hormones have a pro-inflammatory and an anti-inflammatory role under different conditions. The current study explored this phenomenon in the context of septic inflammation. This study involved 48 female albino rats. E(2) (4 mg/100 g body weight (b.w.) and P (5 mg/kg b.w.) were administered to ovariectomized (OVX) rats after systemic inflammation (SI) induced by puncturing the caecum I cm from its end with a single hole by using a 21-gauge needle. Key indices of inflammation and apoptosis were evaluated. OVX animals subjected to SI showed significantly increased levels of serum tumor necrosis factor-alpha (TNF-u), C reactive protein (CRP) and alanine aminotransferase (ALT). They also showed higher levels of expression of the enzyme inducible nitric oxide synthase (iN OS); 312 ± 43 mg/ml; in the liver, and the activity of both cyclooxygenase 2 (COX-2); 59.4 ± 3.2 U/ml; and caspase 3 enzymes; 6.3 ± 0.54 ng/ml; when compared to non-OVX animals subjected to (SI), (180 ± 3 mg/ml, 16.4 ± 1.69 U/ml, 0.98 ± 0.23 ng/ml respectively). Administration of E(2) resulted in a significant reduction of all serum and liver tissue parameters of inflammation (e.g.decreased iNOS; 193 ± 28 mg/ml and COX-2; 27.6 ± 3.91 U/ml) and decreased apoptosis (Caspase 3; 1.18 ± 0.21 ng/ml). In contrast, OVX animals injected with P before induction of SI showed a significant rise of all measured parameters. E(2) and Pin physiological levels have contrasting though complementary roles in regulation of the immune system possibly allowing a limited inflammatory response while preventing excessive damage to the tissues.

AETIQ: A Novel Synthetic Compound with Anti-inflammatory Properties in Activated Microglia

Neuroinflammation is believed to be involved in the pathogenesis of neurodegenerative diseases. Our novel synthetic compound 2-acetyl-1-ethyl-7-hydroxy-6-methoxy-1, 2, 3, 4-tetrahydroisoquinoline (AETIQ) was tested for its anti-inflammatory properties in activated microglial BV-2 cells. AETIQ attenuated nitric oxide (NO) and reactive oxygen species generation. It also downregulated the production of the proinflammatory enzymes inducible NO synthase, cyclooxygenase-2, and matrix metalloproteinase-3 at both mRNA and protein levels. Furthermore, AETIQ suppressed generation of the proinflammatory cytokines IL-1β and TNF-α as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA assays. AETIQ attenuated NFκB signaling by downregulating NFκB nuclear translocation. The compound was stable against the liver enzymes in the microsomal and S9 fractions, indicative of good bioavailability. These results suggested that AETIQ might be utilized towards development of a therapy for neuroinflammation-related diseases.

Lancemaside A from Codonopsis lanceolata modulates the inflammatory responses mediated by monocytes and macrophages.

In this study, we aimed to examine the cellular and molecular mechanisms of lancemaside A from Codonopsis lanceolata (Campanulaceae) in the inflammatory responses of monocytes (U937 cells) and macrophages (RAW264.7 cells). Lancemaside A significantly suppressed the inflammatory functions of lipopolysaccharide- (LPS-) treated RAW264.7 cells by suppressing the production of nitric oxide (NO), the expression of the NO-producing enzyme inducible NO synthase (iNOS), the upregulation of the costimulatory molecule CD80, and the morphological changes induced by LPS exposure. In addition, lancemaside A diminished the phagocytic activity of RAW264.7 cells and boosted the neutralizing capacity of these cells when treated with the radical generator sodium nitroprusside (SNP). Interestingly, lancemaside A strongly blocked the adhesion activity of RAW264.7 cells to plastic culture plates, inhibited the cell-cell and cell-fibronectin (FN) adhesion of U937 cells that was triggered by treatment with an anti-β1-integrin (CD29) antibody and immobilized FN, respectively. By evaluating the activation of various intracellular signaling pathways and the levels of related nuclear transcription factors, lancemaside A was found to block the activation of inhibitor of κB kinase (IKK) and p65/nuclear factor- (NF-) κB. Taken together, our findings strongly suggest that the anti-inflammatory function of lancemaside A is the result of its strong antioxidative and IKK/NF-κB inhibitory activities.

Antiinflammatory effects of essential oil from the leaves of Cinnamomum cassia and cinnamaldehyde on lipopolysaccharide-stimulated J774A.1 cells.

Cassia oil (CO) from different parts of Cinnamomum cassia have different active components. Very few pharmacological properties of cassia leaf oil have been reported. This study investigated and compared effects of cassia leaf oil and cinnamaldehyde on lipopolysaccharide (LPS)-activated J774A.1 cells. Volatile compositions in cassia leaf oil were identified by gas chromatography-mass spectrometry (MS)/MS. Effects of CO and cinnamaldehyde on LPS-activated J774A.1 cells were investigated by determining nitric oxide (NO) production using Griess reaction assay; expression of pro-inflammatory cytokines, enzymes involve in inflammatory mediators; antiinflammatory cytokines, and iron exporter ferroportin1 (Fpn1) using reverse transcription-polymerase chain reaction; and production of tumor necrosis factor (TNF-α) and interleukin (IL)-10 using ELISA. The main component of CO was cinnamaldehyde. Both oils at 1-20 μg/ml markedly inhibited NO production in LPS-activated J774A.1 cells with IC50 value of 6.1 ± 0.25 and 9.97 ± 0.35 μg/ml, respectively. They similarly inhibited mRNA expression of pro-inflammatory cytokines and chemokines. These mediators included TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α in LPS-activated cells. They also significantly decreased expression of inducible enzymes inducible nitric oxide synthase, cyclooxygenase-2, microsomal prostaglandin-E synthase-1. In the opposite way, they increased mRNA expression and the production of antiinflammatory cytokines IL-10 and transforming growth factor-β. In addition, they promoted the expression of Fpn1. These results demonstrated that inhibitory effects of cassia leaf oil from C. cassia mainly came from cinnamaldehyde. This compound not only inhibited inflammatory mediators but also activated antiinflammatory mediators in LPS-activated J774A.1 cells. It may also have an effect on iron regulatory proteins in activated macrophages.

Amomum tsao-ko suppresses lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages via Nrf2-dependent heme oxygenase-1 expression.

Amomum tsao-ko Crevost et Lemaire, used as a spice in Asia, is an important source of Chinese cuisine and traditional Chinese medicines. A. tsao-ko is reported to exert a variety of biological and pharmacological activities, including anti-proliferative, anti-oxidative and neuroprotective effects. In this study, NNMBS227, consisting of the ethanol extract of A. tsao-ko, exhibited potent anti-inflammatory activities in RAW264.7 macrophages. We investigated the effect of NNMBS227 in the suppression of pro-inflammatory mediators, including pro-inflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2) and cytokines (tumor necrosis factor-α and interleukin-1β) in LPS stimulated macrophages. NNMBS227 also inhibited the phosphorylation and degradation of IκB-α, as well as the nuclear translocation of nuclear factor kappa B (NF-κB) p65 caused by stimulation with LPS. In addition, NNMBS227 induced heme oxygenase (HO)-1 expression through the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in macrophages. Using tin protoporphyrin (SnPP), an HO activity inhibitor, we confirmed an association between the anti-inflammatory effects of NNMBS227 and the up-regulation of HO-1. These findings suggest that Nrf2-dependent increases in the expression of HO-1 induced by NNMBS227 conferred anti-inflammatory activities in LPS stimulated RAW264.7 macrophages.

The anti-inflammatory effect of Indonesian Areca catechu leaf extract in vitro and in vivo.

BACKGROUND/OBJECTIVESOverproduction of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) enzyme can cause inflammation. Cyclooxygenase-2 (COX-2) is also involved in the inflammatory response through regulation of nuclear factor-kappa B (NF-κB). Areca catechu is one of the known fruit plants of the Palmaceae family. It has been used for a long time as a source of herbal medicine in Indonesia. In this study, we explored the effect of Indonesian Areca catechu leaf ethanol extract (ACE) in lipopolysaccharide (LPS)-induced inflammation and carrageenan-induced paw edema models. Recently, this natural extract has been in the spotlight because of its efficacy and limited or no toxic side effects. However, the mechanism underlying its anti-inflammatory effect remains to be elucidated.MATERIALS/METHODSWe measured NO production by using the Griess reagent, and determined the expression levels of inflammation-related proteins, such as iNOS, COX2, and NF-κB, by western blot. To confirm the effect of ACE in vivo, we used the carrageenan-induced paw edema model.RESULTSCompared to untreated cells, LPS-stimulated RAW 264.7 cells treated with ACE showed reduced NO generation and reduced iNOS and COX-2 expression. We found that the acute inflammatory response was significantly reduced by ACE in the carrageenan-induced paw edema model.CONCLUSIONTaken together, these results suggest that ACE can inhibit inflammation and modulate NO generation via downregulation of iNOS levels and NF-κB signaling in vitro and in vivo. ACE may have a potential medical benefit as an anti-inflammation agent.

Local Arginase 1 Activity Is Required for Cutaneous Wound Healing

Chronic nonhealing wounds in the elderly population are associated with a prolonged and excessive inflammatory response, which is widely hypothesized to impede healing. Previous studies have linked alterations in local L-arginine metabolism, principally mediated by the enzymes arginase (Arg) and inducible nitric oxide synthase (iNOS), to pathological wound healing. Over subsequent years, interest in Arg/iNOS has focused on the classical versus alternatively activated (M1/M2) macrophage paradigm. Although the role of iNOS during healing has been studied, Arg contribution to healing remains unclear. Here, we report that Arg is dynamically regulated during acute wound healing. Pharmacological inhibition of local Arg activity directly perturbed healing, as did Tie2-cre-mediated deletion of Arg1, revealing the importance of Arg1 during healing. Inhibition or depletion of Arg did not alter alternatively activated macrophage numbers but instead was associated with increased inflammation, including increased influx of iNOS+ cells and defects in matrix deposition. Finally, we reveal that in preclinical murine models reduced Arg expression directly correlates with delayed healing, and as such may represent an important future therapeutic target.

Mice genetically inactivated in interleukin‐17A receptor are defective in long‐term control of Mycobacterium tuberculosis infection

Interleukin-17A (IL-17A), a pro-inflammatory cytokine acting on neutrophil recruitment, is known to play an important role during Mycobacterium tuberculosis infection, but the role of IL-17A receptor signalling in immune defence against this intracellular pathogen remains poorly documented. Here we have analysed this signalling using C57BL/6 mice genetically inactivated in the IL-17 receptor A subunit (IL-17RA(-/-) ). Although early after infection bacterial growth was controlled to the same extent as in wild-type mice, IL-17RA(-/-) mice were defective in exerting long-term control of M.tuberculosis infection, as demonstrated by a progressively increasing pulmonary bacterial burden and shortened survival time. Compared with infected wild-type mice, IL-17RA(-/-) mice showed impaired recruitment of neutrophils to the lungs at the early but not the late stage of infection. Pulmonary tumour necrosis factor-α, IL-6 and particularly IL-10 levels were decreased in the absence of IL-17RA signalling, whereas IL-1β was increased. CD4(+) -mediated and γδ-mediated IL-17A production was dramatically increased in IL-17RA(-/-) mice (confirming part of their phenotype), whereas production of interferon-γ and expression of the bactericidal enzyme inducible nitric oxide synthase were not affected. Collectively, our data suggest that early but not late neutrophil recruitment is essential for IL-17A-mediated long-term control of M.tuberculosis infection and that a functional interferon-γ response is not sufficient to control M.tuberculosis growth when the IL-17RA pathway is deficient. As treatment of auto-immune diseases with anti-IL-17A antibodies is actually being tested in clinical studies, our data suggest that caution should be taken with respect to possible reactivation of tuberculosis.

Mice lacking inducible nitric oxide synthase develop exacerbated hepatic inflammatory responses induced by Plasmodium berghei NK65 infection

Infection of mice with Plasmodium berghei NK65 represents a well-recognized malaria model in which infection is accompanied by an intense hepatic inflammatory response. Enzyme-inducible nitric oxide synthase is an important regulator of inflammation and leukocyte recruitment in microvessels, but these functions have yet to be evaluated in experimental malaria. In this study, we assessed the involvement of inducible nitric oxide synthase in inflammatory responses to murine experimental malaria induced by P. berghei NK65. We observed that wild type (WT) and nitric oxide synthase (iNOS)-deficient mice (iNOS−/−) mice showed similar levels of parasitemia following P. berghei NK65 infection, although infected iNOS−/− mice presented early mortality. Inducible nitric oxide synthase deficiency led to increased leukocyte rolling and adhesion to the liver in iNOS−/− mice relative to the WT animals, as observed via intravital microscopy. Infected iNOS−/− mice also exhibited increased hepatic leukocyte migration and subsequent liver damage, which was associated with high serum levels of the cytokines TNF-α, IL-6 and IL-10. Our data suggest potential role for the iNOS enzyme as a regulator of hepatic inflammatory response induced by P. berghei NK65-infection, and its absence leads to exacerbated inflammation and sequential associated-hepatic damage in the animals.

Progesterone reduces the expression of spinal cyclooxygenase‐2 and inducible nitric oxide synthase and prevents allodynia in a rat model of central neuropathic pain

Spinal cord injury (SCI) results in the development of chronic pain that is refractory to conventional treatment. Progesterone, a neuroprotective steroid, may offer a promising perspective in pain modulation after central injury. Here, we explore the impact of progesterone administration on the post-injury inflammatory cascade involving the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at the spinal cord level. We also analyse pain behaviours, the profile of glial cell activation, and IκB-α mRNA levels, as an index of NF-κB transactivation. We used biochemical, immunohistochemical and molecular techniques, as well as behavioural studies, to investigate the effects of progesterone in a well-characterized model of central neuropathic pain. Injured animals receiving progesterone presented reduced mRNA levels of the proinflammatory enzymes, as well as decreased COX-2 activity and nitrite levels, as compared to vehicle-treated injured rats. Further, animals receiving the steroid exhibited lower levels of IκB-α mRNA, suggesting decreased NF-κB transactivation. Progesterone administration also attenuated the injury-induced increase in the number of glial fibrillary acidic protein and OX-42 positive cells both at early and late time points after injury, and prevented the development of mechanical and thermal allodynia. Further, when injured rats received early progesterone administration for a critical period of time after injury, they did not display allodynic behaviours even after the treatment had stopped. Our results suggest that progesterone, by modulating early neuroinflammatory events triggered after SCI, may represent a useful strategy to prevent the development of central chronic pain.

“Click” Dendrimers as Anti-inflammatory Agents: With Insights into Their Binding from Molecular Modeling Studies

These studies explore the relationship between the inhibitory actions of low generation dendrimers in stimulated microglia and dendrimer-enzyme interactions using in silico molecular modeling. Low generation (DG0 and DG1) dendrimers with acetylene and hydroxyl terminal groups were tested for their anti-inflammatory activity in microglia stimulated by lipopolysaccharides (LPS), and the results were compared with those from the established anti-inflammatory agents, ibuprofen and celecoxib. We hypothesized that hydroxyl terminal groups of DG0 and DG1 dendrimers could interact with the active sites of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzymes due to their small size and favorable electrochemical properties. The enzymatic activity of iNOS and COX-2 was determined in the presence of low generation dendrimers using biochemical assays and their values related to dendrimer docking confirmations from in silico molecular modeling. We found that results from the molecular modeling studies correlated well with the in vitro biological data, suggesting that, indeed, hydroxyl terminal groups of low generation dendrimers enable multivalent macromolecular interactions, resulting in the inhibition of both iNOS and COX-2 enzymes.

In vivo modulation of iNOS pathway in hepatocellular carcinoma by Nigella sativa

Nitric oxide (NO) and inducible nitric oxide synthase enzyme (iNOS) have been implicated in various tumors. Hepatocellular carcinoma is a highly aggressive form of solid tumor. The lack of effective therapy necessitates the introduction of novel therapeutic strategies to counter this disease. Nigella sativa (NS) has been shown to have specific health benefits. The aim of this study was to investigate the in vivo modulation of the iNOS pathway by NS ethanolic extract (NSEE) and the implications of this effect as an antitumor therapeutic approach against diethylnitrosamine (DENA)-induced hepatocarcinogenesis. Rats were divided into four groups, normal control, NSEE control, cancer control, and NSEE-DENA groups. The diagnosis of cancer was based on alpha-fetoprotein (AFP) levels and histological variations. Serum NO, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels and serum iNOS activity were measured. Liver iNOS expression was investigated by reverse transcriptase (RT)-PCR and western blot assays. Serum AFP, NO, TNF-α, and IL-6 levels and iNOS enzyme activity were significantly increased in rats treated with DENA. Significant up-regulation of liver iNOS mRNA and protein expression was also observed. Subsequent treatment with NSEE significantly reversed these effects and improved the histopathological changes in malignant liver tissue which appeared after treatment with DENA, without any toxic effect when given alone. These results provide evidence that attenuation of the iNOS pathway and suppression of the inflammatory response mediated by TNF-α, and IL-6 could be implicated in the antitumor effect of NSEE. As such, our findings hold great promise for the utilization of NS as an effective natural therapeutic agent in the treatment of hepatocarcinogenesis.

Association of Sodium Induced High Blood Pressure with Up-Regulation of Inducible Nitric Oxide Synthase (iNOS) Expression and Inhibition of Trimethylation of H3K27 on Its Promoter Region

Nitric oxide (NO), a simple molecule, playing very diverse roles in the regulation of the cardiovascular, immunologic and nervous system, is formed from L-arginine by converting it to L-citrulline via nitric oxide synthase enzymes (NOS) including endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). We previously reported that long term inhibition of iNOS by intravenous administration of aminogunidine (AG) led to an aggravation of hypertension induced by high sodium chloride (NaCl) loading. Also, high blood pressure induced by high NaCl loading is associated with higher NO production in SD rats. The goal of the present study was to clarify whether the change of blood pressure alter iNOS gene expression as well as epigenetic modification of iNOS gene promoter region. Up-regulation of iNOS mRNA and protein were observed in renal medullary tissue of high sodium infused rats. Furthermore, chromatin immunoprecipitation assay evidenced the inhibition of recruitment of methyltransferase enhancer of Zeste homology 2 (EZH2) to, and trimethylation of histone 3 (H3) at lysine 27 (K27) of iNOS gene promoter region. We concluded that blood pressure is a regulator of iNOS gene expression through alteration of histone covalent modification of its genomic DNA.

Association of Sodium Induced High Blood Pressure with Up-Regulation of Inducible Nitric Oxide Synthase (iNOS) Expression and Inhibition of Trimethylation of H3K27 on Its Promoter Region

Nitric oxide (NO), a simple molecule, playing very diverse roles in the regulation of the cardiovascular, immunologic and nervous system, is formed from L-arginine by converting it to L-citrulline via nitric oxide synthase enzymes (NOS) including endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). We previously reported that long term inhibition of iNOS by intravenous administration of aminogunidine (AG) led to an aggravation of hypertension induced by high sodium chloride (NaCl) loading. Also, high blood pressure induced by high NaCl loading is associated with higher NO production in SD rats. The goal of the present study was to clarify whether the change of blood pressure alter iNOS gene expression as well as epigenetic modification of iNOS gene promoter region. Up-regulation of iNOS mRNA and protein were observed in renal medullary tissue of high sodium infused rats. Furthermore, chromatin immunoprecipitation assay evidenced the inhibition of recruitment of methyltransferase enhancer of Zeste homology 2 (EZH2) to, and trimethylation of histone 3 (H3) at lysine 27 (K27) of iNOS gene promoter region. We concluded that blood pressure is a regulator of iNOS gene expression through alteration of histone covalent modification of its genomic DNA.

Possible involvement of galectin-3 in microglial activation in the hippocampus with trimethyltin treatment

Trimethyltin (TMT) is an organotin neurotoxicant with effects that are selectively localized to the limbic system (especially the hippocampus), which produces memory deficits and temporal lobe seizures. Galectin-3 (Gal-3) is a beta-galactoside-binding lectin that is important in cell proliferation and regulation of apoptosis. The present study evaluated the temporal expression of Gal-3 in the hippocampus of adult BALB/c mice after TMT treatment (i.p., 2.5mg/kg). Western blotting analyses showed that Gal-3 immunoreactivity began to increase 2days after treatment; the immunoreactivity peaked significantly within 4days after treatment but significantly declined between days 4 and 8. Immunohistochemical analysis indicated that Gal-3 expression was very rare in the hippocampi of vehicle-treated controls. However, Gal-3 immunoreactivity appeared between 2 and 8days after TMT treatment and was primarily localized to the hippocampal dentate gyrus (DG), in which neuronal degeneration occurred. The immunoreactivity was detected predominantly in most of the Iba1-positive microglia and in some GFAP-positive astrocytes of the hippocampal DG. Furthermore, Gal-3 expression co-localized with the pro-inflammatory enzymes cyclooxygenase-2 and inducible nitric oxide synthase in the hippocampal DG. Therefore, we suggest that Gal-3 is involved in the inflammatory process of neurodegenerative disorder induced by organotin intoxication.

Experimental inhibition of nitric oxide increases Plasmodium relictum (lineage SGS1) parasitaemia

Malaria is a widespread vector-borne disease infecting a wide range of terrestrial vertebrates including reptiles, birds and mammals. In addition to being one of the most deadly infectious diseases for humans, malaria is a threat to wildlife. The host immune system represents the main defence against malaria parasites. Identifying the immune effectors involved in malaria resistance has therefore become a major focus of research. However, this has mostly involved humans and animal models (rodents) and how the immune system regulates malaria progression in non-model organisms has been largely ignored. The aim of the present study was to investigate the role of nitric oxide (NO) as an immune effector contributing to the control of the acute phase of infection with the avian malaria agent Plasmodium relictum. We used experimental infections of domestic canaries in conjunction with the inhibition of the enzyme inducible nitric oxide synthase (iNOS) to assess the protective function of NO during the infection, and the physiological costs paid by the host in the absence of an effective NO response. Our results show that birds treated with the iNOS inhibitor suffered from a higher parasitaemia, but did not pay a higher cost of infection (anaemia). While these findings confirm that NO contributes to the resistance to avian malaria during the acute phase of the infection, they also suggest that parasitaemia and costs of infection can be decoupled.

Expression of inducible nitric oxide synthase and nitric oxide production in the mud-dwelled air-breathing singhi catfish, Heteropneustes fossilis under condition of water shortage

Nitric oxide (NO) is known to be an important regulator molecule for regulating the multiple signaling pathways and also to play diverse physiological functions in mammals including that of adaptation to various stresses. The present study reports on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) enzyme that produces NO from l-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis) while dwelling inside the mud peat under semidry conditions. Desiccation stress, due to mud-dwelling for 2weeks, led to significant increase of NO concentration in different tissues and in plasma of singhi catfish, and also the increase of NO efflux from the perfused liver with an accompanying increase of toxic ammonia level in different tissues. Mud-dwelling also resulted to induction of iNOS activity, expression of iNOS protein in different tissues after 7days with further increase after 14days, which otherwise was not detectable in control fish. Further, mud-dwelling also resulted to a significant expression of iNOS mRNA after 7 days with a more increase of mRNA level after 14days, suggesting that the desiccation stress caused transcriptional regulation of iNOS gene. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues. Desiccation stress also led to activation and nuclear translocation of nuclear factor кB (NFкB) in hepatic cells. These results suggest that the activation of iNOS gene under desiccation-induced stresses such as high ammonia load was probably mediated through the activation of one of the major transcription factors, the NFкB. This is the first report of desiccation-induced induction of iNOS gene, iNOS protein expression leading to more generation of NO while living inside the mud peat under condition of water shortage in any air-breathing teleosts.