Constitutive Nitric Oxide Synthase Research Articles

Constitutive NOS Production Is Modulated by Alzheimer's Disease Pathology Depending on APOE Genotype.

Both the endothelial (eNOS) and the neuronal (nNOS) isoforms of constitutive Nitric Oxide Synthase have been implicated in vascular dysfunctions in Alzheimer's disease (AD). We aimed to explore the relationship between amyloid pathology and NO dynamics by comparing the cerebrospinal fluid (CSF) levels of nNOS and eNOS of 8 healthy controls (HC) and 27 patients with a clinical diagnosis of Alzheimer's disease and isolated CSF amyloid changes, stratified according to APOE ε genotype (APOE ε3 = 13, APOE ε4 = 14). Moreover, we explored the associations between NOS isoforms, CSF AD biomarkers, age, sex, cognitive decline, and blood-brain barrier permeability. In our cohort, both eNOS and nNOS levels were increased in APOE ε3 with respect to HC and APOE ε4. CSF eNOS inversely correlated with CSF Amyloid-β42 selectively in carriers of APOE ε3; CSF nNOS was negatively associated with age and CSF p-tau only in the APOE ε4 subgroup. Increased eNOS could represent compensative vasodilation to face progressive Aβ-induced vasoconstriction in APOE ε3, while nNOS could represent the activation of NO-mediated plasticity strategies in the same group. Our results confirm previous findings that the APOE genotype is linked with different vascular responses to AD pathology.

Scutellaria Baicalensis Georgi Stem and Leaf Flavonoids Ameliorate the Learning and Memory Impairment in Rats Induced by Okadaic Acid.

The objective of this study is to explore the impact and underlying mechanism of Scutellaria baicalensis Georgi stem and leaf flavonoids (SSFs) on cognitive impairment caused by intracerebroventricular injection of okadaic acid (OA) in rats. An experimental model of Alzheimer's disease (AD) was induced in rats by intracerebroventricular injection of OA, resulting in memory impairment. The Morris water maze test was employed to confirm the successful establishment of the memory impairment model. The rats that exhibited significant memory impairment were randomly divided into different groups, including a model group, three SSFs dose groups (25, 50, and 100 mg/kg), and a positive control group treated with Ginkgo biloba tablets (GLT) at a dose of 200 mg/kg. To evaluate the learning and memory abilities of the rats, the Morris water maze test was conducted. Hematoxylin-eosin (HE) staining was used to observe any morphological changes in neurons. Immunohistochemistry (IHC) was performed to measure the expression of choline acetyltransferase (ChAT) protein. Western blotting (WB) was utilized to assess the phosphorylation levels of tau protein at Ser262 and Ser396. The activities of inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (cNOS) were quantified using ultraviolet spectrophotometry. The levels of inflammatory factors, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), were measured using ELISA. In rats, the administration of OA via intracerebroventricular injection resulted in cognitive impairment, neuropathological changes, and alterations in protein expression and activity levels. Specifically, the protein expression of ChAT was significantly reduced (P<0.01), while the phosphorylation levels of tau protein at Ser262 and Ser396 were significantly increased (P<0.01). Moreover, iNOS activity in the hippocampus and cerebral cortex exhibited a significant increase (P<0.01), whereas cNOS activity showed a decrease (P<0.05). Furthermore, the levels of IL-1β and TNF-α in the cerebral cortex were elevated (P<0.01), while the level of IL-6 was decreased (P<0.05). The administration of three doses of SSFs and GLT to rats exhibited varying degrees of improvement in the aforementioned pathological alterations induced by OA. SSFs demonstrated the ability to enhance cognitive function and mitigate memory deficits in rats following intracerebroventricular injection of OA. This beneficial effect may be attributed to the modulation of ChAT protein expression, tau hyperphosphorylation, NOS activity, and inflammatory cytokine levels by SSFs.

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.

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.

FORMATION OF REACTIVE OXYGEN AND NITROGEN SPECIES IN RAT LIVER TISSUES UNDER CONDITIONS OF SURGICAL TRAUMA REPRODUCED ON THE BACKGROUND OF EXPERIMENTAL MODEL OF POST-TRAUMATIC STRESS DISORDER

In an experiment on 42 white rats, the mechanisms of oxidative-nitrosative stress in liver tissues under conditions of surgical trauma reproduced on the background of post-traumatic stress disorder were studied. It has been shown that the reproduction of an experimental model of posttraumatic stress disorder (single prolonged stress) causes the development of oxidative-nitrosative stress in rat liver tissues: it increases the rate of superoxide anion radical generation by microsomes, mitochondria and leukocyte NADPH oxidase, increases NO-synthase activity by activating the inducible isoform, reduces the activity and conjugation of constitutive NO-synthases, which is accompanied by an increase in the concentration of peroxynitrites of alkaline and alkaline-earth metals. On the 7th day after laparotomy against the background of reproduction of the experimental model of post-traumatic stress disorder, the indicators of oxidative-nitrosative stress in rat liver tissues, such as the production of superoxide anion radical by microsomes, microsomes and leukocyte NADPH oxidase total and inducible NO-synthase activity and concentration of peroxynitrites of alkaline and alkaline-earth metals, significantly exceed their values after a single laparotomy and after a sham operation against the background of simulated single prolonged stress.

Nasal Nitric Oxide in Children: A Review of Current Outreach in Pediatric Respiratory Medicine.

Nasal nitric oxide (nNO) is a gas synthesized by the inducible and constitutive NO synthase (NOS) enzyme in the airway cells of the nasal mucosa. Like lung nitric oxide, it is thought to be associated with airway inflammation in various respiratory diseases in children. The aim of our review was to investigate the current state of use of nNO measurement in children. A comprehensive search was conducted using the Web of Science and PubMed databases specifically targeting publications in the English language, with the following keywords: nasal NO, children, allergic rhinitis, chronic rhinosinusitis, acute rhinosinusitis, primary ciliary dyskinesia (PCD), and cystic fibrosis (CF). We describe the use of nNO in pediatric allergic rhinitis, chronic rhinosinusitis, acute rhinosinusitis, PCD, and CF based on the latest literature. nNO is a noninvasive, clinically applicable test for use in pediatric allergic rhinitis, chronic rhinosinusitis, acute rhinosinusitis, PCD, and CF. It can be used as a complementary method in the diagnosis of these respiratory diseases and as a monitoring method for the treatment of allergic rhinitis and acute and chronic rhinosinusitis.

Enhancing Muscle Intracellular Ca2+ Homeostasis and Glucose Uptake: Passive Pulsatile Shear Stress Treatment in Type 2 Diabetes.

Type 2 diabetes mellitus (T2D) is a significant global public health problem that has seen a substantial increase in the number of affected individuals in recent decades. In a murine model of T2D (db/db), we found several abnormalities, including aberrant intracellular calcium concentration ([Ca2+]i), decreased glucose transport, increased production of reactive oxygen species (ROS), elevated levels of pro-inflammatory interleukins and creatine phosphokinase (CK), and muscle weakness. Previously, we demonstrated that passive pulsatile shear stress, generated by sinusoidal (headward-forward) motion, using a motion platform that provides periodic acceleration of the whole body in the Z plane (pGz), induces the synthesis of nitric oxide (NO) mediated by constitutive nitric oxide synthase (eNOS and nNOS). We investigated the effect of pGz on db/db a rodent model of T2D. The treatment of db/db mice with pGz resulted in several beneficial effects. It reduced [Ca2+]i overload; enhanced muscle glucose transport; and decreased ROS levels, interleukins, and CK. Furthermore, pGz treatment increased the expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), and neuronal nitric oxide synthase (nNOS); reduced inducible nitric oxide synthase (iNOS); and improved muscle strength. The cytoprotective effects of pGz appear to be mediated by NO, since pretreatment with L-NAME, a nonspecific NOS inhibitor, abolished the effects of pGz on [Ca2+]i and ROS production. Our findings suggest that a non-pharmacological strategy such as pGz has therapeutic potential as an adjunct treatment to T2D.

Role of sulfide anion in the development of chronic alcoholic hepatitis under the conditions of modulation of adenosine monophosphate kinase – a correlational study

Introduction and aim. Hydrogen sulfide (H 2 S) has attracted the attention of researchers as a novel signaling molecule that affects vascular metabolism, immune function, stress and inflammation. It plays an important role in pathophysiological disorders under the conditions of the development of obesity, diabetes, non-alcoholic fatty liver disease and cardiovascular diseases. The purpose of this work is to establish correlation ratios of H 2 S concentration with markers of oxidative-nitrosative stress and extracellular matrix metabolism of the liver during chronic alcoholic hepatitis modeling and AMPK modulation by phenformin and doxorubicin. Material and methods. The experiments were performed on 36 white, sexually mature male Wistar rats, weighing 180-220 g. Alcoholic hepatitis was modelled by alcohol administration, on the background of alcoholic hepatitis animals received phenformin orally at a dose of 10 mg/kg or doxorubicin at a dose of 1.25 mg/kg intraperitoneally. Statistical processing of the results of biochemical studies was carried out using the non-parametric method of Spearman to determine correlations. Results. H 2 S during alcoholic hepatitis inversely proportionally strongly correlates with the concentration of nitrites, oxyproline and arginase activity. Phenformin administration during alcoholic hepatitis leads to formation of inversely proportionally strongly correlation of H 2 S with the production of superoxide anion radical, the concentration of malondialdehyde, activities of constitutive NO-synthases, nitrite reductases, nitrate reductases, and arginase. Doxorubicin administration during alcoholic hepatitis leads to formation of directly proportional strongly correlation of H 2 S with the activity of constitutive NO-synthases, nitrite reductases, nitrate reductases. Conclusion. Administration of phenformin or doxorubicin expands correlations between H 2 S and indicators of oxidative-nitrosative stress.

OXIDATIVE MECHANISMS UNDERLYING BRAIN CELL DAMAGE IN RATS UNDER CHANGES IN DURATION OF THE LIGHTDARK CYCLES

One of the main mechanisms underlying cell damage is lipid peroxidation, which is characterized by increased levels of free radical, products of lipid peroxidation chain reactions, and alterations in the activity of the antioxidant system in tissues. Therefore, the objective of this study is to investigate the proand antioxidant balance in rat brain homogenate under conditions of disrupted "light-dark" cycles. The study involved 28 white Wistar rats weighing 150-200 gm, divided into two groups: control (15) and the group of animals exposed to acute desynchronosis simulation (13). To simulate desynchronosis, a normal "light-dark" cycle of 12 hours of light and 12 hours of darkness was initially established for three weeks, followed by a shift of the "light-dark" phases backward by six hours over the next three days. The animals were kept in standard conditions with a balanced diet in the vivarium, and the study adhered to the principles of biomedical ethics.&#x0D; The results obtained indicate a significant increase in the activity of superoxide anion radical production in the brain tissues of the rats with modelled desynchronosis by 75.8% compared to the control group. This increase is attributed to the NADPH-oxidase mechanism by 20.2% and to xanthine oxidase mechanisms by 28%. The concentration of TBA-active products increased by 36.8%, the growth of TBA-active products elevated by 30%, and the activity of the key antioxidant enzymes, superoxide dismutase and catalase, decreased by 53% and 14%, respectively, in the second group of animals. Furthermore, in the brain tissues of rats with disrupted "light-dark" cycles, there were found elevated levels of nitrites by 23.8%, nitrosothiols by 19%, and peroxynitrites by 69%, along with a reduction in the activity of total NO-synthase by 59.1%, inducible NO-synthase by 61.1%, and constitutive NO-synthase by 9% compared to the control group. The activity of ornithine decarboxylase increased by 26.5% in the brain tissues of the rats with simulated desynchronosis compared to the control group.&#x0D; In conclusion, the disruption of "light-dark" cycles leads to the development of oxidative stress in brain tissues of animals and is characterized by increased production of reactive oxygen species and diminished antioxidant protection. Acute desynchronosis decreases nitric oxide production through the NO-synthase pathway, while increasing the accumulation of toxic nitric oxide metabolites in the brain tissues of rats, thereby posing a potential risk of nitrosative stress. Furthermore, alterations in the normal light regimen of the day activate the arginase pathway of L-arginine metabolism and result in increased glutamate production in the brain tissues of rats, potentially leading to oxidative stress.

Hydrogen sulfide alleviates heart failure with preserved ejection fraction in mice by targeting mitochondrial abnormalities via PGC-1α

AimIncreasing evidence has proposed that mitochondrial abnormalities may be an important factor contributing to the development of heart failure with preserved ejection fraction (HFpEF). Hydrogen sulfide (H2S) has been suggested to play a pivotal role in regulating mitochondrial function. Therefore, the present study was designed to explore the protective effect of H2S on mitochondrial dysfunction in a multifactorial mouse model of HFpEF. MethodsWild type, 8-week-old, male C57BL/6J mice or cardiomyocyte specific-Cse (Cystathionine γ-lyase, a major H2S-producing enzyme) knockout mice (CSEcko) were given high-fat diet (HFD) and l-NAME (an inhibitor of constitutive nitric oxide synthases) or standardized chow. After 4 weeks, mice were randomly administered with NaHS (a conventional H2S donor), ZLN005 (a potent transcriptional activator of PGC-1α) or vehicle. After additional 4 weeks, echocardiogram and mitochondrial function were evaluated. Expression of PGC-1α, NRF1 and TFAM in cardiomyocytes was assayed by Western blot. ResultsChallenging with HFD and l-NAME in mice not only caused HFpEF but also inhibited the production of endogenous H2S in a time-dependent manner. Meanwhile the expression of PGC-1α and mitochondrial function in cardiomyocytes were impaired. Supplementation with NaHS not only upregulated the expression of PGC-1α, NRF1 and TFAM in cardiomyocytes but also restored mitochondrial function and ultrastructure, conferring an obvious improvement in cardiac diastolic function. In contrast, cardiac deletion of CSE gene aggravated the inhibition of PGC-1α-NRF1-TFAM pathway, mitochondrial abnormalities and diastolic dysfunction. The deleterious effect observed in CSEcko HFpEF mice was partially counteracted by pre-treatment with ZLN005 or supplementation with NaHS. ConclusionOur findings have demonstrated that H2S ameliorates left ventricular diastolic dysfunction by restoring mitochondrial abnormalities via upregulating PGC-1α and its downstream targets NRF1 and TFAM, suggesting the therapeutic potential of H2S supplementation in multifactorial HFpEF.

ROLE OF NO IN SOFT PERIODONTAL TISSUES OF RATS DURING STRESS AND INFLAMMATION

Aim. To evaluate the activity of NO-synthase isoforms, the concentration of peroxynitrites and nitrosothiols in the soft tissues of the periodontium of rats under the conditions of modeling chronic stress against the background of lipopolysaccharide-induced inflammation. Methods. Experimental studies were performed on 24 male Wistar rats weighing 190–240 g. The animals were divided into 4 groups: 1 — control, 2 — chronic stress (ChrStr group), animals were kept above water for 1 hour every day for 30 days, 3 — animals that were intraperitoneally injected with 0.4 μg/kg of bacterial LPS of S. typhi (pyrogenal) (LPS group); 4 — animals that were simultaneously simulated chronic stress as in group 2 and administered LPS as in group 3 (ChrStr+LPS). The activity of inducible NO-synthase (iNOS), constitutive NO-synthase (cNOS), the concentration of nitrosothiols (S-NO), and concentration of peroxynitrites of alkali and alkaline earth metals (ONOO) were studied in the homogenate of the periodontal soft tissues of rats. Results. The activity of NOS in the soft periodontal tissues of rats under chronic stress simulation conditions, LPS administration, combined exposure to chronic stress and LPS was increased compared to control group. The concentration of ONOO- in the soft periodontal tissues of rats under chronic stress simulation conditions, LPS administration, combined exposure to chronic stress and LPS was increased compared to the control group. The concentration of nitrosothiols in the soft periodontal tissues of rats under the conditions of chronic stress simulation, LPS administration, combined exposure to chronic stress and LPS was decreased compared to the control group. Conclusions. The combined effect of bacterial lipopolysaccharide and chronic stress leads to increased production of nitrogen monoxide from inducible NO-synthase and elevates concentration of reactive forms of nitrogen, which creates possibility for development of nitrosative stress in the soft periodontal tissues.

Вплив модуляторів факторів транскрипції NF-κB і Nrf2 на показники оксидативно–нітрозативного стресу в скелетних м’язах щурів за умов хронічної гіпомелатонінемії та вуглеводно-ліпідної дієти

This article describes the effect of NF-κB inhibitor ammonium pyrrolidine dithiocarbamate and Nrf2 inducer dimethylfumarate on indicators of oxidative-nitrosative stress in skeletal muscles of rats with chronic hypomelatoninemia, induced by roundthe-clock illumination with an intensity of 1500 lux for 30 days, against the background of a carbohydrate-lipid diet (20% fructose solution and appropriate food) for 60 days. The study demonstrated that the administration of ammonium pyrrolidine dithiocarbamate and dimethylfumarate under the experimental conditions impedes the development of hypomelatoninemia, this was accompanied by a two-fold increase in the blood serum melatonin concentration compared to the control group. Moreover, the administration of ammonium pyrrolidine dithiocarbamate and dimethylfumarate significantly reduces the production of superoxide anion radical by electron transport chains of mitochondria (by 47.9 and 51.3%) and sarcoplasmic reticulum (by 48.6 and 52.0%) in the homogenate of femoral muscles, the total activity of NO synthase (by 37.2 and 36.2%) and its inducible isoform (by 41.1 and 40.0%), the concentration of peroxynitrites of alkaline and alkaline earth metals (by 37.2 and 41.0 %), while the activity of constitutive NO-synthases (by 2.9 and 2.8 times) and their conjugation index (by 5.2 and 5.4 times) increases compared to the respective control values. We suggest that modulators of redox-sensitive transcription factors NF-κB and Nrf2 under conditions of chronic hypomelatoninemia and the exposure to the carbohydrate-lipid diet are effective means to restrain the development of oxidative-nitrosative stress in skeletal muscles.

Ecdysterone treatment restores constitutive NO synthesis and alleviates oxidative damage in heart tissue and mitochondria of streptozotocin-induced diabetic rats

Constitutive NO synthases (cNOS) are the primary targets of diabetes mellitus and the impairment of cNOS functioning in cardiovascular system is one of the hallmarks of this disease. The aim of this work was to study the effect of a plant sterol ecdysterone (20-β-hydroxyecdysterone) on the NO synthases functioning and RNS metabolism in heart mitochondria and the heart tissue in the rat model of streptozotocin-induced type I diabetes. Diabetes development resulted in cNOS dysfunction both in heart mitochondria and heart tissue. cNOS activity was dramatically suppressed, but 3-fold and 6-fold rise of iNOS activity was observed in mitochondria and heart tissue respectively. Also, in mitochondria there was ~2.5 time’s increase in urea content and the activity of arginase 2 (ARG2), which could compete with NOS for the common substrate L-arginine. Total RNS production was dramatically elevated in mitochondria of diabetic animals, which well agreed with iNOS activation. Unlike this, in heart tissue dramatic increase of iNOS activity increased the content of nitrosothiols (RSNO), while total RNS production remained close to control. Both in the heart tissue and mitochondria, there was dramatic augmentation of superoxide production that correlated with sharp elevation of iNOS activity and steep rise of diene conjugates (DC) content, which indicated strong lipid oxidation. Ecdysterone treatment resulted in the reduction of iNOS activity and twofold elevation of mtNOS activity as compared to control. However, in the whole heart tissue eNOS was restored only by half of control level, which indicated specific action of ecdysterone on mtNOS isoform. RNS production returned to control in mitochondria, and was by half reduced in the heart tissue, which indicated the abolition of nitrosative stress. Correlation dependence between iNOS activity and superoxide production was found in mitochondria, which could indicate iNOS uncoupling. The restoration of cNOS activity and the reduction of iNOS activity to control level after ecdysterone treatment well correlated with the reduction of superoxide production and indicated possible ‘iNOS re-coupling‘, which resulted in the reduction of DC formation to control level. So, STZ-induced type I diabetes dramatically up-regulated iNOS activity and suppressed cNOS activity. Ecdysterone treatment reduced iNOS activity and restored constitutive NO biosynthesis to control level, which abolished oxidative and nitrosative stress in cardiac mitochondria and heart tissue of STZ-induced diabetic animals. Possible pathways involved in ecdysterone action on constitutive NO biosynthesis were discussed.

Galangin protects gastric mucosa from indomethacin induced injury via scavenging ROS and inhibition of PKC, Akt, and NF κB protein expressions

Purpose: To investigate the mechanism of action of galangin (GAL) against indomethacin (Indo)-induced gastric injury.&#x0D; Methods: Ten micromolar GAL with 2 mM Indo or 0.5 mM L-NAME or 2 mM propargylglycine were co-administered to human gastric mucosal epithelial cells GES-1 for 24 h. Cell viability assay and reactive oxygen species (ROS) test were conducted with commercial kits. Protein expressions of constitutive and inducible nitric oxide synthase (cNOS and iNOS, respectively), and cystathionine-γ-lyase (CSE) in GSE-1 cells were investigated by western blot. After 100 - 300 mg/kg, GAL was daily administered to rats for 5 days, and 30 mg/kg Indo was administered to induce gastric injury. Protein expressions of endothelial NOS (eNOS), iNOS, CSE, nuclear factor (NF)-κB, PKC, Akt, phospho-eNOS (Ser-1177), and phospho-eNOS (Thr-495) were investigated by western blot.&#x0D; Results: Galangin ameliorated the proliferative inhibitions of Indo, propargylglycine, and L-NAME on GSE-1 cells (p &lt; 0.05), rapidly scavenged ROS, and increased cNOS protein expressions (p &lt; 0.05). Galangin (300 mg/kg) inhibited protein expressions of iNOS (p &lt; 0.01), NF-κB, Akt, and PKC (p &lt; 0.05), inhibited the Ser-1177 and Thr 495 phosphorylation of eNOS, elevated eNOS and CSE protein expressions (p &lt; 0.001).&#x0D; Conclusion: Galangin protects gastric mucosa from Indo by scavenging ROS and inhibiting protein expressions of Akt and PKC. The inhibition leads to Ser-1177 and Thr 495 dephosphorylation of eNOS, thereby prolonging eNOS activity. Besides, GAL inhibits iNOS-produced NO by inhibiting NF-κB to ameliorate the inflammatory reaction induced by Indo.

A Clinical Trial on the Welfare Effects of Administering Meloxicam to 10-21 Day Dairy Calves Following Caustic Paste Disbudding.

Caustic paste disbudding (CPD) is widely utilized for calves, which has been known to result in adverse effects on the calves and ethical concerns related to animal welfare, despite the use of local anesthetics. The administration of meloxicam has been demonstrated to provide benefits in alleviating pain and inflammation in juvenile calves under nine days old and subjected to CPD. Nonetheless, there is a scarcity of literature documenting the beneficial impact of meloxicam in alleviating pain in calves aged over nine days that have undergone CPD. Therefore, the objective of this clinical trial was to evaluate the efficacy of administering meloxicam and lidocaine for cornual nerve block together in mitigating the deleterious effects of CPD, as opposed to using lidocaine alone in calves older than nine days. Thirty Holstein calves, aged between 10 and 21 days, were enrolled and randomly assigned to 1 of 2 treatments: lidocaine alone (Placebo), lidocaine and normal saline treatment before CPD, and lidocaine plus meloxicam (MX), lidocaine and 0.5mg/kg of meloxicam treatment prior to CPD. The researchers were blind to the treatment of calves to control the subjective error. The occurrences of actions associated with pain, which included head shaking, head rubbing, ear flicking, tail flicking, kicking, and head passing through the fence, were recorded. Physiological performance, including the respiration rate, heart rate, rectal temperature, mechanical nociceptive threshold (MNT), food intake, and daily activity level, was monitored. Hematological conditions were ascertained through the use of routine blood tests and enzyme-linked immunosorbent assay. The generalized linear mixed model (GLMM) was employed to analyze the data. The research findings revealed that applying the CPD procedure significantly elevated the frequencies of tail flicking, head shaking, and kicking, resulted in increases in respiratory rate, heart rate, daily active steps, and food intake and a decrease in MNT, and led to alterations in hematological markers, including platelet counts, mean platelet volume, prostaglandin E2, constitutive nitric oxide synthase, and hydroxyl radical. Considerable benefits, such as lower heart rates, higher food intake, and MNTs, as well as lower levels of white blood cell counts, lymphocyte counts, hemoglobin, mean platelet volume, prostaglandin E2, tumor necrosis factor-α, constitutive nitric oxide synthase, malondialdehyde, and hydroxyl radical, were observed in the calves that received meloxicam treatment in response to CPD. The findings of the study indicate that the co-administration of lidocaine and meloxicam provides obvious benefits in mitigating pain, inflammation, and oxidative stress in calves aged over nine days and undergoing CPD. This endorses the use of meloxicam during the disbudding and dehorning procedures of calves.

The double-hit protocol induces HFpEF and impairs myocardial ubiquitin-proteasome system performance in FVB/N mice.

Heart failure with preserved ejection fraction (HFpEF) is a leading cause of death and disability, with its prevalence surpassing that of heart failure with reduced ejection fraction. Obesity and hypertension are often associated with HFpEF. HFpEF can be modeled through simultaneous metabolic and hypertensive stresses in male C57BL/6N mice provoked by a combination treatment of a high-fat diet (HFD) and constitutive nitric oxide synthase inhibition by Nω-nitro-L-arginine methyl-ester (L-NAME). Ubiquitin-proteasome system (UPS) dysfunction was detected in many forms of cardiomyopathy, but whether it occurs in HFpEF remains unknown. We report successful modeling of HFpEF in male FVB/N mice and, by taking advantage of a transgenic UPS reporter mouse, we have detected myocardial UPS functioning impairment during HFpEF, suggesting a pathogenic role for impaired protein degradation in the development and progression of HFpEF.

PS-B02-12: EFFECTS OF ULTRAVIOLET LIGHT ON BLOOD PRESSURE AND LEFT VENTRICULAR FUNCTION

Ultraviolet light (UV), induces the activation of constitutive nitric oxide synthases, which produces nitric oxide (NO) of vascular relaxation factor. We hypothesize that UV-induced production of NO could have beneficial effect on hypertension. Twelve 6-week-old salt-sensitive hypertensive rats (Dahl-Iwai salt-sensitive DIS/Eis, Japan Society for the Study of Hypertension, Shizuoka, Japan) were used in this study. These rats were fed a high-salt diet containing 8%NaCl until 10 weeks of age and were then randomly divided into three groups i.e. baseline group (group non-UV), 0 h post UV (group UV 0 h), and 6 h post UV (group UV 6 h). The rats were irradiated with UVA and UVB lamps at an intensity of 100 mJ/cm2. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by using tail-cuff method (BP-98A; Softron Co., Ltd., Tokyo, Japan). We evaluated left ventricular (LV) systolic and diastolic function using echocardiography with a 12 MHz transducer connected to a Xario ultrasound system (Toshiba Medical Systems Co., Tochigi, Japan). Interventricular septal thickness, LV posterior wall thickness, and LV end-diastolic and end-systolic diameters (LVDd and LVDs) were obtained from a short-axis view. Percent LV fractional shortening (%LVFS) was calculated as an index of LV systolic function. Peak flow velocities at the mitral level during rapid filling (E) and deceleration time (Dct), and peak early diastolic mitral annular velocity which indicate LV diastolic function, were measured using pulsed and tissue Doppler echocardiography. Histological skin tissue sections were fixed with 4% paraformaldehyde phosphate buffer solution for 24 h at room temperature, embedded in paraffin, sectioned transversely (thickness, 3 μm), and stained with hematoxylin-eosin to evaluate the impairments of skin. The SBP and DBP were lower in group UV 6 h than group non-UV and group UV 0 h. In contrast, there were no significant differences in heart rate and body weight among the three groups. The peak early diastolic mitral annular velocity was greater in group UV 6 h than group non-UV and group UV 0 h. LVDd, LVDs, and %LVFS were not significantly different among the three groups. We did not find the impairments in skin after UV irradiation. UVA and UVB may play an important role in regulating not only blood pressure but also LV function.

Asymmetric dimethylarginine accumulation under hyperglycemia facilitates β-cell apoptosis via inhibiting nitric oxide production

Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) has been shown to suppress apoptosis in pancreatic β-cells. In the present study, the influence of asymmetric dimethylarginine (ADMA), the major endogenous inhibitor of NOS, on the apoptosis-suppressive effect of NO was investigated. The expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2), an ADMA-metabolizing enzyme, in INS-1 β-cells and in mouse pancreatic islets was drastically reduced by in vitro exposure to high-concentration glucose (20 mM) and by in vivo treatment of mice with the insulin receptor blocker S661, which resulted in hyperglycemia, respectively. In line with this, a higher ADMA level was observed in INS-1 cells exposed to 20 mM glucose. The treatment of INS-1 cells with ADMA, similarly to with the NOS inhibitor NG-nitro-L-arginine methyl ester, significantly facilitated 20 mM glucose-induced increase in cleaved caspase-3 protein expression. Furthermore, increased protein expression of cleaved caspase-3 and CHOP was observed in INS-1 cells with knockdown of DDAH2. These results suggest that ADMA accumulation through a decrease in DDAH2 expression in β-cells, which is induced under hyperglycemic conditions, facilitates β-cell apoptosis through suppression of cNOS-mediated NO production.

Abstract 15701: Histone Deacetylase 6Inhibition Demonstrates Comparable Efficacy as Empagliflozin in a Mouse Model of Heart Failure With Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFpEF) is associated with high morbidity and mortality, yet there are few effective therapies for HFpEF. Previously, we have demonstrated that TN-301, a highly selective small molecule inhibitor of histone deacetylase 6 (HDAC6), works directly on the heart and on systemic metabolism and inflammation in mouse models of HFpEF. Recently, empagliflozin, a selective inhibitor of sodium-glucose transport protein (SGLT2) inhibitor, was approved in the U.S. for patients with HFpEF. Purpose: We aimed to compare the efficacy of HDAC6i and SGLT2i in a mouse model of established diastolic dysfunction with preserved ejection fraction. Methods and Results: To recapitulate systemic and cardiovascular features of HFpEF in humans, we induced diastolic dysfunction with a combination of high fat diet and inhibition of constitutive nitric oxide synthase using N ω -nitrol-arginine methyl ester (L-NAME). After the HFpEF phenotypes were established, mice received TYA-11018 (TN-301-like HDAC6i) or empagliflozin, or vehicle orally once daily for 9 weeks. We found that TYA-11018 reduced left ventricular hypertrophy and restored diastolic function with comparable efficacy to empagliflozin. In addition, TYA-11018 reduced fasting glucose and improved glucose tolerance in HFpEF mice, to levels similar to empagliflozin. Preliminary data suggested TYA-11018 corrects hyperglycemia independently of glucose urine excretion, which is distinct from SGLT2i. Using whole transriptome RNA-seq, TYA-11018 showed greater effects on correcting expression of gene sets associated with inflammation, fibrosis and mitochondrial energy production compared to empagliflozin in HFpEF hearts. Conclusion: Our results show that with its distinct HDAC6 inhibition mechanism, TYA-11018 reverses preexisting hypertrophy, diastolic dysfunction and glucose tolerance, demonstrated similar efficacy to that with a SGLT2 inhibitor in HFpEF model. The comparable efficacy seen in this HFpEF model with TYA-11018 and empagliflozin provides early but encouraging evidence of the potential translatability of these findings to clinical development. We are developing TN-301 for treating HFpEF patients.

Protective effects of curcumin on ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury is a term used to describe phenomena connected to the dysfunction of various tissue damage due to reperfusion after ischemic injury. While I/R may result in systemic inflammatory response syndrome or multiple organ dysfunction syndrome, there is still a long way to improve therapeutic outcomes. A number of cellular metabolic and ultrastructural alterations occur by prolonged ischemia. Ischemia increases the expression of proinflammatory gene products and bioactive substances within the endothelium, such as cytokines, leukocytes, and adhesion molecules, even as suppressing the expression of other "protective" gene products and substances, such as thrombomodulin and constitutive nitric oxide synthase (e.g., prostacyclin, nitric oxide [NO]). Curcumin is the primary phenolic pigment derived from turmeric, the powdered rhizome of Curcuma longa. Numerous studies have shown that curcumin has strong antiinflammatory and antioxidant characteristics. It also prevents lipid peroxidation and scavenges free radicals like superoxide anion, singlet oxygen, NO, and hydroxyl. In our study, we highlight the mechanisms of protective effects of curcumin against I/R injury in various organs.