Nitric Oxide Synthase Activity Research Articles

Modulation of the activation of endothelial nitric oxide synthase and nitrosative stress biomarkers by aspirin triggered lipoxins: A possible mechanism of action of aspirin in the antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is characterized by the clinical manifestation of vascular thrombosis (VT) or pregnancy morbidity (PM) and antiphospholipid antibodies (aPL) that can modify the nitric oxide production. Low-dose aspirin is used in the prevention and treatment of diverse alterations of pregnancy. One of the mechanisms of action of aspirin is to induce the production of aspirin-triggered-lipoxins (ATL). The aim of this study was to evaluate the modulatory effect of ATL over the activation of endothelial nitric oxide synthase (eNOS) and nitrosative stress biomarkers induced by aPL. We used polyclonal IgG and sera from women with aPL and PM/VT or VT only, and from women with PM only and positive for non-criteria aPL (SN-OAPS). In these sera, biomarkers of nitrosative stress (nitrites and nitrotyrosine) were measured. The protein expression of nitrotyrosine and the phosphorylation of eNOS (at Ser1177) were estimated in human umbilical vein endothelial cells (HUVECs) stimulated with polyclonal IgG with or without ATL. Women with SN-OAPS showed increased circulating levels of nitrites and nitrotyrosine. Likewise, polyclonal IgG from either SN-OAPS or VT patients stimulated nitrotyrosine expression in HUVECs. ATL decreased the nitrotyrosine expression induced by polyclonal IgG from the SN-OAPS group. ATL also recovered the reduced eNOS phosphorylation at Ser1177 in HUVECs stimulated with polyclonal IgG from women with PM/VT or SN-OAPS. Increased nitrosative stress present in serum of women with SN-OAPS is associated with IgG-mediated impaired endothelial NO synthesis in endothelial cells. ATL prevent these cellular changes.

Targeting the adenosine monophosphate-activated protein kinase signalling pathway by bempedoic acid attenuates Angiotensin II-induced cardiac remodelling in renovascular hypertension in rats

The crosstalk between the renin-angiotensin system and Adenosine monophosphate-activated protein kinase (AMPK) gained significant interest due to their involvement in the pathogenesis of several cardiovascular diseases. Angiotensin II (Ang II) plays a crucial role in developing cardiac remodelling by inducing energy imbalance, inflammation, oxidative and endoplasmic reticulum stress, and transforming growth factor-β (TGF-β)-induced fibrosis. Ang II directly or through extracellular signal-regulated kinase (ERK) activation impairs AMPK signalling with well-known antioxidant, anti-inflammatory, and anti-fibrotic effects. AimThis study aimed to investigate the role of bempedoic acid, a novel antihyperlipidemic drug, in attenuating hypertension-induced cardiac remodelling in rats by modulating Ang II-induced damage and activating the AMPK signalling pathway. MethodSixty adult male Sprague Dawley rats were randomly allocated into the Sham control group, Hypertensive group, Captopril group (30 mg/kg), and Bempedoic acid group (30 mg/kg). Hypertension was induced by left renal artery ligation in all groups except the Sham control group. Treatment with captopril and bempedoic acid started 14 days post-surgy and lasted two weeks. Finally, Hemodynamic measurements and electrocardiographic examination were done followed by heart tissue samples collection for biochemical, histopathological, and immunohistochemical examinations. Key findingsBempedoic acid preserved the cardiac function and electrocardiogram patterns. It inhibited endoplasmic reticulum stress, exhibited antioxidant activity, and increased endothelial nitric oxide synthase activity. Bempedoic acid interfered with ERK signalling pathways, including nuclear factor-κB and TGF-β, exerting anti-inflammatory and anti-fibrotic effects. SignificanceThese findings indicate the cardioprotective and antihypertrophic activity of bempedoic acid, which are suggested to result from energy-independent AMPK downstream signalling activation.

Phenotypic screening of novel probiotics with potential anti-neuroinflammation activity based on cell and zebrafish models

Neuroinflammation, a major pathological factor contributing to various neurodegenerative diseases, is closely associated with gut microbiota imbalance. The aim of this study was to establish a rapid and efficient screening method to obtain potential anti-neuroinflammatory strains. Firstly, 94 strains isolated from different sources were screened against neuroinflammation in lipopolysaccharide (LPS)-activated BV-2 cells model to identify potential strains quickly. In vitro cellular experiments revealed that 21 strains significantly inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity, which were selected for further evaluation of gastrointestinal tolerance and adhesion properties. Among them, 18 strains with promising probiotic properties were further confirmed for anti-neuroinflammatory effects in vivo using the CuSO4-induced zebrafish model. The results showed that eight strains significantly mitigated the migratory behavior of zebrafish neutrophils and the mRNA expression level of pro-inflammatory cytokines IL-1β compared to other strains. Additionally, Lactiplantibacillus plantarum and Limosilactobacillus fermentum were identified as the main species by sequencing their 16S rDNA. In conclusion, this cell-zebrafish model is promising for high-throughput screening of novel probiotics with potential anti-neuroinflammation activity.

Effect of Pre-Slaughter Transport Stress on Protein S-Nitrosylation Levels of Pork during Postmortem Aging.

This study aimed to investigate the effect of pre-slaughter transport stress on the protein S-nitrosylation levels of pork during 0, 3, and 6 days of aging. Pigs (N = 16) were randomly selected and divided into two treatments: 3 h transport (transport stress, TS) and 3 h transport followed by 3 h resting (control, CON). Results showed that the TS group at 0 and 3 days presented a higher nitric oxide synthase (NOS) activity and neuronal NOS (nNOS) expression than the CON group (P < 0.05). Additionally, nNOS was not only abundantly localized in the membrane but also observed in small amounts in the cytoplasm. The immunoblot of overall S-nitrosylated protein showed that protein S-nitrosylation levels in the TS group were greater than in the CON group during postmortem aging (P < 0.05). This work can deliver novel insights into the mechanism of meat quality changes in response to pre-slaughter stress.

Alcohol and vascular endothelial function: Biphasic effect highlights the importance of dose.

Alcohol (ethanol) consumption has different influences on arterial disease, being protective or harmful depending on the amount and pattern of consumption. The mechanisms mediating these biphasic effects are unknown. Whereas endothelial cells play a critical role in maintaining arterial health, this study compared the effects of moderate and high alcohol concentrations on endothelial cell function. Human coronary artery endothelial cells (HCAEC) were treated with levels of ethanol associated with either low-risk/moderate drinking (i.e., 25 mM) or high-risk/heavy drinking (i.e., 50 mM) after which endothelial function was assessed. The effect of ethanol's primary metabolite acetaldehyde (10 and 25 μM) was also determined. Moderate ethanol exposure (25 mM) improved HCAEC barrier integrity as determined by increased transendothelial electrical resistance (TEER), inhibited cell adhesion molecule (CAM) mRNA expression, decreased inflammatory cytokine (interferon-γ and interleukin 6) production, inhibited monocyte chemotactic protein-1 (MCP-1) expression and monocyte adhesion, and increased homeostatic Notch signaling. In contrast, exposure to high-level ethanol (50 mM) decreased TEER, increased CAM expression and inflammatory cytokine production, and stimulated MCP-1 and monocyte adhesion, with no effect on Notch signaling. Reactive oxygen species (ROS) generation and endothelial nitric oxide synthase activity were increased by both alcohol treatments, and to a greater extent in the 50 mM ethanol group. Acetaldehyde-elicited responses were generally the same as those of the high-level ethanol group. Ethanol has biphasic effects on several endothelial functions such that a moderate level maintains the endothelium in a nonactivated state, whereas high-level ethanol causes endothelial dysfunction, as does acetaldehyde. These data show the importance of dose when considering ethanol's effects on arterial endothelium, and could explain, in part, the J-shaped relationship between alcohol concentration and atherosclerosis reported in some epidemiologic studies.

Nitrosative stress-based specific evaluation of creatine use in combination with aerobic running exercise at different speeds: a preclinical study in mice.

In acute and chronic aerobic exercise, skeletal muscle and liver are the main organs that adapt and regulate metabolic activity. The levels of nitrosative stress caused by exercise in these organs are extremely important in the continuity of exercise, its health-promoting benefits, and the evaluation of therapeutic efficacy. In this study, nitrosative stress levels were investigated in musculus quadriceps femoris tissue and liver tissue of mice that were given low and high-speed aerobic running exercise and also received Cr supplementation. In this study, nitrosative stress levels were investigated in the muscle/liver tissue of 42 BALB/c mice that were given low and high-speed aerobic running exercise and creatine monohydrate (Cr) (40 mg/kg of diet daily) supplementation with exercise. The study included six groups with and without Cr supplementation, low-speed aerobic running, high-speed aerobic running, and no exercise. The mice in groups with low-speed and high-speed aerobic exercise with and without Cr supplementation were run on the treadmill for 8 weeks. Then, nitric oxide (NO·), nitric oxide synthase (NOS), and peroxynitrite (ONOO-) levels in muscle/liver tissue were measured by spectrophotometric method. It was found that the nitrosative stress level in the groups that did low and high-speed aerobic running exercises increased compared to the group that did not exercise. It was found that NO· decreased NOS activity and ONOO- level increased in muscle tissues of low and high-speed aerobic exercise groups that received Cr supplementation compared to those that did not. However, NO· and ONOO- levels in liver tissue decreased while NOS activity did not change. The lowest level of nitrosative stress in both muscle and liver tissue was found in the low-speed exercise group receiving Cr supplementation. Although supplements in exercise are an important component, the simultaneously measured nitrosative stress level is critical in determining the optimal exercise.

Sodium Selenite Modulates Global Activation of Proinflammatory M1-like Macrophages, Necroinflammation and M1-like/M2-like Dichotomy at the Onset of Human Type 1 Diabetes.

The study aims to show that sodium selenite (Ss) would have an immunomodulatory effect on the functional activity of proinflammatory macrophages (Mφs) during their extended extracellular activation at the onset of human type 1 diabetes (T1D). Exacerbated activation of proinflammatory "M1" macrophages (Mφs) can promote chronic local pancreatic islet inflammation and T1D development. We investigated the ex vivo effects of Ss on the immune modulation of global/extended activation of human proinflammatory M1-like Mφs. Experiments were carried out on primary monocytes-derived Mφs (MDMs). The levels of IL-1β, TNF-α, H2O2 and intracellular free calcium ions (ifCa2+), and the ratios of IL-1β-to-IL-10 and TNF-α-to-IL-10 were markedly increased in T1D Mφs than in healthy control Mφs. Conversely, both IL-10 production and arginase 1 (ARG1) activity were downregulated in T1D Mφs. Additionally, Ss treatment induced a marked downregulation of respiratory burst, ifCa2+ levels, M1-like Mφ-associated inducible nitric oxide (NO) synthase (iNOS) activity, cell necrosis and related necroinflammation biomarkers, including IL-1β and TNF-α, CD14 expression, and the ratios of iNOS-to-ARG1, IL-1β-to-IL-10, and TNF-α-to-IL-10. Moreover, Ss upregulated anti-inflammatory "M2-like" Mφ activity as demonstrated by ARG1 activity and IL-10 production, as well as phagocytosis capacity. Ss exerts a potent immunomodulatory role on functional activities of human proinflammatory T1D M1-like Mφs subjected to extended activation, as well as on the M1-like/M2-like dichotomy. Additionally, the current study provides a novel therapeutic approach using Ss to promote the anti-inflammatory function of Mφs at the onset of T1D.

Molecular Study on Twin Cohort with Discordant Birth Weight.

The increased rate of twinning has pointed out newer challenges in clinical practices related to gestational complications, intrauterine growth restriction, perinatal mortality, and comorbidities. As a twin pregnancy progresses, the increased demand for oxygen supply can easily disrupt the redox homeostasis balance and further impose a greater challenge for the developing fetuses. A substantial birth-weight difference acts as an indicator of a deficit in oxygenation or blood flow to one of the fetuses, which might be related to a low bioavailable nitric oxide level. Therefore, in this study, we focused on networks involved in the adjustment of oxygen supply, like the activation of inducible and endothelial nitric oxide synthase (NOS3) along with free radical and lipid peroxide formation in mature twin pairs with high birth-weight differences. The selected parameters were followed by immunofluorescence staining, fluorescence-activated cell sorting analysis, and biochemical measurements in the umbilical cord vessels and fetal red blood cells. Based on our data set, it is clear that the lower-weight siblings are markedly exposed to persistent intrauterine hypoxic conditions, which are connected to a decreased level in NOS3 activation. Furthermore, the increased level of peroxynitrite aggravates lipid peroxidation and induces morphological and functional damage and loss in redox homeostasis.

A Mannose Receptor from Litopenaeus vannamei Involved in Innate Immunity by Pathogen Recognition and Inflammation Regulation.

Mannose receptor, as a member of the C-type lectin superfamily, is a non-canonical pattern recognition receptor that can internalize pathogen-associated ligands and activate intracellular signaling. Here, a mannose receptor gene, LvMR, was identified from the Pacific white shrimp Litopenaeus vannamei. LvMR encoded a signal peptide, a fibronectin type II (FN II) domain, and two carbohydrate-recognition domains (CRDs) with special EPS and FND motifs. LvMR transcripts were mainly detected in the hepatopancreas, and presented a time-dependent response after pathogen challenge. The recombinant LvMR (rLvMR) could bind to various PAMPs and agglutinate microorganisms in a Ca2+-dependent manner with strong binding ability to D-mannose and N-acetyl sugars. The knockdown of LvMR enhanced the expression of most NF-κB pathway genes, inflammation and redox genes, while it had no obvious effect on the transcription of most phagocytosis genes. Moreover, the knockdown of LvMR caused an increase in reactive oxygen species (ROS) content and inducible nitric oxide synthase (iNOS) activity in the hepatopancreas after Vibrio parahaemolyticus infection. All these results indicate that LvMR might perform as a PRR in immune recognition and a negative regulator of inflammation during bacterial infection.

Protective effects and mechanisms of N-acetylcysteine on indomethacin-induced intestinal injury in a porcine model

This study aimed to investigate the effect of N-acetylcysteine (NAC) on indomethacin (IDMT)-induced intestinal injury in a piglet model and explore the underlying molecular mechanisms. Piglets were randomly divided into 3 treatment groups: (1) control group; (2) IDMT group; (3) NAC+IDMT group. The results showed that NAC administration significantly increased the average daily gain of piglets, attenuated the intestine hyperemia, and restored normal jejunal morphology. Further studies indicated that NAC administration significantly increased plasma citrulline concentration and jejunal villin expression, but decreased the content of proinflammatory cytokines in plasma and jejunum of IDMT-stimulated piglets. NAC administration selectively decreased the proportion of eosinophils but not neutrophils in plasma. Furthermore, NAC administration significantly increased the activities of superoxide dismutase and catalase in plasma but decreased the concentrations of hydrogen peroxide (plasma) and malondialdehyde (plasma and jejunum), as well as the activity of myeloperoxidase (jejunum) when comparing NAC+IDMT group with IDMT group. Gene Ontology analysis showed that the significantly enriched molecular function term was “ubiquitin-like protein ligase binding” for NAC+IDMT versus IDMT differentially regulated genes. In the biological process category, differentially regulated genes of NAC+IDMT versus IDMT were mainly enriched in immune-related terms. The major enrichments for differentially regulated proteins (DRPs) of NAC+IDMT versus IDMT were terms involved in lipid metabolism and immune response. KEGG pathway enrichment analysis showed that “arginine biosynthesis” was a significant enrichment term for the DRPs of NAC+IDMT versus IDMT. Further studies demonstrated that NAC administration up-regulated argininosuccinate synthase 1 mRNA expression and down-regulated arginase mRNA expression in the jejunum of IDMT-stimulated piglets. Moreover, the content of nitric oxide was restored to a normal level with the reduction of nitric oxide synthase activity. NAC administration ameliorated intestinal injury in IDMT-challenged piglets by enhancing antioxidant and anti-inflammatory functions and modulating arginine metabolism in the small intestine.

Dynamics of CD68 receptor expression in macrophages of the interstitial space of the rat testis under triptorelin prolonged administration

Testosterone, the production of which is stimulated by the release of luteinizing hormone (LH) has a remarkable anti-inflammatory and immunomodulatory effect, and in conditions of testosterone deficiency tissue damage can occur due to excessive differentiation of macrophages into a pro-inflammatory M1 phenotype. The aim of this study was to determine the spread of CD68 receptorn as the marker of inflammation on the cells in the interstitial space and testicular vessels under LH synthesis blockade with tryptorelin. Sexually mature white male rats were randomly divided into control (10 animals) and experimental (25 animals) groups. Animals in the experimental group were injected with a tryptorelin acetate solution (0.3 mg/kg). Immunochemical analysis of CD68+ expression was estimated at Olympus FV10i-LIV laser scanning confocal microscope using fluorescent labeling dye hilyte flour 488. It was shown that luteinizing hormone deprivation led to an increase in the distribution of the CD68 receptor in the interstitial space and in the testicular vessels from day 30 to 180 of the experiment, associated with the increase of inducible NO synthase activity in testis tissue. Keywords: CD68, luteinizing hormone, macrophage M1 phenotype, testis, testosterone, tryptorelin

#3145 ASSOCIATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE POLYMORPHISM WITH ENDOTHELIAL DYSFUNCTION IN DIABETIC NEPHROPATHY AMONG AZERBAIJANIS

Abstract Background and Aims Vascular endothelial dysfunction resulting from impaired nitric oxide synthase (NOS) activity in the endothelial cells of blood vessels has been suggested as playing an important role in the pathogenesis of Diabetic nephropathy (DN). It regulates endothelial function and maintains endothelial-dependent vasodilation in multiple organs, including the kidney. The study was carried out to investigate the association of T-786C single nucleotide polymorphisms with the flow mediated dilation (FMD) in DN among type 2 diabetic subjects. Method 90 patients (45 male and 45 female) with diabetic nephropathy were recruited from Nephrology and Endocrinology departments of the Azerbaijan Medical University hospital. As a control group the results were compared with the findings in 100 healthy Azerbaijani individuals (50 males and 50 females). The study was approved by ethics committee and informed consent was obtained from all patients in written form. Brachial endothelial function was determined by brachial artery vasodilator response or brachial FMD in the dominant arm according to previously validated techniques. T786C polymorphism was detected using polymerase chain reaction-restriction fragment length polymorphism analysis. Results The results showed significant differences between DN group and control group regarding the genotype and allele distributions of the T786C single nucleotide polymorphisms. The CC genotype was significantly more frequent in diabetics nephropathy patients than in control group (42% vs. 8%, p = 0.01). FMD in allele homozygote T786C(TT), T786C(CC) and heterozygote T786C(CT) were respectively 13.5 ±0.16%, 12.3±0.19%,13.9 ±0,28%. In the study groups, Azerbaijani patients showed dominance of the C allele and a mutant homozygous genotype CC of the T786C gene of eNOS. Subjects with the 786C(CC) (n = 48) showed significantly reduced FMD with those with T786C (TT) (n = 7) (p = 0.021) and T786C (CT) (n = 35) (p = 0.023), whereas there was no significant difference in endothelial-independent vasodilation between these groups. Although subjects with homozygote T786C(TT), did not show significantly reduced levels of FMD. Who were carriers of the T786C allele demonstrated markedly reduced levels of FMD compared with noncarriers (p = 0.019). Conclusion The data suggest that in diabetic nephropathy, the eNOS T786C allele may be involved in endothelial dysfunction. Azerbaijani patients showed dominance of the C allele and a mutant homozygous genotype CC of the T786C gene of eNOS.

Transport of nitrite from large arteries modulates regional blood flow during stress and exercise.

Acute cardiovascular stress increases systemic wall shear stress (WSS)-a frictional force exerted by the flow of blood on vessel walls-which raises plasma nitrite concentration due to enhanced endothelial nitric oxide synthase (eNOS) activity. Upstream eNOS inhibition modulates distal perfusion, and autonomic stress increases both the consumption and vasodilatory effects of endogenous nitrite. Plasma nitrite maintains vascular homeostasis during exercise and disruption of nitrite bioavailability can lead to intermittent claudication. During acute cardiovascular stress or strenuous exercise, we hypothesize enhanced production of nitric oxide (NO) by vascular endothelial cells raises nitrite concentrations in near-wall layers of flowing blood, resulting in cumulative NO concentrations in downstream arterioles sufficient for vasodilation. Utilizing a multiscale model of nitrite transport in bifurcating arteries, we tested the hypothesis for femoral artery flow under resting and exercised states of cardiovascular stress. Results indicate intravascular transport of nitrite from upstream endothelium could result in vasodilator-active levels of nitrite in downstream resistance vessels. The hypothesis could be confirmed utilizing artery-on-a-chip technology to measure NO production rates directly and help validate numerical model predictions. Further characterization of this mechanism may improve our understanding of symptomatic peripheral artery occlusive disease and exercise physiology.

Effect of amifostine on pulmonary contusion from blunt chest trauma in rats

Purpose: To investigate the efficacy of different doses of amifostine (AMI) and dexamethasone (DXM) on bilateral pulmonary contusion in rats.&#x0D; Methods: Forty-two Sprague Dawley rats were divided into six groups of seven animals each (control, pulmonary contusion (PC), PC + AMI 400 mg/kg, PC + AMI 200 mg/kg, PC + AMI 400 mg/kg + DXM, and PC + DXM-treated groups). RelAssay commercial kit was used to determine total oxidant status (TOS), total antioxidant status (TAS), and oxidative stress index (OSI). Nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) activity associated with blunt trauma induced pulmonary damage were determined by enzyme-linked immunosorbent assay (ELISA). Blood gas was determined using a blood gas analyzer, while histopathological examinations were performed on the tissues by hematoxylin and eosin (H &amp; E) staining method.&#x0D; Results: Significant improvement in biochemical parameters and histopathological findings were observed in the groups treated with dexamethasone and high-dose amifostine (400 mg/kg; p &lt; 0.05). However, low-dose amifostine (200 mg/kg) and a combination of dexamethasone with amifostine (400 g/kg) was ineffective in balancing the biochemical changes due to trauma (p &gt; 0.05).&#x0D; Conclusion: High-dose amifostine combined with dexamethasone mitigates trauma-induced damage in rat by preventing the elevation of key inflammatory markers. Further preclinical and clinical studies are required to determine the efficacy of amifostine in humans and to compare its activity with other agents.

Nutritional Content and Benefits of Coconut Water for the Diabetes Metabolism: a Narrative Review

Background: Coconut water is one of the natural food products that is widely available around the world. This commodity provides nutritional content that could increase rehydration status and improve health. The application of coconut water to health, including improving type 2 diabetes metabolism, has gotten research interest in recent years. Objectives: This narrative review paper aimed to summarize several articles about the nutritional content and the mechanism of the potential bioactive component in coconut water to improve type 2 diabetes metabolism. Methods: This study design was a narrative review of several articles from five databases search: PubMed/MEDLINE, Scielo, PsycINFO, Microsoft Academic, and Google Scholar. Discussion: Coconut water includes several biologically active components such as protein, amino acid, fatty acid, mineral, vitamin, and phenolic compounds. The recent discovery of nutrient content, especially antioxidant properties, and arginine in coconut water, signifies a good potential in improving type 2 diabetes mellitus. Antioxidant and arginine altered blood glucose, glycated hemoglobin, serum creatinine, blood urea, albumin, albumin/globulin ratio, liver function enzyme, lipid profile, antioxidant status, and lipid peroxidation without any significant hepatocellular damage. Arginine enhanced the nitric oxide synthase activity in the liver and arginine levels in the plasma. In mammals, the arginine-nitric oxide system, which includes AMPK, cGMP, PGC-1α, and PI3K, can maintain blood glucose homeostasis, increase insulin sensitivity, and prevent diabetes-induced oxidative stress. Conclusion: Coconut water can help to improve metabolism in type 2 diabetes mellitus conditions through antioxidant activity and arginine content.

Endothelial MRs mediate Western diet-induced lipid disorders and skeletal muscle insulin resistance in females.

Consumption of a Western diet (WD) consisting of excess fat and carbohydrates activates the renin-angiotensin-aldosterone system, which has emerged as an important risk factor for systemic and tissue insulin resistance. We recently discovered that activated mineralocorticoid receptors (MRs) in diet-induced obesity induces CD36 expression, increases ectopic lipid accumulation, and results in systemic and tissue insulin resistance. Here, we further investigated whether endothelial cell (EC) specific MR (ECMR) activation participates in WD-induced ectopic skeletal muscle lipid accumulation, insulin resistance and dysfunction. Six-week-old female ECMR knockout (ECMR-/-) and wild type (ECMR+/+) mice were fed either a WD or a chow diet for 16 weeks. ECMR-/- mice were found to have decreased WD-induced in vivo glucose intolerance and insulin resistance at 16-weeks. Improved insulin sensitivity was accompanied by increased glucose transporter type 4 (Glut4) expression in conjunction with improved soleus insulin metabolic signaling in phosphoinositide 3-kinases/protein kinase B and endothelial nitric oxide synthase activation. Additionally, ECMR-/- also blunted WD-induced increases in CD36 expression and associated elevations in soleus free fatty acid, total intramyocellular lipid content, oxidative stress, and soleus fibrosis. Moreover, in vitro and in vivo activation of ECMR increased EC derived exosomal CD36 that was further taken up by skeletal muscle cells, leading to increased skeletal muscle CD36 levels. These findings indicate that in the context of an obesogenic WD, enhanced ECMR signaling increases EC derived exosomal CD36 resulting in increased uptake and elevated concentrations of CD36 in skeletal muscle cells, contributing to increased lipid metabolic disorders and soleus insulin resistance.

CHLOROGENIC ACID CAN INHIBIT CATECHOLAMINE SECRETION FROM THE ADRENAL MEDULLA

Objective: Chlorogenic acid (CGA) is one of the most available phenolic acid compounds in foods, such as coffee and tea, which is known to be the most effective agent in lowering blood pressure and helpful in treating hypertension-related disorders. Thus the present study was aimed to determine the characteristics of CGA on secretion of catecholamines (CA) in the perfused model of the rat adrenal medulla, and also to validify its mechanism of action. Design and method: he adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: CGA, administered into an adrenal vein for 90 min, reduced ACh -induced CA secretion in a dose- and time-dependent manner. CGA also time-dependently depressed the CA secretion induced by McN-A-343, DMPP, and angiotensin II . CGA itself failed to influence spontaneous CA secretion. Also, During the perfusion of CGA for 90 min, the CA secretory responses induced by high K+, veratridine, cyclopiazonic acid, Bay-K-8644 were suppressed. However, in the simultaneous presence of CGA and L-NAME, the CA secretory responses induced by ACh, Ang II, Bay-K-8644 and veratridine was restored nearly to the control level compared to those of the inhibitory effects of CGA-treatment alone. Virtually, the adrenal NO release was significantly enhanced by CGA-treatment. Also, in the coexistence of CGA and olmesartan, ACh-induced CA secretion was markedly reduced compared to that of olmesartan-treatment alone. Conclusions: Taken together, we present the first evidence that CGA suppresses the CA secretion induced by activation of cholinoceptors as well as AT1-receptors. This CGA-induced inhibitory effect seems to be exerted by reducing both the influx of Na+ and Ca2+ through their ionic channels into adrenomedullary chromaffin cells as well as by suppressing the Ca2+ release from the cytoplasmic calcium store, through the elevated NO release by the activation of NO synthase, which is associated to the blockade of neuronal cholinonoceptors and AT1-receptors. Based on these results, the ingestion of CGA may be helpful to alleviate or prevent the cardiovascular diseases, via reduction of adrenal CA secretion and consequent decreased CA level in circulation.

The influence of nanodispersed cerium oxide on the development of oxidative stress and the production of nitric oxide in patients with type 2 diabetes mellitus

Background. In the pathogenesis of complications of diabetes mellitus (DM), in addition to glucotoxicity, the development of oxidative stress plays a leading role. Antioxidants have been of great interest for physicians in recent years. Contemporary diabetology have been focused on the search and practical implementation of pathogenetic medications that can affect the main chains of DM and prevent its negative consequences. Purpose of the study is to determine the effect of nanodispersed cerium oxide (NCO) on the production of nitric oxide (NO), the activity of antioxidant enzymes and the intensity of lipid peroxidation in the blood of patients with type 2 diabetes mellitus. Materials and methods. Seventy-two patients aged 36 to 66 years, average age of 55.20±6.82 years, who received treatment at the Municipal Clinical Hospital 2 in Poltava from July to December 2022 have been involved in the study. They have been divided into 2 groups: controls (n=35), which included people without diabetes; experimental group (n=37), which consisted of patients diagnosed with type 2 DM. Results. The use of NCO in patients with type 2 DM significantly reduces the activity of inducible NO synthase in the blood by 34.70% and the activity of arginases by 52.17% compared to the levels before treatment. The use of nanodispersed cerium oxide in the treatment of type 2 DM increases the activity of superoxide dismutase in the blood by 102.74%, and the activity of catalase by 103.04% compared to same indicators in the experimental group before therapy. Notably, blood malondialdehyde was significantly lower (by 2.35 times) compared to the same indicator before treatment. Conclusions. The use of NCO in patients with type 2 diabetes mellitus leads to an increase in antioxidant protection and a decrease in the intensity of lipid peroxidation in blood. NCO reduces the production of nitric oxide from the inducible NO synthase and weakens the competition between NO synthases and arginases for the reaction substrate. The findings of the study justify the need to include antioxidants in the pathogenetic therapy of diabetes mellitus and its complications.

Glyphosate induces autophagy in hepatic L8824 cell line through NO-mediated activation of RAS/RAF/MEK/ERK signaling pathway and energy metabolism disorders

Glyphosate is an herbicide commonly used worldwide, and its substantial use causes widespread pollution with runoff. However, research on glyphosate toxicity has mostly remained at the embryonic level and existing studies are limited. In the present study, we investigated whether glyphosate can induce autophagy in hepatic L8824cells by regulating energy metabolism and rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular regulated protein kinases (ERK) signaling by activating nitric oxide (NO). First, we selected 0, 50, 200, and 500μg/mL as the challenge doses, according to the inhibitory concentration of 50% (IC50) of glyphosate. The results showed that glyphosate exposure increased the enzyme activity of inducible nitric oxide synthase (iNOS), which in turn increased the NO content. The activity and expression of enzymes related to energy metabolism, such as hexokinase (HK)1, HK2, phosphofructokinase (PFK), phosphokinase (PK), succinate dehydrogenase (SDH), and nicotinamide adenine dinucleotide with hydrogen (NADH), were inhibited, and the RAS/RAF/MEK/ERK signaling pathway was activated. This led to the negative expression of mammalian target of rapamycin (mTOR) and P62 in hepatic L8824cells and the activation of the autophagy marker genes microtubule-associated proteins light chain 3 (LC3) and Beclin1 to induce autophagy. The above results were dependent on glyphosate concentration. To verify whether autophagy can be excited by the RAS/RAF/MEK/ERK signaling pathway, we treated L8824cells with the ERK inhibitor U0126 and found that the autophagy gene LC3 was reduced due to the inhibition of ERK, thus demonstrating the reliability of the results. In conclusion, our results demonstrate that glyphosate can induce autophagy in hepatic L8824cells by activating NO, thus regulating energy metabolism and the RAS/RAF/MEK/ERK signaling pathway.

Impact of Short-Term (+)-JQ1 Exposure on Mouse Aorta: Unanticipated Inhibition of Smooth Muscle Contractility.

(+)-JQ1, a specific chemical inhibitor of bromodomain and extraterminal (BET) family protein 4 (BRD4), has been reported to inhibit smooth muscle cell (SMC) proliferation and mouse neointima formation via BRD4 regulation and modulate endothelial nitric oxide synthase (eNOS) activity. This study aimed to investigate the effects of (+)-JQ1 on smooth muscle contractility and the underlying mechanisms. Using wire myography, we discovered that (+)-JQ1 inhibited contractile responses in mouse aortas with or without functional endothelium, reducing myosin light chain 20 (LC20) phosphorylation and relying on extracellular Ca2+. In mouse aortas lacking functional endothelium, BRD4 knockout did not alter the inhibition of contractile responses by (+)-JQ1. In primary cultured SMCs, (+)-JQ1 inhibited Ca2+ influx. In aortas with intact endothelium, (+)-JQ1 inhibition of contractile responses was reversed by NOS inhibition (L-NAME) or guanylyl cyclase inhibition (ODQ) and by blocking the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. In cultured human umbilical vein endothelial cells (HUVECs), (+)-JQ1 rapidly activated AKT and eNOS, which was reversed by PI3K or ATK inhibition. Intraperitoneal injection of (+)-JQ1 reduced mouse systolic blood pressure, an effect blocked by co-treatment with L-NAME. Interestingly, (+)-JQ1 inhibition of aortic contractility and its activation of eNOS and AKT were mimicked by the (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains. In summary, our data suggest that (+)-JQ1 directly inhibits smooth muscle contractility and indirectly activates the PI3K/AKT/eNOS cascade in endothelial cells; however, these effects appear unrelated to BET inhibition. We conclude that (+)-JQ1 exhibits an off-target effect on vascular contractility.