Nitric Oxide Synthase Activity Research Articles

Anthocyanins as an element of nutritional support for athletes: effects and molecular mechanisms of action

Antioxidants are among the common components of sports nutrition designed to counteract oxidative stress that develops during intense physical activity. One of the promising antioxidants are anthocyanins which belong to polyphenolic compounds of plant origin (class of flavonoids). The purpose of the research was to analyze the results of prospective controlled studies on the effect of anthocyanins on physical performance, and to consider the possible molecular mechanisms of their action. Material and methods. Sources were searched in PubMed, Google Scholar, and CyberLeninka databases of peer-reviewed scientific literature without restrictions on the year of publication using the following keywords: anthocyanins, physical performance, recovery, sport and exercise nutrition, oxidative stress, inflammation. Results. The main data set on the effects of anthocyanins in athletes was obtained using extracts of blackcurrant and Montmorency tart cherry. Volunteers received anthocyanins at a dose of 86-547 mg per day for 1 to 10 days with subsequent evaluation of their performance in cycling, running and fitness activities. The possibility of favorable effect of anthocyanins on physical performance and acceleration of its recovery after exertion has been shown. The source, dose and duration of intake did not significantly influence the established effects of anthocyanins. Acting as exogenous regulators of metabolism, anthocyanins can activate several mechanisms of performance enhancement, including influence on antioxidant and immune status and apoptosis intensity. Anthocyanins prevent the formation of reactive oxygen species, neutralize electrophilic compounds by direct interaction or through activation of Nrf2 factor, which regulates the transcription of antioxidant enzyme genes. The basis of the anti-inflammatory action of anthocyanins is their ability to inhibit MARK and NF-κB mediated signal transduction. Inclusion of bilberry and blackcurrant extract in the diet prevented the intensification of myocyte apoptosis and suppression of cellular immunity induced by exhausting exercise. An additional mechanism of anthocyanin action on physical performance may be an increase in blood supply of organs and tissues due to vascular dilation caused by activation of endothelial nitric oxide synthase. Conclusion. The intake of plant extracts with a high anthocyanin content can increase physical performance and improve recovery after physical exertion, which may be due to the antioxidant and anti-inflammatory effects of anthocyanins, their ability to regulate apoptosis processes and improve blood supply to organs and tissues.

Ранні маркери гострої доксорубіциніндукованої кардіотоксичності та механізми її розвитку

Arrhythmias and congestive heart failure, which pose the greatest risk of toxic cardiomyopathy, are the clinically limiting side effects of doxorubicin, the main highly active anticancer agent. The difficulty of early diagnosis of cardiomyopathy and timely detection of cardiac dysfunction associated with chemotherapy remains a significant medical problem. The aim of our study was to identify early signs of acute doxorubicin-induced cardiotoxicity in adult rats by assessing ECG changes and biochemical parameters. Acute cardiotoxicity was modelled by short-term intraperitoneal injection of doxorubicin at a total dose of 15 mg/kg. On the 5th day of the experiment, visual fluctuations of electrocardiogram (ECG) waveforms, duration and amplitude of the main teeth and intervals, as well as heart rate (HR) in the control and experimental groups of rats were studied to determine early ECG signs of cardiotoxicity. The most significant ECG changes were a doubling of the QT interval duration and significant ST-segment elevation in the rats of the experimental group. In experiments on isolated aortic rings, we demonstrated doxorubicin-induced disruption of both vascular relaxation and contraction mechanisms. The endothelium-dependent relaxation of vascular preparations of animals after administration of doxorubicin to acetylcholine (0.1 μmol/l) was 47% less than in the control group. The vascular ring contractions in rats under the influence of norepinephrine (10 μmol/l) were 59% lower than in control rats. After doxorubicin administration, oxidative stress developed against the background of cardiovascular disorders. Thus, the content of diene conjugates and malondialdehyde increased by 4 and 2.5 times, respectively. At the same time, in isolated cardiac mitochondria, the activity of inducible NO synthase increased 3.7-fold with a simultaneous significant 4.8-fold inhibition of constitutive NO synthase. An increase in the content of acute-phase biochemical parameters that are markers of damage, namely alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine phosphokinase myocardial fraction (CPK MB) by 2.2, 1.4 and 1.5 times, respectively, was detected. Thus, the acute cardiotoxic effect of doxorubicin results in changes in the systolic-diastolic function of the left ventricle of the heart and its conduction, automaticity and contractility, as well as impaired relaxation and contraction of isolated preparations of the aorta against the background of increased activity of inducible and decreased constitutive NO synthesis, accompanied by oxidative stress and increased content of biochemical markers of myocardial damage.

Caveolae and caveolin-1 as targets of dietary polyphenols for protection against vascular endothelial dysfunction.

Caveolae, consisting of caveolin-1 proteins, are ubiquitously present in endothelial cells and contribute to normal cardiovascular functions by acting as a platform for cellular signaling pathways as well as transcytosis and endocytosis. However, caveolin-1 is thought to have a proatherogenic role by inhibiting endothelial nitric oxide synthase activity and Nrf2 activation, or by promoting inflammation through NF-κB activation. Dietary polyphenols were suggested to exert anti-atherosclerotic effects by a mechanism involving the inhibition of endothelial dysfunction, by which they can regulate redox-sensitive signaling pathways in relation to NF-κB and Nrf2 activation. Some monomeric polyphenols and microbiota-derived catabolites from monomeric polyphenols or polymeric tannins might be responsible for the inhibition, because they can be transferred into the circulation from the digestive tract. Several polyphenols were reported to modulate caveolin-1 expression or its localization in caveolae. Therefore, we hypothesized that circulating polyphenols affect caveolae functions by altering its structure leading to the release of caveolin-1 from caveolae, and attenuating redox-sensitive signaling pathway-dependent caveolin-1 overexpression. Further studies using circulating polyphenols at a physiologically relevant level are necessary to clarify the mechanism of action of dietary polyphenols targeting caveolae and caveolin-1.

LAPAROTOMY ENHANCES THE PRODUCTION OF REACTIVE NITROGEN SPECIES IN THE HEARTS OF RATS EXPOSED TO A SINGLE PROLONGED STRESS

The aim of this study was to assess the production of reactive nitrogen species in the hearts of rats subjected to surgical trauma simulated against the background of post-traumatic stress disorder (PTSD). Materials and methods.The study was conducted on 42 white Wistar rats weighing 210-230 g, then divided into 6 groups: Group 1 consisted of intact animals, Group 2 included animals exposed to the induction of the PTSD model through single-prolonged stress (SPS), Group 3 – rats subjected to a sham operation, Group 4 – animals undergoing laparotomy, Group 5 – rats undergoing a sham surgical operation following SPS, and Group 6 – animals undergoing laparotomy under modeled SPS. NO synthase activity in the heart homogenate was determined spectrophotometrically. Results. The formation of peroxynitrite was assessed by the content of peroxynitrite of alkaline and alkaline-earth metals. SPS exposure significantly increases the production of reactive nitrogen species in the hearts of rats, in particular, it enhances NO synthase activity by activating the inducible isoform and reduces the activity of constitutive NO synthases, which is accompanied by the growth in the concentration of peroxynitrites. On the 7th day after laparotomy against the background of the experimental PTSD model, the indicators of nitrosative stress in the hearts of rats (total and inducible NO synthase activity and peroxynitrite concentration) significantly exceeded their values in the groups subjected to a single laparotomy and to a sham operation against the background of simulated SPS.

Endothelium-Mimicking Bilayer Vascular Grafts with Dual-Releasing of NO/H2S for Anti-Inflammation and Anticalcification.

Vascular complications caused by diabetes impair the activities of endothelial nitric oxide synthase (eNOS) and cystathionine γ-lyase (CSE), resulting in decreased physiological levels of nitric oxide (NO) and hydrogen sulfide (H2S). The low bioavailability of NO and H2S hinders the endothelialization of vascular grafts. In this study, endothelium-mimicking bilayer vascular grafts were designed with spatiotemporally controlled dual releases of NO and H2S for in situ endothelialization and angiogenesis. Keratin-based H2S donor was synthesized and electrospun with poly(l-lactide-co-ε-caprolactone) (PLCL) as the outer layer of the graft to release H2S. Hyaluronic acid, one of the major glycosaminoglycans in endothelial glycocalyx, was complexed with Cu ions as the inner layer to mimic glutathione peroxidase (GPx) and maintain long-term physiological NO flux. The synergistic effects of NO and H2S of bilayer grafts selectively promoted the regeneration and migration of human umbilical vascular endothelial cells (HUVECs), while inhibiting the overproliferation of human umbilical artery smooth muscle cells (HUASMCs). Bilayer grafts could effectively prevent vascular calcification, reduce inflammation, and alleviate endothelial dysfunction. The in vivo study in a rat abdominal aorta replacement model for 1 month showed that the graft had a good patency rate and had potential for vascular remodeling in situ.

Rapid Screening of Phenolic Compounds with Anti-Enteritis Activity from Camellia oleifera Oil Using a Smurf Drosophila Model and Molecular Docking Methods.

Screening and identifying the active compounds in foods are important for the development and utilization of functional foods. In this study, the anti-enteritis activity of ethanol extract from Camellia oleifera oil (PECS) was quickly evaluated using a Smurf Drosophila model and the metabolomics approach, combined with molecular docking techniques, were performed to rapidly screen and identify compounds with potential anti-enteritis activity in PECS. PECS showed good anti-enteritis activity and inhibited the activity of 5-lipoxygenase (LOX), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). In particular, wighteone and p-octopamine were newly identified in C. oleifera oil and were proven to have good anti-enteritis activity. The inhibitory activity of kaempferitrin (IC50 = 0.365 mmol L-1) was higher than that of wighteone (IC50 = 0.424 mmol L-1) and p-octopamine (IC50 = 0.402 mmol L-1). Of note, the IC50 value of salazosulfapyridine was 0.810 mmol L-1. Inhibition of LOX activity is likely one of the anti-enteritis mechanisms of PECS. These new findings lay the foundation for further investigations into the underlying mechanisms of anti-enteritis activity in C. oleifera oil.

The Role of β-Defensin 1 Against Porphyromonas gingivalis Lipopolysaccharide-Mediated Inflammation in the THP-1 Cell Line.

Introduction Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) is one of the crucial virulence factors of periodontitis. Antimicrobial peptides (AMPs) are emerging as alternatives or adjuncts to antibiotics in the treatment of microbial infections. In this study, cytotoxicity, anti-inflammatory activity, anti-oxidative stress, cell cycle analysis, and apoptosis properties of AMP, β-defensin 1, were studied in Pg-LPS-stimulated THP-1 (Tohoku Hospital Pediatrics - 1) cell line. Methods The cytotoxic nature of Pg-LPS and β-defensin 1 was studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The cytotoxic effect of β-defensin 1 on Pg-LPS-stimulated THP-1 cells was also studied by the same method. The anti-inflammatory role of β-defensin 1 against cyclooxygenase (COX), lipoxygenase (LOX), myeloperoxidase (MPO), and inducible nitric oxide synthase activities were studied. The anti-oxidative nature of β-defensin 1 was analyzed by measuring reactive oxygen species (ROS) generation by dichlorodihydrofluorescein diacetate (DCFDA) assay. Cell cycle distribution and apoptosis were studied by flow cytometry. The hemolytic nature of β-defensin 1 was predicted using the HemoPred web tool. Results The results of the study demonstrated that Pg-LPS showed dose-dependent cytotoxicity to THP-1 cells. β-Defensin 1 had dose-dependent cytotoxicity to THP-1 cells and showed a protective effect on THP-cells up to 1 µg/mL of Pg-LPS, beyond which cell viability decreased. β-Defensin 1 inhibited COX, LOX, MPO, and inducible nitric oxide synthase activities in a concentration-dependent manner. β-Defensin 1 showed anti-oxidative activity by suppressing the generation of ROS measured through fluorescence intensity. From the cell cycle analysis, it was found that β-defensin 1 was able to reduce the Pg-LPS-induced cell cycle arrest at the G0/G1 phase. From the apoptosis profile, β-defensin 1 was found to increase the live cells when compared to THP-1 cells stimulated only with Pg-LPS, indicating that β-defensin 1 provided a protective role to THP-1 cells. β-Defensin 1 was found to be hemolytic in nature by the HemoPred web tool. Conclusion β-Defensin 1 exerted multifunctional activities and can be considered a promising agent for controlling periodontitis.

Synergistic gastroprotective and antioxidative effects of natural olive oil and usnic acid isolated from Usnea longissima, a lichen species in Anatolia (Türkiye), in the indomethacin ulcer model created in rats

Usnea longissima, a medically important lichen growing up in forests in Anatolia (Turkey). In this study, the gastroprotective effect of usnic acid (UA) was investigated using an indomethacin (IND)-induced gastric ulcer model in rats. While 25, 50, 100 and 200 mg/kg UA doses were dissolved in 2 ml of olive oil (OO) and administered to rats, only OO was given to one group. In addition, lansoprazole (LAN) and ranitidine (RAN) and IND were dissolved in water and administered to rat groups. IND administration caused very high levels of damage to rat stomachs. On the other hand, when four doses of UA, OO, RAN and LAN were administered, it was determined that hyperemias in the stomach of rats was significantly reduced. After macroscopic analysis of gastric tissues, the activities of superoxide dismutase (SOD), catalase (CAT), myeloperoxidase (MPO) and nitric oxide synthase (iNOS and cNOS) enzymes as well as glutathione (GSH) and lipid peroxidation (LPO) levels were determined in these tissues. After IND application, it was detected increases in MPO, CAT and iNOS activities in gastric tissues and decreases in SOD, cNOS and GSH amounts. Four doses of UA, OO, RAN and LAN applications reversed the trend, bringing them closer to healthy levels.

Modification-Specific Proteomic Analysis Reveals Cysteine S-Nitrosylation Mediated the Effect of Preslaughter Transport Stress on Pork Quality Development.

This study aimed to explore the effects of preslaughter transport stress on protein S-nitrosylation levels and S-nitrosylated proteome in post-mortem pork longissimus thoracis (LT) muscle. Pigs (N= 16) were randomly divided into 3 h transport (high-stress group, HS) and 3 h transport followed by 3 h resting treatments (low-stress control group, LS). Results demonstrated that high transport stress levels induced nitric oxide (NO) overproduction by promoting NO synthase (NOS) activity and neuronal NOS (nNOS) expression, which thereby notably increased protein S-nitrosylation levels in post-mortem muscle (p < 0.05). Proteomic analysis indicated that 133 S-nitrosylation-modified cysteines belonging to 85 proteins were significantly differential, of which 101 cysteines of 63 proteins were higher in the HS group (p < 0.05). Differential proteins including cytoskeletal and calcium-handling proteins, glycolytic enzymes, and oxidoreductase were mainly involved in the regulation of muscle contraction and energy metabolism that might together mediate meat quality development. Overall, this study provided direct evidence for changes in S-nitrosylation levels and proteome in post-mortem muscle in response to preslaughter transport stress and revealed the potential impact of S-nitrosylated proteins on meat quality.

Inhibition of nitric oxide synthase modulates learned helplessness response in female rats

Learned helplessness is the condition of humans or animals that have learned to behave helplessly, failing to respond even though there are opportunities to help themselves by avoiding unpleasant circumstances. It was investigated the participation of nitric oxide (NO) in female Wistar rats in learned helplessness tests. The animals received acute treatment with N–nitro-l-arginine methyl ester (L-NAME, 10 mg/kg or 100 mg/kg), an inhibitor of NO synthase, 30 minutes before random inescapable shock sessions lasting 25 minutes. On the test day, the animals that received L-NAME of 10 mg/kg or 100 mg/kg 24 hours beforehand, demonstrated reduced difference between stress and non-stress groups compared with control groups. The activity of the neuronal nitric oxide synthase was lower in animals pretreated with L-NAME, especially at a dose of 100 mg/kg. These results indicate that acute inhibition of NO synthase improved learned helplessness response, suggesting that NO is involved with stress and depression.

Influence of organism stimulation with bacterial lipopolysaccharide on nitric oxide production and metabolism in rat heart on the background of metabolic syndrome

Aim. The aim of the study was to establish the changes in nitric oxide production and metabolism in rat heart during combined influence of organism stimulation with bacterial lipopolysaccharide (LPS) and modeling of metabolic syndrome (MetS).&#x0D; Materials and methods. The study was conducted on 24 mature male Wistar rats weighing 200–260 g. Experiment lasted 60 days. The animals were divided into 4 groups of 6 animals each: control group, MetS group, LPS stimulation group, LPS + MetS group. MetS was reproduced by using a 20 % fructose solution as the only source of drinking water. LPS of Salmonella typhi was administered at a dose of 0.4 μg/kg intraperitoneally. Animals from LPS + MetS group received a 20 % fructose solution as the only source of drinking water and were administered LPS. In 10 % tissue homogenate of rat heart we studied: total activity of NO-synthases (NOS), activity of constitutive (cNOS) and inducible (iNOS) isoforms, activity of nitrate (NaR) and nitrite (NiR) reductases, concentration of peroxynitrites (ONOO-), nitrites, nitrosothiols and hydrogen sulfide.&#x0D; Results. Combination of MetS and stimulation of organism with LPS led to increase in total NOS activity by 32.72 % compared to control group. Activity of cNOS did not change compared to control group. Activity of iNOS increased by 33.76 %. Arginase activity decreased by 23.53 %. NaR activity and NiR activity were increased by 86.67 % and by 149.29 %, respectively. Combination of MetS and stimulation of organism with LPS led to decrease in nitrite and nitrosothiols concentration by 38.73 % and by 54.79 %, respectively. Under these conditions concentration of ONOOelevated by 398.0 % compared to control group. Concentration of H S decreased by 27.56 %.&#x0D; Conclusions. Combination of metabolic syndrome and stimulation of organism with bacterial lipopolysaccharide leads to prevalence of peroxynitrite formation during increased nitric oxide production NO-synthase-dependent and nitrate-nitrite-NO pathways in rat heart.

Ghrelin misbalance affects mice embryo implantation and pregnancy success by uterine immune dysregulation and nitrosative stress.

In a previous study we found that ghrelin (Ghrl) misbalance during the peri-implantation period significantly impaired fetus development. In this study we aimed to evaluate the putative mechanisms underlying these effects, including embryo implantation success, uterine nitric oxide synthase (NOS) activity, nitric oxide synthesis and the inflammatory/immune uterine profile. Ghrelin misbalance was induced by injecting 4nmol/animal/day of Ghrl (hyperghrelinemia) or 6nmol/animal/day of a Ghrl antagonist (Ant: (D-Lys3)GHRP-6) from day 3 to 8 of pregnancy. Control animals (C) were injected with de vehicle. Females were euthanized at pregnancy day 8 and their uteri excised in order to evaluate: the percentage of reabsorbed embryos (microscopically), eNOS, iNOS and nytrotirosine expression (by immunohistochemistry), nitrite synthesis (by Griess technique), VEGF, IL-10, IL-17, IL-6, MMP9 and GM-CSF expression (by qPCR) and leukocyte infiltration by flow cytometry (evaluating T cells, NK cells, granulocytes, dendritic cells and macrophages). Ant-treatment significantly increased the percentage of reabsorbed embryos and the uterine expression of eNOS, iNOS and nytrotirosine. (D-Lys3)GHRP-6-treatment increased also the expression of the inflammatory cytokines IL-6, IL-17 and MMP9, and decreased that of IL-10 (anti-inflammatory). Moreover, Ant-treatment increased also the NK cells population and that of CD11b+ dendritic cells; and decreased T cells percentages. Similarly, hyperghrelinemia showed a significant increase vs. C on eNOS, iNOS and nytrotirosineuterine expression and a decrease in T cells percentages. Ghrl misbalance during the peri-implantation period induces pro-inflammatory changes and nitrosative stress in the gravid uterus, impairing significantly embryo implantation and/or development.

Oral Vitamin C Intake Ameliorates Crude Oil-Polluted Water-Induced Jejunal Contractile Dysfunctions in Wistar rats

Inhabitants of rural Niger-Delta oil communities in Nigeria often, unintentionally, consume crude oil polluted water (COCW) due to crude oil spills in the region. The impact of vitamin C supplementation during COCW ingestion on the contractile mechanism of the jejunum is not fully known. Hence, this study investigates the outcomes of COCW water and vitamin C intake on the smooth muscle activity of the jejunum in Wistar rats using standard techniques. Data obtained showed that jejunal tissue SOD concentration was significantly reduced, while jejunal tissue MDA concentration was significantly increased in the COCW-only treated group. Contraction mediated by acetylcholine was significantly increased in the COCW-only treated rats. Calcium and potassium ion influx significantly increased jejunal contraction in the COCW-only rats. Incubation of the jejunal tissue in nifedipine, indomethacin, and L-NAME significantly increased acetylcholine-mediated contractions in the jejunal tissue of the COCW-only treated rats when compared to other groups. COCW causes free radical-induced jejunal damage that results in impaired jejunal contractile activity mediated by the M2 muscarinic receptor, nitric oxide synthase activity, voltage-gated large-conductance calcium channels, potassium channels, and prostaglandins. However, the oral intake of Vitamin C supplementation significantly ameliorated impairments by enhancing jejunal antioxidant activity.&#x0D;

Pre and postharvest spraying of arginine enhanced the stress resistance and promoted wound healing in broccoli during storage

Broccoli rapidly yellows and dehydrates post-harvest, causing a short storage life. Although arginine post-harvest has been shown to enhance broccoli’s storage quality and shelf life, it remains unclear whether pre-harvest treatment yields similar benefits. The results showed that pre-harvest spraying of 5 mM arginine reduces the respiration rate and delays electrolyte leakage during broccoli’s storage period, minimizing weight loss and yellowing and chlorophyll degradation. It also inhibits the degradation of ascorbic acid (ASA) and promotes the synthesis of total phenols, thereby increasing the total antioxidant capacity. Further studies showed that pre-harvest arginine spraying enhanced the activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and nitric oxide synthase (NOS). This accelerated the nitric oxide (NO) and the polyamine (PA) accumulation. Additionally, arginine aids in reactive oxygen species (ROS) balance and wound healing. Notably, pre-harvest arginine spraying was found to inhibit the hydrogen peroxide (H2O2) accumulation and enhance the activities of superoxide dismutase (SOD) and catalase (CAT), while also activating the phenylalanine ammonia lyase (PAL) and cinnamic acid 4-hydroxylase (C4H) activities. In conclusion, pre-harvest arginine spraying extends the broccoli’s storage life and improves its storage quality by promoting endogenous arginine catabolism, ROS metabolism, and the wounds healing. Therefore, this study establishes a theoretical foundation for pre-harvest handling in post-harvest broccoli storage.

Internal ribosomal entry site-mediated translational activity of nitric oxide synthase 2

ABSTRACT The internal ribosome entry site (IRES) is a unique structure found in the 5’ untranslated region (5'-UTR) of specific messenger RNAs (mRNAs) that allows ribosomes to bind and initiate translation without the need for a cap structure. In this study, we investigated the presence and functional properties of the IRES activity of nitric oxide synthase 2 (NOS2) mRNA, which encodes an enzyme that produces nitric oxide in response to various stimuli such as inflammation. Nitric oxide is a signaling molecule that plays a crucial role in various physiological processes, including immune responses and neuronal signaling. Our results showed the existence of IRES activity in the 5'-UTR of Nos2 mRNA in various cell types. IRES-mediated translation of NOS2 mRNA was higher in neuronal cells and its activity increased in response to lipopolysaccharide (LPS). Despite inhibition of cap-dependent translation, nitrite production was partially maintained. These results demonstrate the presence of IRES activity in the 5'-UTR of NOS2 mRNA and suggest that IRES-mediated translation plays a key role in controlling nitric oxide production in response to LPS, an inflammatory stimulus.

Human omentin-1 reduces vascular insulin resistance and hypertension in Otsuka Long-Evans Tokushima Fatty rats.

Hypertension is one of the major risk factors for renal failure and cardiovascular diseases, and is caused by various abnormalities including the contractility of blood vessels. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which mimic human type 2 diabetes, are frequently used to study obesity-induced insulin resistance (IR) and hypertension. Human omentin-1 is one of the recently identified adipocytokines. We previously demonstrated that human omentin-1 not only caused vasodilation in rat isolated blood vessels, but also prevented inflammatory responses, a possible mechanism relating IR, in human vascular endothelial cells. Taken together, we hypothesized that human omentin-1 may reduce obesity-induced IR and hypertension in OLETF rats. OLETF rats were intraperitoneally administered with human omentin-1 for 7days. Human omentin-1 had no influence on overweight, hyperglycemia, urinary glucose extraction, hyperinsulinemia, and systemic IR in OLETF rats. Human omentin-1 decreased systolic blood pressure in OLETF rats. The measurement of isometric contraction revealed that human omentin-1 had no influence on the agonist-induced contractile and relaxant responses in isolated thoracic aorta from OLETF rats. However, the relaxant response mediated by human insulin was converted into the contractile response in thoracic aorta from OLETF rats, which was prevented by human omentin-1. The Western blotting revealed that human omentin-1 improved the decrease in endothelial nitric oxide synthase activation in isolated thoracic aorta from OLETF rats. In summary, we for the first time revealed that human omentin-1 partly reduces vascular IR and thereby inhibits hypertension in OLETF rats.

Effects of injecting L-Arginine on immune responses and intestinal structure of the hybrid grouper (Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus ♂)

To study the effects of L-Arginine (Arg) on immune responses and intestinal structure in a hybrid grouper (Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus ♂), intraperitoneal injection of different Arg doses (0, 50, 100, 200 mg per kg body weight of grouper) was administered once every seven days, repeating the process three times in the present study. A total of 216 groupers (initial weight of 349.42 ± 0.67 g) were randomly divided into four groups with three replicates, and each of the replicates comprised 18 fish. Serum was sampled at the seven, 14, and 21 days post-injection. The results showed that injection of Arg had a significant effect on serum albumin, immunoglobulin M (IgM), total protein (TP) and nitric oxide (NO) content, as well as nitric oxide synthase (NOS) and lysozyme activity (P < 0.05). With an increase of Arg dose, serum NO, TP, and albumin levels initially increased and subsequently decreased, and were significantly higher in fish injected with Arg at doses of 50 and 100 mg/kg in comparison to the control group. On days seven and 14, the highest serum albumin, TP, and IgM levels were observed in the 100 mg/kg group. With an increase in injection time and frequency, the 50 mg/kg group showed the optimal immune status after the third injection. Based on the results of the intestinal digestive enzyme activity and morphology, the injection of Arg at 50 mg/kg was the most beneficial, followed by the 100 mg/kg dose. Injection of 200 mg/kg Arg, especially after three injections, had adverse effects on immune responses and intestinal structure. These findings indicated that the intraperitoneal injection of L-Arg at a certain concentration can enhance the immunity and intestinal morphology of groupers. Notably, the most significant benefits were observed after three consecutive injections of Arg at 50 mg/kg in this experiment.

Bile acids promote endothelial cell homeostasis via the regulation of cell metabolism and quiescence

Abstract Background Endothelial cell (EC) homeostasis is characterized by a quiescent and low-metabolic state. Obesity leads to EC dysfunction and CVD. We demonstrated that bariatric surgery improved obesity-induced EC dysfunction and increased fasting and post-prandial plasma levels of bile acids (BAs)(in particular, cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)). BAs are emerging as metabolic signaling molecules in several tissues, but their effect on ECs has not been clearly established yet. Purpose We investigate the effect of BA on the maintenance of EC quiescence. Methods Human Aortic Endothelial Cells (HAECs) were treated for 24h with vehicle (0.1% DMSO), CA, CDCA and DCA (50uM each) in 2%FBS EGM-2 media. Percentage of apoptotic cells was assessed with AnnexinV/PI. Seahorse Mitostress test, Mitoplate and glucose uptake with 0.5µCi/ml of 14C-2-deoxy-D-glucose were used to evaluate changes of HAEC metabolism. Immunostaining for Ki67, Crystal Violet staining and scratch assay were performed to assess HAEC proliferation and migration. NO production and endothelial NO synthase (eNOS) activity were measured via DAF-2 probe and Western Blot, respectively. By qPCR genes involved in Notch pathway were evaluated. Mesoscale vascular injury panel, scratch assay, Ki67 immunostaining and glucose uptake were also performed in HAECs pre-treated with 50uM of BAs and stimulated with 0.5ng/mL TNF-α for 24h. Results HAECs treated with BAs increased NO production compared to vehicle and a reduction in both basal and maximal mitochondrial respiration and extracellular acidification rate was shown by Seahorse Mitostress test. Mitoplate assay displayed an overall reduction in the metabolic rate of HAECs treated with CA and DCA and a peculiar metabolic pattern in cells treated with CDCA. Reduction in glucose uptake was also observed after BA treatment, suggesting that BAs are able to decrease EC metabolism. Immunostaining for Ki67 and Crystal Violet highlighted that BAs reduced cell proliferation. The scratch assay also displayed a reduction in EC migration after BA treatment. BAs also reduced EC apoptosis, as significant differences in the percentage of AnnexinV/PI positive cells was observed in BA-treated cells compared to vehicle. qPCR showed increased expression of genes involved in Notch pathway promoting quiescence after BA treatment. Glucose uptake, scratch assay and Ki67 immunostaining performed in TNF-α stimulated HAECs suggested a BA-dependent endothelial protection in conditions of EC pro-inflammatory activation. We did not observed a reduction in pro-inflammatory biomarkers released in the supernatant of HAECs pre-treated with BA and stimulated with TNF-α. Conclusions Our results suggest that BAs may be involved in the maintenance of EC quiescence by regulating EC metabolism and gene expression profile. These findings highlight a potential role of BAs as endothelial-protective molecules.

Abstract 16373: Pyridoxamine (Vitamin B6) Mitigation of Protein Glycation and Deleterious Platelet-Material Interactions in Bioprosthetic Heart Valve Leaflets

Introduction: Though bioprosthetic heart valves (BHV) most often do not require anticoagulation, it has been reported that 12% of BHVs have significant leaflet thrombosis. We identified Vitamin B6 (pyridoxamine, abbr. PYR) as a pretreatment agent to reduce advanced glycation end-product (AGE) accumulation within bovine pericardial (BP) BHVs. AGEs contribute to valvular degeneration through collagen crosslinking and immune signaling. PYR is also known to inhibit platelet aggregation. Hypothesis: PYR pretreatment and supplementation mitigate BHV thrombosis. Methods: Glutaraldehyde fixed collagen coated (GFC) PVC tubing was used as a leaflet surface model and methylglyoxal (MGO) pretreatment to simulate glycation. PYR pretreated collagen surfaces and perfusate PYR supplementation were compared to untreated controls. These studies were repeated with glycated GFC. Fresh citrated human whole blood from healthy donors was perfused over the surfaces for 4 hours at 37 °C and arterial shear using the Chandler loop apparatus. Post perfusion platelet activation (CD62P expression via flow cytometry) and nitric oxide synthase (NOS) activity were measured. Platelets adhered to the tubing sections were visualized and quantitated via scanning electron microscopy. Results: PYR pretreatment of GFC surfaces reduced platelet activation after perfusion compared to the control (Fig1a). Surface glycation with MGO resulted in increased platelet activation, but PYR pretreatment of the glycated surface mitigated the effect of glycation on platelet activation (Fig1b). NOS activity was impaired by surface glycation and PYR treatment appears to rescue function in glycated environments (Fig1c). Platelet adhesion to GFC was increased by glycation (Fig1d-f). Conclusions: Glycated surfaces, even in euglycemia, are prothrombotic when compared to non-glycated surfaces. Vitamin B6 reduces platelet activation and adhesion in both glycated and non-glycated environments.

MECHANISMS OF NITROSATIVE STRESS AND DESTRUCTION OF THE ORGANIC MATRIX OF THE RAT MANDIBLE IN THE RECOVERY PERIOD AFTER ITS INCOMPLETE FRACTURE UNDER CONDITIONS OF CHRONIC ALCOHOL INTOXICATION

The mechanisms of nitrosative stress and depolymerization of biopolymers of the mandibular bone tissue after its incomplete fracture under conditions of chronic alcohol intoxication were investigated. The experiments were conducted on 20 white Wistar rats weighing 190-240 g, divided into 4 groups: Group 1 - intact animals, Group 2 - after reproduction of chronic alcohol intoxication, Group 3 - after modeling of mandibular fracture, Group 4 - after reproduction of mandibular fracture in the setting of chronic alcohol intoxication. The activity of total NO synthase, its constitutive and inducible isoforms, ornithine decarboxylase, concentrations of peroxynitrites of alkaline and alkaline-earth metals, free hydroxyproline, N-acetylneuraminic and hexuronic acids were determined in the homogenate of the standard mandible area by spectrophotometric method. It has been shown that the reproduction of chronic alcohol intoxication significantly increases the total and inducible NO synthase activity in the mandibular homogenate, accompanied by an increase in the concentration of the key marker of nitrosative stress – peroxynitrite. On the 14th day after the reconstruction of a mandibular fracture on the background of chronic alcohol intoxication, NO synthase activity and the content of peroxynitrites of alkaline and alkaline-earth metals in the bone homogenate exceed the results of groups with separate action of pathogenic factors. Under these conditions, the activity of the key enzyme of polyamine biosynthesis, ornithine decarboxylase, significantly decreases, and the depolymerization of bone biopolymers (collagen, glycoproteins, and proteoglycans) increases, which creates prerequisites for disruption of reparative processes in the fracture site.