Increase In Nitric Oxide Synthase Research Articles

DYNAMICS OF THE LEVELS OF ASYMMETRIC DIMETHYLARGININE AND PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 IN PATIENTS WITH ACUTE ST-SEGMENT ELEVATION MYOCARDIAL INFARCTION AND TYPE 2 DIABETES MELLITUS DEPENDING ON THE REPERFUSION STRATEGY

Introduction. Despite the major successes achieved in the treatment of acute coronary syndromes (ACS), acute myocardial infarction (AMI) remains the main cause of death among the working-age population of Ukraine. The means of treatment of interventional cardiology can actually reduce the mortality of patients with ACS, improve the course of the acute period of the disease and ensure less reduction in the functional capabilities of the heart in the future. Among the many pathogenetic mechanisms of vascular inflammation in coronary heart disease and type 2 diabetes, endothelial dysfunction is the determining factor.
 The aim of the study. To evaluate the levels of plasminogen activator inhibitor type 1, asymmetric dimethylarginine and endothelial nitric oxide synthase on the 10-14th day in patients depending on the presence or absence of concomitant diabetes type 2 and the type of reperfusion therapy.
 Materials and methods. 130 patients with acute myocardial infarction were examined, who were divided into 2 groups: 1 group consisted of patients with acute myocardial infarction with accompanying type 2 diabetes (n=73), 2 group – patients with acute myocardial infarction without type 2 diabetes (n =57). The quantitative content of the plasminogen activator inhibitor type 1 (PAI-1) was determined by the immunoenzymatic method using a commercial test system manufactured by Technoclone PAI-1 ELISA Kit (Austria), endothelial nitric oxide synthase (NOS) – Enzyme-Linked Immunosorbent Assay (ELISA) Kit For Nitric Oxide Synthase Endothelial, asymmetric dimethylarginine (ADMA) – Immunodiagnostik ADMA ELISA Kit (Austria).
 Results. Percutaneous coronary intervention (PCI) contributes to a more significant decrease in the content of the marker of endothelial dysfunction – ADMA and an increase in NOS on the 10-14th day of acute myocardial infarction in comparison with standard therapy. During PCI, the level of PAI-1 did not reliably change during treatment due to post-inflammatory and post-traumatic activation of platelets in the vascular wall.
 Conclusions. In patients with acute myocardial infarction with type 2 diabetes mellitus, percutaneous coronary intervention contributes to a significant decrease in the content of asymmetric dimethylarginine and an increase in NOS on the 10-14th day of acute myocardial infarction, but was not accompanied by a significant decrease in the level of PAI-1, which in general indicates positive effect of performed myocardial revascularization.

MLN-4760 Induces Oxidative Stress without Blood Pressure and Behavioural Alterations in SHRs: Roles of Nfe2l2 Gene, Nitric Oxide and Hydrogen Sulfide.

Reduced angiotensin 1-7 bioavailability due to inhibition of angiotensin-converting enzyme 2 (ACE2) may contribute to increased mortality in hypertensive individuals during COVID-19. However, effects of ACE2 inhibitor MLN-4760 in brain functions remain unknown. We investigated the selected behavioural and hemodynamic parameters in spontaneously hypertensive rats (SHRs) after a 2-week s.c. infusion of MLN-4760 (dose 1 mg/kg/day). The biochemical and molecular effects of MLN-4760 were investigated in the brainstem and blood plasma. MLN-4760 had no effects on hemodynamic and behavioural parameters. However, MLN-4760 increased plasma hydrogen sulfide (H2S) level and total nitric oxide (NO) synthase activity and conjugated dienes in the brainstem. Increased NO synthase activity correlated positively with gene expression of Nos3 while plasma H2S levels correlated positively with gene expressions of H2S-producing enzymes Mpst, Cth and Cbs. MLN-4760 administration increased gene expression of Ace2, Sod1, Sod2, Gpx4 and Hmox1, which positively correlated with expression of Nfe2l2 gene encoding the redox-sensitive transcription factor NRF2. Collectively, MLN-4760 did not exacerbate pre-existing hypertension and behavioural hyperactivity/anxiety in SHRs. However, MLN-4760-induced oxidative damage in brainstem was associated with activation of NO- and H2S-mediated compensatory mechanisms and with increased gene expression of antioxidant, NO- and H2S-producing enzymes that all correlated positively with elevated Nfe2l2 expression.

Nitric oxide synthase activity: A novel potential biomarker for predicting Alopecia areata.

Alopecia areata is a dermatological disease characterized by nonscarring type hair loss. The cause of Alopecia areata not known exactly but studies support that it has an autoimmune etiology in which oxidative stress play an important role. This study was conducted to evaluate the level of nitrosative stress in Alopecia areata and to investigate the predictive power of nitrosative stress parameters for Alopecia areata. Thirty patients diagnosed with Alopecia areata, and 30 healthy controls were included in a prospective, cross-sectional study. In both groups, nitric oxide (NO· ), peroxynitrite (ONOO- ), and nitric oxide synthase (NOS) activity as nitrosative stress markers were measured spectrophotometrically in serum samples. The predictive power of nitrosative stress parameters in Alopecia areata and control groups was compared with binary logistic regression and Receiver Operating Characteristic analysis. NO· , ONOO- , and NOS activity were significantly higher in patients with Alopecia areata than in the control group (p=0.001; p < 0.001; p < 0.001, respectively). A positive correlation was found between the parameters. Significantly, binary logistic regression modeling suggested that increases in NOS (p=0.003, OR=1.305, 95% CI=1.095-1.556) activity were associated with Alopecia areata. According to the data obtained from the present study, patients with Alopecia areata were exposed to potent nitrosative stress. In particular, peroxynitrite, which acts as a bridge between reactive oxygen species and reactive nitrogen species, caused the expansion of the oxidative stress cascade. Nitrosative stress might play a role in the etiopathogenesis of Alopecia areata. Nitrosative stress parameters, particularly NOS activity, may be potential markers for Alopecia areata.

Developmental Stage, Solid Food Introduction, and Suckling Cessation Differentially Influence the Comaturation of the Gut Microbiota and Intestinal Epithelium in Rabbits

BackgroundIn mammals, the establishment around weaning of a symbiotic relationship between the gut microbiota and its host determines long-term health. ObjectivesThe aim of this study was to identify the factors driving the comaturation of the gut microbiota and intestinal epithelium at the suckling-to-weaning transition. We hypothesized that the developmental stage, solid food ingestion, and suckling cessation contribute to this process. MethodsFrom birth to day 18, Hyplus rabbits were exclusively suckling. From day 18 to day 25, rabbits were 1) exclusively suckling; 2) suckling and ingesting solid food; or 3) exclusively ingesting solid food. The microbiota (16S amplicon sequencing), metabolome (nuclear magnetic resonance), and epithelial gene expression (high-throughput qPCR) were analyzed in the cecum at days 18 and 25. ResultsThe microbiota structure and metabolic activity were modified with age when rabbits remained exclusively suckling. The epithelial gene expression of nutrient transporters, proliferation markers, and innate immune factors were also regulated with age (e.g., 1.5-fold decrease of TLR5). Solid food ingestion by suckling rabbits had a major effect on the gut microbiota by increasing its α diversity, remodeling its structure (e.g., 6.3-fold increase of Ruminococcaceae), and metabolic activity (e.g., 4.6-fold increase of butyrate). Solid food introduction also regulated the gene expression of nutrient transporters, differentiation markers, and innate immune factors in the epithelium (e.g., 3-fold increase of nitric oxide synthase). Suckling cessation had no effect on the microbiota, while it regulated the expression of genes involved in epithelial differentiation and immunoglobulin transport (e.g., 2.5-increase of the polymeric immunoglobulin receptor). ConclusionsIn rabbits, the maturation of the microbiota at the suckling-to-weaning transition is driven by the introduction of solid food and, to a lesser extent, by the developmental stage. In contrast, the maturation of the intestinal epithelium at the suckling-to-weaning transition is under the influence of the developmental stage, solid food introduction, and suckling cessation.

Transient production of H2O2 and NO induced by ascorbic acid coincides with promotion of antioxidant enzyme activity and reduction of pericarp browning of harvested longan fruit

Ascorbic acid (AsA) plays an important role in the protection against oxidative stress in plants. Although the underlining mechanism is yet to be fully elucidated, it is believed that hydrogen peroxide (H2O2) and nitric oxide (NO) play a key role as a signaling agent. This study investigated the relationship between exogenously applied AsA and the activation of antioxidant defense through these signaling molecules, which coincided with reduced pericarp browning of harvested longan (Dimocarpus longan Lour. cv. Daw). It was found that the level of H2O2 in longan pericarp increased immediately after treatment with 2.5 mM AsA, reaching maximum 9 h afterward. The expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and the activity of NOX simultaneously peaked after the AsA treatment and remained so for the next 9 h. NO content, on the other hand, did not rise until H2O2 peak was reached, attaining the highest level 3 h later. The change in NO content correlated well with the increase in nitric oxide synthase (NOS) gene expression and enzyme activity. Interestingly, the AsA treatment differently altered gene expression and activities of some vital antioxidant enzymes including superoxide dismutase, catalase, ascorbate peroxidase and glutathione peroxidase during the first 4 d of storage. With the exception of catalase, the treatment positively affected both the expression and the activity. Catalase activity remained constant for the first 9 h before rising afterward. These changes in the activity of the antioxidant enzymes were closely associated with the reduction in pericarp browning. These results suggested that H2O2 and NO generation triggered by AsA may activate the antioxidant defense mechanism in longan pericarp. This, in turn, helps overcome subsequent stressed-induced H2O2 and NO production, thereby reducing the pericarp browning.

Estradiol and the feeding state modulate the interaction between leptin and the nitrergic system in female rats

Leptin mediates the interaction between reproductive function and energy balance. However, leptin receptors are not expressed in neurons that produce gonadotropin-releasing hormone (GnRH), likely indicating an indirect action through interneurons. Among likely neurons that modulate the secretion of GnRH are NO (nitric oxide) neurons. We assessed whether estradiol and feeding conditions modulate a possible interaction between leptin and NO in brain areas related to the control of reproductive function. Estradiol-treated and untreated ovariectomized rats were normally fed or fasted for 48 h. Then, saline (control) or leptin (3 μg/1 μl) intracerebroventricular microinjections were administered, and after thirty minutes, the brains collected subsequent to the decapitation or transcardially perfusion. Leptin and estradiol increased NO synthase (nNOS) gene expression (RT-PCR) and content (Western blotting) in the medial preoptic area (MPOA) and medial basal hypothalamus (MBH) only in fasted rats. Leptin increased: 1-phosphorylated-signal transducer and activator of transcription-3(pSTAT3) (immunohistochemistry) in the MPOA and various hypothalamic nuclei [arcuate (ARC); ventromedial (VMH); dorsal/ventral dorsomedial (dDMH/vDMH); premammilar ventral (PMV)], effects potentiated by estradiol/fasting interaction; 2- nNOS/pSTAT3 coexpression in the MPOA only in estradiol-treated, fasted rats; 3- nNOS-immunoreactive cell expression in the VMH, DMH and PMV (areas related to reproductive function control) of estradiol -treated rats. Thus, when leptin is reduced during fasting, leptin replacement effectively increased the expression of nitric oxide, which activated the HPG axis only in the presence of estradiol. Estradiol modulates the nitrergic system, leptin sensitivity and consequently leptin's effects on the nitrergic system in hypothalamus and in particular vDMH and PMV.

As3+ or/and Cu2+ exposure triggers oxidative stress imbalance, induces inflammatory response and apoptosis in chicken brain

Arsenic (As) and copper (Cu) are common environmental pollutants in nature. When they are excessively present in living organisms, they can cause heavy metal poisoning. There were relatively few studies of the toxicological concentrations of As and Cu in the brain using chicken as a model. Therefore, in this study, arsenic trioxide or/and copper sulfate were added to chicken diets for a 12-week toxicity test. The test results showed that excessive intake of As or/and Cu led to a significant reduction in the total antioxidant capacity (T-AOC), catalase (CAT) and hydroxyl radicals. And significant increase in nitric oxide synthase (NOS) indicates an imbalanced oxidation reaction. In addition, the increase in heat shock protein (HSPs), the increase of NF-κB pathway-related pro-inflammatory mediators, the change of apoptosis factors on the death receptor and mitochondrial apoptosis pathway show that, As or/and Cu exposure induced chicken brain has heat shock response (HSP), tissue inflammation and apoptosis. This damage is inseparable from the oxidative imbalance. It is worth noting that these injury changes are time-dependent, and the combined effect of these two metals is more severe than that of a single group of injuries. Our findings can inform the regulation of animal feed additives and avoid agricultural economic losses or biological health damage.

THU0084 TOFACITINIB REVERSED ENDOTHELIAL DYSFUNCTION IN RHEUMATOID ARTHRITIS: MECHANISTIC INSIGHTS FROM THE RAT ADJUVANT-INDUCED ARTHRITIS MODEL.

Background:Tofacitinib, an inhibitor of JAK3 and JAK1, is approved for the treatment of rheumatoid arthritis (RA)1. Cardiovascular (CV) risk2and events3in RA patients treated with Tofacitinib is a matter of debate, but the vascular mechanisms involved are unknown.Objectives:The aim of this study was to investigate whether Tofacitinib improves endothelial dysfunction (ED) and if so to explore the underlying mechanisms in the model of adjuvant-induced arthritis (AIA) in rats.Methods:AIA was induced by injection ofMycobacterium butyricumin the tail of male Lewis rats. A group of rats without arthritis served as controls. At the first signs of arthritis, AIA received Tofacitinib (10 mg/kg twice daily, s.c.) or 33%DMSO/PEG300(Vehicle). Arthritis score was daily evaluated. After 21 days, preconstricted isolated aortic rings were relaxed with acetylcholine (Ach, 10-11-10-4moles/liter) in the presence or not of inhibitor of nitric oxide (NO) synthase (L-NAME), cyclooxygenase-2 (NS398), arginase (nor-NOHA), endothelium-derived hyperpolarizing factor (EDHF) (apamin/charybdotoxin) and superoxide anions production (Tempol). Endothelium-denuded rings were used to determine the vasorelaxant response to the NO-donor sodium nitroprussiate (SNP, 10-11-10-4moles/liter). Blood pressure and heart rate were measured by invasive method. A radiographic score was attributed to hind paws.Results:Compared to AIA-Vehicle, Tofacitinib dramatically reduced arthritis (-76%) and radiographic (-73%) scores (p&lt;0.001), and improved Ach-induced vasorelaxation (p&lt;0.05). Of note, Ach-induced vasorelaxation was not different between Tofacitinib-AIA and control rats. The response to SNP was not different between groups. The effect of Tofacitinib on ED was mediated by decreased cyclooxygenase-2 and arginase activities, decreased superoxide anions production, increased NO synthase activity and EDHF synthesis. As compared to AIA-Vehicle, Tofacitinib changed neither blood pressure nor heart rate.Conclusion:The present results demonstrated that Tofacitinib reversed arthritis-induced ED, through the correction of all the acknowledged impaired endothelial pathways in the AIA model. As ED is theprimum movensof atherogenesis, these data provide a mechanistic explanation to the potential benefits of Tofacitinib on the cardiovascular comorbidities associated to RA.

Autonomic and Cognitive Function Response to Normobaric Hyperoxia Exposure in Healthy Subjects. Preliminary Study.

Background and objective: This is the first study to investigate the effect of high-flow oxygen therapy, using a normobaric chamber on cognitive, biochemical (oxidative stress parameters and the level of neurotrophins), cardiovascular and autonomic functioning. Materials and methods: 17 healthy volunteers, eight males and nine females, with a mean age of 37.5 years, were examined. The experimental study involved ten two-hour exposures in a normobaric chamber with a total pressure of 1500 hPa, in air adjusted to 37% oxygen, 1.079% carbon dioxide and 0.44% hydrogen. Cognitive function was assessed by using Trail Making Test parts A, B and difference in results of these tests (TMT A, TMT B and TMT B-A); California Verbal Learning Test (CVLT); Digit symbol substitution test (DSST); and Digit Span (DS). Fatigue (Fatigue Severity Scale (FSS)), cardiovascular, autonomic and baroreceptor functioning (Task Force Monitor) and biochemical parameters were measured before and after intervention. Results: After 10 sessions in the normobaric chamber, significant decreases in weight, caused mainly by body fat % decrease (24.86 vs. 23.93%, p = 0.04 were observed. TMT part A and B results improved (p = 0.0007 and p = 0.001, respectively). In contrast, there was no statistically significant influence on TMT B-A. Moreover, decrease in the number of symbols left after a one-minute test in DSST was noted (p = 0.0001). The mean number of words correctly recalled in the CVLT Long Delay Free Recall test improved (p = 0.002), and a reduction in fatigue was observed (p = 0.001). Biochemical tests showed a reduction in levels of malondialdehyde (p < 0.001), with increased levels of Cu Zn superoxide dismutase (p < 0.001), Neurotrophin 4 (p = 0.0001) and brain-derived neurotrophic factor (p = 0.001). A significant increase in nitric oxide synthase 2 (Z = 2.29, p = 0.02) and Club cell secretory protein (p = 0.015) was also noted. Baroreceptor function was significantly improved after normobaric exposures (p = 0.003). Significant effect of normobaric exposures and BDNF in CVLT Long Delay Free Recall was noted. Conclusions: This study demonstrates that 10 exposures in a normobaric chamber have a positive impact on visual information and set-shifting processing speed and increase auditory-verbal short-term memory, neurotrophic levels and baroreceptor function. A response of the respiratory tract to oxidative stress was also noted. There is a need to rigorously examine the safety of normobaric therapy. Further studies should be carried out with physician examination, both pre and post treatment.

Effects of Oral Supplementation With Nitrate-Rich Beetroot Juice in Patients With Pulmonary Arterial Hypertension—Results From BEET-PAH, an Exploratory Randomized, Double-Blind, Placebo-Controlled, Crossover Study

BackgroundThe nitrate–nitrite–nitric oxide (NO) pathway may represent a potential therapeutic target in patients with pulmonary arterial hypertension (PAH). We explored the effects of dietary nitrate supplementation, with the use of nitrate-rich beetroot juice (BRJ), in patients with PAH. Methods and ResultsWe prospectively studied 15 patients with PAH in an exploratory randomized, double-blind, placebo-controlled, crossover trial. The patients received nitrate-rich beetroot juice (∼16 mmol nitrate per day) and placebo in 2 treatment periods of 7 days each. The assessments included; exhaled NO and NO flow–independent parameters (alveolar NO and bronchial NO flux), plasma and salivary nitrate and nitrite, biomarkers and metabolites of the NO-system, N-terminal pro–B-type natriuretic peptide, echocardiography, ergospirometry, diffusing capacity of the lung for carbon monoxide, and the 6-minute walk test. Compared with placebo ingestion of BRJ resulted in increases in; fractional exhaled NO at all flow-rates, alveolar NO concentrations and bronchial NO flux, and plasma and salivary levels of nitrate and nitrite. Plasma ornithine levels decreased and indices of relative arginine availability increased after BRJ compared to placebo. A decrease in breathing frequency was observed during ergospirometry after BRJ. A tendency for an improvement in right ventricular function was observed after ingestion of BRJ. In addition a tendency for an increase in the peak power output to peak oxygen consumption ratio (W peak/VO2 peak) was observed, which became significant in patients reaching an increase of plasma nitrite >30% (responders). ConclusionsBRJ administered for 1 week increases pulmonary NO production and the relative arginine bioavailability in patients with PAH, compared with placebo. An increase in the W peak/VO2 peak ratio was observed after BRJ ingestion in plasma nitrite responders. These findings indicate that supplementation with inorganic nitrate increase NO synthase–independent NO production from the nitrate-nitrite-NO pathway.

IL‐10 Supplementation Decreases BP in Male, But Not Female, Spontaneously Hypertensive Rats

Interleukin 10 (IL‐10) is an anti‐inflammatory cytokine that induces nitric oxide (NO) production. IL‐10 supplementation has previously been shown to lower blood pressure (BP) in male hypertensive mice, but the effect of exogenous IL‐10 in hypertensive females has not yet been studied. For the current study, we hypothesized that chronic infusion of IL‐10 in hypertensive rats would lower BP concomitant with an increase in NO synthase (NOS) activity. First, endogenous basal levels of renal IL‐10+ cells and the IL‐10 receptor were measured in a subset of male and female spontaneously hypertensive rats (SHR). In a second set of rats, baseline BP was recorded by tail cuff plethsmography, and rats were placed in metabolic cages for 24‐hr urine collection prior to initiating infusion of IL‐10. IL‐10 was infused by subcutaneous minipump (3.5 ug/kg/d) in half of the male and female SHR, with the remaining rats serving as sham controls (n=4–6 per treatment group per sex). After 2 weeks of treatment, final BP and metabolic cage timepoints were collected, and tissues were harvested. Female SHR had greater IL‐10+ renal cells than males (flow cytometry, 4.6±0.4 vs 1.9±0.4%, p=0.003, n=8) and greater expression of the IL‐10 receptor (Western blot, 0.4±0.1 vs 0.13±0.1 densitometric units, p=0.03, n=4). BP did not change in female SHR treated with IL‐10 (2‐way ANOVA, p=0.3), but BP significantly decreased following IL‐10 infusion in male rats (p=0.04, Table); BP was higher in males than females in both groups (p&lt;0.001). Urinary protein excretion (Bradford assay) did not differ between treatment and control groups in either sex (Table); but excretion was higher in males than in females (p=&lt;0.001). Contrary to our hypothesis, NOS enzymatic activity (measured by the conversion of radio‐labeled arginine to radio‐labeled citrulline) significantly decreased with IL‐10 treatment in both the inner medulla (p=0.004) and the renal cortex (p=0.003) of both sexes (Table). In conclusion, despite the finding that male SHR have less IL‐10 and IL‐10 receptor expression in the kidney compared to females, BP was selectively decreased in males. We hypothesize that the higher endogenous levels of IL‐10 in the female SHR contribute to the observed sex difference in BP and prevent further decreases in BP with exogenous IL‐10 infusion. Furthermore, our data suggests that decreases in BP in male SHR with IL‐10 is not dependent on upregulating NOS activity in the kidney, although the effect of IL‐10 on NO bioavailability was not assessed. Future studies will further explore the role of endogenous IL‐10 on BP control in female SHR. Baseline BP (mmHg) Final BP (mmHg) Baseline Proteinuria (mg/d) Final Proteinuria (mg/d) Inner Medulla NOS activity (pmol/mg) Cortex NOS activity (pmol/mg) Male CON 211±1 221±5 10±1 15±2 780±123 17287±42772 Male IL‐10 213±5 200±4* 12±1 16±2 284±29* 7316±1917* Female CON 160±8# 169±7# 2±0.4# 3±1# 1296±213# 20260±2207 Female IL‐10 163±2# 169±9# 1±0.3# 2±0# 650±65*# 8279±1215* p&lt;0.05 vs CON same sex, p&lt;0.05 vs male same treatment

Dietary Nitrate and the Epidemiology of Cardiovascular Disease: Report From a National Heart, Lung, and Blood Institute Workshop.

In view of continuing unanswered questions regarding the geographical and demographic distribution of cardiovascular disease, and recent discoveries about the effects of dietary nitrate on cardiovascular physiology, the National Heart, Lung, and Blood Institute (NHLBI) convened a workshop to

Neuroprotective effect of melatonin in experimental optic neuritis in rats.

Optic neuritis (ON) is an inflammatory, demyelinating, and neurodegenerative condition of the optic nerve, which might induce permanent vision loss. Currently, there are no effective therapies for this disorder. We have developed an experimental model of primary ON in rats through a single microinjection of 4.5 μg of bacterial lipopolysaccharide (LPS) into the optic nerve. Since melatonin acts as a pleiotropic therapeutic agent in various neurodegenerative diseases, we analyzed the effect of melatonin on LPS-induced ON. For this purpose, LPS or vehicle were injected into the optic nerve from adult male Wistar rats. One group of animals received a subcutaneous pellet of 20 mg melatonin at 24 hr before vehicle or LPS injection, and another group was submitted to a sham procedure. Melatonin completely prevented the decrease in visual evoked potentials (VEPs), and pupil light reflex (PLR), and preserved anterograde transport of cholera toxin β-subunit from the retina to the superior colliculus. Moreover, melatonin prevented microglial reactivity (ED1-immunoreactivity, P < 0.01), astrocytosis (glial fibrillary acid protein-immunostaining, P < 0.05), demyelination (luxol fast blue staining, P < 0.01), and axon (toluidine blue staining, P < 0.01) and retinal ganglion cell (Brn3a-immunoreactivity, P < 0.01) loss, induced by LPS. Melatonin completely prevented the increase in nitric oxide synthase 2, cyclooxygenase-2 levels (Western blot) and TNFα levels, and partly prevented lipid peroxidation induced by experimental ON. When the pellet of melatonin was implanted at 4 days postinjection of LPS, it completely reversed the decrease in VEPs and PLR. These data suggest that melatonin could be a promising candidate for ON treatment.

Sevoflurane post-conditioning reduces rat myocardial ischemia reperfusion injury through an increase in NOS and a decrease in phopshorylated NHE1 levels.

The protective effects of sevoflurane post-conditioning against myocardial ischemia/reperfusion (I/R) injury (MIRI) have been previously reported. However, the mechanisms responsible for these protective effects remain elusive. In this study, in order to investigate the molecular mechanisms responsible for the protective effects of sevoflurane post-conditioning on isolated rat hearts subjected to MIRI, Sprague-Dawley rat hearts were randomly divided into the following 6 groups: i) the sham-operated control; ii) 2.5% sevoflurane; iii) ischemia/reperfusion (I/R); iv) 2.5% sevoflurane post-conditioning plus I/R; v) 2.5% sevoflurane post-conditioning + NG-nitro-L-arginine methyl ester (L-NAME) plus I/R; and vi) L-NAME plus I/R. The infarct size was measured using 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Additionally, the myocardial nitric oxide (NO), NO synthase (NOS) and nicotinamide adenine dinucleotide (NAD+) levels were determined. Autophagosomes and apoptosomes in the myocardium were detected by transmission electron microscopy. The levels of Bcl-2, cleaved caspase-3, Beclin-1, microtubule-associated protein light chain 3 (LC3)-I/II, Na+/H+ exchanger 1 (NHE1) and phosphorylated NHE1 protein were measured by western blot analysis. NHE1 mRNA levels were measured by reverse transcription-quantitative polymerase chain reaction. Compared with the I/R group, 15 min of exposure to 2.5% sevoflurane during early reperfusion significantly decreased the myocardial infarct size, the autophagic vacuole numbers, the NHE1 mRNA and protein expression of cleaved caspase-3, Beclin-1 and LC3-I/II. Post-conditioning with 2.5% sevoflurane also increased the NO and NOS levels and Bcl-2 protein expression (P<0.05 or P<0.01). Notably, the cardioprotective effects of sevoflurane were partly abolished by the NOS inhibitor, L-NAME. The findings of the present study suggest that sevoflurane post-conditioning protects the myocardium against I/R injury and reduces the myocardial infarct size. The underlying protective mechanisms are associated with the inhibition of mitochondrial permeability transition pore opening, and with the attenuation of cardiomyoctye apoptosis and excessive autophagy. These effects are mediated through an increase in NOS and a decrease in phopshorylated NHE1 levels.

Catechin-7-O-β-D-glucopyranoside isolated from the seed of Phaseolus calcaratus Roxburgh ameliorates experimental colitis in rats

The seeds of Phaseolus calcaratus Roxburgh (PHCR) are common legumes that comprise part of the daily diet in Chinese and Korean culture. Recent findings highlight anti-inflammatory and anti-septic potentials of catechin-7-O-β-D-glucopyranoside (CGP) isolated from PHCR seeds. We investigated the intestinal anti-inflammatory activity and associated mechanisms of CGP using a rat model of trinitrobenzenesulfonic acid (TNBS)-induced colitis. Oral treatment with CGP (10mg/kg body weight) suppressed body weight loss and intestinal inflammatory damages in TNBS-induced colitic rats. This treatment reduced myeloperoxidase activity and malondialdehyde level, but increased glutathione level in the TNBS colitic rats. CGP treatment also inhibited the TNBS-mediated increases in nitric oxide synthase, cyclooxygenase-2, interleukin-1β, tumor necrosis factor-α, intercellular adhesion molecule-1, and monocyte chemotactic protein-1 proteins or mRNA levels. This inhibition was accompanied by the increased mRNA levels of mucins MUC2 and MUC3. The CGP treatment prevented phosphorylation of p38 mitogen-activated protein kinase, IκB-α, and DNA-nuclear factor-κB binding, all of which were increased in the inflamed colons of TNBS-treated rats. Furthermore, oral administration with a crude PHCR butanol extract (100mg/kg body weight) which contains 1.5% of CGP showed intestinal anti-inflammatory potentials similar to that of CGP. Collectively, our current findings suggest that CGP or CGP-containing PHCR seeds may have favorable effects on intestinal inflammatory diseases.

Nitric Oxide Mediates 5-Aminolevulinic Acid-Induced Antioxidant Defense in Leaves of Elymus nutans Griseb. Exposed to Chilling Stress.

Nitric oxide (NO) and 5-aminolevulinic acid (ALA) are both extremely important signalling molecules employed by plants to control many aspects of physiology. In the present study, the role of NO in ALA-induced antioxidant defense in leaves of two sources of Elymus nutans Griseb. (Damxung, DX and Zhengdao, ZD) was investigated. Chilling stress enhanced electrolyte leakage, accumulation of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide radical in two E. nutans, which were substantially alleviated by exogenous ALA and NO application. Pretreatment with NO scavenger PTIO or NOS inhibitor L-NNA alone and in combination with ALA induced enhancements in electrolyte leakage and the accumulation of MDA, H2O2 and superoxide radical in leaves of DX and ZD exposed to chilling stress, indicating that the inhibition of NO biosynthesis reduced the chilling resistance of E. nutans and the ALA-enhanced chilling resistance. Further analyses showed that ALA and NO enhanced antioxidant defense and activated plasma membrane (PM) H+-ATPase and decreased the accumulation of ROS induced by chilling stress. A pronounced increase in nitric oxide synthase (NOS) activity and NO release by exogenous ALA treatment was found in chilling-resistant DX plants exposed to chilling stress, while only a little increase was observed in chilling-sensitive ZD. Furthermore, inhibition of NO accumulation by PTIO or L-NNA blocked the protective effect of exogenous ALA, while both exogenous NO treatment and inhibition of endogenous NO accumulation did not induce ALA production. These results suggested that NO might be a downstream signal mediating ALA-induced chilling resistance in E. nutans.

Reduction of Nitrosative Stress by Vitamin C: a New Approach for Mitigation of Doxorubicin‐induced Cardiomyopathy

BackgroundDespite the effectiveness of Doxorubicin (Dox) as an anti‐cancer drug, the dose dependent cardiotoxic side effects are serious life threatening concerns associated with its use. In addition to the oxidative stress, there is increasing evidence of involvement of nitrosative stress in Dox‐induced cardiotoxicity. Previously, Vitamin C (Vit C) has been shown to reduce oxidative stress and improved survival of Dox‐treated cardiomyocytes. However there is no evidence on the effect of Vit C on nitrosative stress. The objective of this study was to investigate the effect of Vit C on nitrosative stress in Dox treated cardiomyocytes.Methods and ResultsCardiomyocytes isolated from adult Sprague‐Dawley rats were treated with predetermined doses of Vit C (25 µM), Dox (10 µM) or Vit C (25 µM) + Dox (10 µM) for 24 hours. Dox treatment increased levels of nitrate in cardiomyocytes as well as released in media leading to increased production of peroxynitrite. Vit C significantly reduced the levels of cellular and released nitrate, cellular peroxynitrite as well as the nitrosylation of proteins caused by Dox. Dox induced increase in Nitric oxide synthase (NOS) activity in cardiomyocytes was blunted by Vit C treatment. Western blot analysis revealed an upregulation of inducible NOS (iNOS) and phosphorylation of inhibitory site of endothelial NOS (eNOS) by Dox. In contrast Vit C downregulated iNOS and phosphorylated activating site of eNOS. Dox induced activation of apoptosis was prevented by Vit C.ConclusionThese results provide a hope and rationale for use of Vit C as an adjuvant therapy to mitigate cardiotoxic side effects of Dox by attenuating oxidative and nitrosative stress.Support: CIHR

Иммуногистохимическое исследование с использованием моноклональных антител к NO-синтазе 2 и ФНО-α ткани печени мышей при токсическом поражении тетрахлорметаном

С помощью иммуногистохимического анализа получены данные, которые свидетельствуют о значимой роли NO-синтазы 2 и цитокина ФНО-α в патогенезе CCl4-индуцированного поражения печени. Это проявлялось в том, что действие гепатотоксина приводило к повышению содержания и NO-синтазы 2, и ФНО-α в ткани печени. Применение как исследуемых фитоадаптогенов (экстракта элеутерококка и настойки женьшеня) в дозах 48,5 и 47 мг/кг сухого вещества, соответственно, так и препарата сравнения карсила в дозе 100 мг/кг ежедневно в течение 5 недель, в значительной степени предупреждало накопление как фермента NO-синтазы 2, так и цитокина ФНО-α в ткани печени, что, по всей видимости, является одним из механизмов их гепатопротекторного действия.

Propolis ameliorates tumor nerosis factor-α, nitric oxide levels, caspase-3 and nitric oxide synthase activities in kainic acid mediated excitotoxicity in rat brain.

Increased nitric oxide (NO), neuronal inflammation and apoptosis have been proposed to be involved in excitotoxicity plays a part in many neurodegenerative diseases. To understand the neuro-protective effects of propolis, activities of Nitric oxide synthase (NOS) and caspase-3 along with NO and tumor necrosis factor-α (TNF-α) levels were studied in cerebral cortex (CC), cerebellum (CB) and brain stem (BS) in rats supplemented with propolis prior to excitotoxic injury with kainic acid (KA). Male Sprague-Dawley rats were divided into four groups (n=6 rats per group) as Control, KA, Propolis and KA+Propolis. The control group and KA group have received vehicle and saline. Propolis group and propolis + KA group were orally administered with propolis (150 mg/kg body weight), five times every 12 hours. KA group and propolis +KA group were injected subcutaneously with kainic acid (15 mg/kg body weight) and were sacrificed after 2 hrs. CC, CB and BS were separated, homogenized and used for estimation of NOS, caspase-3, NO and TNF-α by commercial kits. Results were analyzed by one way ANOVA, reported as mean + SD (n=6 rats), and p<0.05 was considered statistically significant. The concentration of NO, TNF-α, NOS and caspase-3 activity were increased significantly (p<0.001) in all the three brain regions tested in KA group compared to the control. Propolis supplementation significantly (p<0.001) prevented the increase in NOS, NO, TNF-α and caspase-3 due to KA. Results of this study clearly demonstrated that the propolis supplementation attenuated the NOS, caspase-3 activities, NO, and TNF-α concentration and in KA mediated excitotoxicity. Hence propolis can be a possible potential protective agent against excitotoxicity and neurodegenerative disorders.

PI3K/Akt-independent NOS/HO activation accounts for the facilitatory effect of nicotine on acetylcholine renal vasodilations: modulation by ovarian hormones.

We investigated the effect of chronic nicotine on cholinergically-mediated renal vasodilations in female rats and its modulation by the nitric oxide synthase (NOS)/heme oxygenase (HO) pathways. Dose-vasodilatory response curves of acetylcholine (0.01–2.43 nmol) were established in isolated phenylephrine-preconstricted perfused kidneys obtained from rats treated with or without nicotine (0.5–4.0 mg/kg/day, 2 weeks). Acetylcholine vasodilations were potentiated by low nicotine doses (0.5 and 1 mg/kg/day) in contrast to no effect for higher doses (2 and 4 mg/kg/day). The facilitatory effect of nicotine was acetylcholine specific because it was not observed with other vasodilators such as 5′-N-ethylcarboxamidoadenosine (NECA, adenosine receptor agonist) or papaverine. Increases in NOS and HO-1 activities appear to mediate the nicotine-evoked enhancement of acetylcholine vasodilation because the latter was compromised after pharmacologic inhibition of NOS (L-NAME) or HO-1 (zinc protoporphyrin, ZnPP). The renal protein expression of phosphorylated Akt was not affected by nicotine. We also show that the presence of the two ovarian hormones is necessary for the nicotine augmentation of acetylcholine vasodilations to manifest because nicotine facilitation was lost in kidneys of ovariectomized (OVX) and restored after combined, but not individual, supplementation with medroxyprogesterone acetate (MPA) and estrogen (E2). Together, the data suggests that chronic nicotine potentiates acetylcholine renal vasodilation in female rats via, at least partly, Akt-independent HO-1 upregulation. The facilitatory effect of nicotine is dose dependent and requires the presence of the two ovarian hormones.