Non-selective Nitric Oxide Synthase Inhibitor Research Articles

Effects of Cross-Sex Hormone Therapy on Metabolic Parameters and Aortic Relaxation in Castrated Male Rats

With the rising awareness, the number of individuals identifying themselves as transgender in the US is increasing over the years. Cross-sex hormone therapy (CSHT) may alter numerous cardiometabolic parameters leading to the increased risk of cardiovascular diseases (CVD) in transgender population. Epidemiological studies suggest that transgender females (male to female, MtF) on estrogen therapy develop higher risk of CVD compared to transgender males (female to male, FtM) taking testosterone. Underlying mechanisms of impaired cardiovascular functions in MtF are not well documented. This study was undertaken to examine the effects of 17β-estradiol (E2) on certain cardiometabolic parameters and aortic reactivity in castrated (CAS) male Sprague Dawley (SD) rats. The 8-10-weeks old CAS rats were subcutaneously implanted with either 1.5 mg of E2 (CAS+E2) or placebo (CAS+PL) containing pellets for ~35 days. The age-matched SD intact male (IM) rats were also included in the experimental groups. After performing the glucose tolerance test, animals were sacrificed, and the blood was collected for later measurements of the HbA1C and triglyceride (TG) levels. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh) in aortic rings pre-contracted with phenylephrine (PE) was measured before and after treatment with indomethacin (a cyclooxygenase inhibitor), followed by L-NAME [a nonselective nitric oxide (NO) synthase (NOS) inhibitor]. Contractile responses to PE before and after L-NAME in aortic rings and sodium nitroprusside (SNP)-induced relaxation responses in endothelium-denuded rings were also measured. Here, we report the decreased body weight and visceral white adipose tissue, and improved HbA1C and glucose tolerance in E2-treated CAS rats. However, the E2-treated CAS rats exhibited elevated plasma level of TG. Furthermore, the vascular relaxation to ACh was significantly impaired in aortas from this group compared to those in CAS+PL and IM rats. The impairment of ACh responses in CAS+E2 rats was accompanied by a significant decrease in NO contribution to ACh vasorelaxation. The basal NO levels and the smooth muscle sensitivity to NO were not altered, whereas the maximum contractile responses to PE were enhanced in aortas of CAS+E2 group compared to IM. In conclusion, our data suggests that the glucose homeostasis was improved in CAS+E2 rats. Regardless, this group exhibited increased level of TG in plasma and decreased aortic vasorelaxation to ACh. The impairment of EDV in E2-treated CAS rats might be due to reduced NO-mediated vasorelaxation or increased contractile responses observed in this group. Additional studies are needed to document the underlying mechanisms for improvement of glucose homeostasis and impairment of EDV in CAS+E2 rats. NIDA/NIGMS, 1SC1DA052120-01 to MF and Bridge Grant from University of the Pacific to RR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Improvement Prospects of Lycopene Supplementation in the L-NAME Toxicity on the Male Wistar Rat Reproductive System

Non-selective nitric oxide synthase inhibitors, such as N (G)-nitro-L-arginine Methyl Ester (L-NAME), are typically prescribed to treat spinal injuries and hypotension. Many physiological processes, including the regulation of reproductive hormones, are ty pically linked to it. Bioactivity in physiological systems is influenced by the antioxidant lycopene. This study sought to determine the imp act of lycopene supplementation and L-NAME administration on gonadal hormones and sperm parameters. Twenty male Wistar rats were used for this study and were divided into four groups containing 5 rats each. L-NAME (5mg/kg) was administered through drinking water and Lycopene (5mg/kg) was supplemented through oral gavage for four weeks. Using established protocols with Elisa Kits, plasma hormone levels were ascertained. Estradiol did not differ substantially across all groups, however, the group that received L-NAME had lower plasma concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and nitric oxide (NO). Lycopene supplements raised testosterone, FSH, LH, and NO levels while not affecting estradiol. The sperm profile was altered with L-NAME but ameliorated in Lycopene supplementation. We conclude that lycopene has the potential to protect against hormonal imbalance and male infertility resulting from Nitric oxide synthase (NOS) inhibition.

Anti-allodynic and anti-hyperalgesic activity of (±)-licarin A in neuropathic rats via NO-cyclic-GMP-ATP-sensitive K+ channel pathway.

The study aimed to examine the effect of intraperitoneal and intrathecal (±)-licarin A in neuropathic pain induced by L5 and L6 spinal nerve ligation (SNL) in male Wistar rats and the possible involvement of the NO-cGMP-ATP-sensitive K+ channel pathway. Neuropathic pain signs (allodynia and hyperalgesia) were evaluated on postoperative Day 14 using von Frey filaments. Single intraperitoneal (0.01, 0.1, 1, and 10 mg/kg) and intrathecal (0.01, 0.1, 1, and 10 µg/rat) administration of (±)-licarin A improved allodynia and hyperalgesia. The (±)-licarin A-induced anti-allodynic and anti-hyperalgesic activity was prevented by the intrathecal injection of l-NAME (100 µg/rat; nonselective nitric oxide synthase inhibitor), ODQ (10 µg/rat; guanylate cyclase inhibitor), and glibenclamide (50 µg/rat; adenosine triphosphate (ATP)-sensitive K+ channel blocker). The data suggest that (±)-licarin A exerts its anti-allodynic and anti-hyperalgesic activity by activating the NO-cGMP-ATP-sensitive K+ channel pathway.

Role of K+ and Ca2+ Channels in the Vasodilator Effects of Plectranthus barbatus (Brazilian Boldo) in Hypertensive Rats.

Plectranthus barbatus, popularly known as Brazilian boldo, is used in Brazilian folk medicine to treat cardiovascular disorders including hypertension. This study investigated the chemical profile by UFLC-DAD-MS and the relaxant effect by using an isolated organ bath of the hydroethanolic extract of P. barbatus (HEPB) leaves on the aorta of spontaneously hypertensive rats (SHR). A total of nineteen compounds were annotated from HEPB, and the main metabolite classes found were flavonoids, diterpenoids, cinnamic acid derivatives, and organic acids. The HEPB promoted an endothelium-dependent vasodilator effect (~100%; EC50 ~347.10 μg/mL). Incubation of L-NAME (a nonselective nitric oxide synthase inhibitor; EC50 ~417.20 μg/mL), ODQ (a selective inhibitor of the soluble guanylate cyclase enzyme; EC50 ~426.00 μg/mL), propranolol (a nonselective α-adrenergic receptor antagonist; EC50 ~448.90 μg/mL), or indomethacin (a nonselective cyclooxygenase enzyme inhibitor; EC50 ~398.70 μg/mL) could not significantly affect the relaxation evoked by HEPB. However, in the presence of atropine (a nonselective muscarinic receptor antagonist), there was a slight reduction in its vasorelaxant effect (EC50 ~476.40 μg/mL). The addition of tetraethylammonium (a blocker of Ca2+-activated K+ channels; EC50 ~611.60 μg/mL) or 4-aminopyridine (a voltage-dependent K+ channel blocker; EC50 ~380.50 μg/mL) significantly reduced the relaxation effect of the extract without the interference of glibenclamide (an ATP-sensitive K+ channel blocker; EC50 ~344.60 μg/mL) or barium chloride (an influx rectifying K+ channel blocker; EC50 ~360.80 μg/mL). The extract inhibited the contractile response against phenylephrine, CaCl2, KCl, or caffeine, similar to the results obtained with nifedipine (voltage-dependent calcium channel blocker). Together, the HEPB showed a vasorelaxant effect on the thoracic aorta of SHR, exclusively dependent on the endothelium with the participation of muscarinic receptors and K+ and Ca2+ channels.

Increased pro-remodeling and pro-thrombotic responses in the left compared to the right atrial appendage: role of low-grade inflammation and the AT1R/NADPH oxidases/SGLT2 pro-oxidant pathway

Abstract Introduction In atrial fibrillation (AF), there is a complex interplay between arrhythmia burden and cardiovascular risk factors leading to left atrial remodeling, an increased risk of stroke and heart failure. Most thrombi get formed in the atrial appendage. Several studies suggested that sodium-glucose cotransporter 2 inhibitors (SGLT2i), besides showing major benefits on heart failure, might lower the risk of incident AF. Therefore, we examined the expression level of SGLT2 in human right and left atrial appendages (RAA, LAA) and determined its role in pro-oxidant, pro-fibrotic and pro-thrombotic responses. Methods Human RAA and LAA were collected from patients undergoing cardiac surgery at a university hospital. The LAA was cut into a proximal part (low stasis, high shear) and a distal part (high stasis, low shear). The level of reactive oxygen species (ROS) was determined using dihydroethidium, mRNA and protein expression levels by RT-qPCR, and Western blot analysis and immunofluorescence, respectively, and the level of fibrosis by Sirius red staining. Results The distal LAA had higher mRNA levels of ICAM-1 and p53, and protein levels of ICAM-1, AT1R, p53, p21 and tissue factor than the proximal LAA. LAA displayed higher mRNA and protein levels of SGLT2 and SGLT1, pro-adhesive, pro-thrombotic, pro-remodeling, pro-fibrotic and senescence markers, and components of the angiotensin system, in addition to IL-1 mRNA levels and p-p65 NF-kB protein levels compared to RAA. These responses were associated with higher levels of oxidative stress, nitrotyrosine and fibrosis. SGLT2 immunofluorescence signals in distal LAA were colocalized with those of CD31, CD68, TNF-α and troponin T. Oxidative stress levels in distal and proximal LAA and RAA were reduced by inhibitors of NADPH oxidases, ACE1 and SGLT2, an AT1R antagonist, and by a TNF-α neutralizing antibody whereas NG-nitro-L-Arginine methyl ester, the non-selective NO synthase inhibitor, inhibited ROS in distal and proximal LAA without affecting RAA. In addition, the level of TNF-α in LAA was positively correlated to the indexed LA volume indicating a link between inflammation and remodeling. Conclusions The distal part of the LAA of patients undergoing heart surgery showed greater impaired endothelial function and senescence associated with more pronounced pro­-oxidant, pro­-remodeling and pro-­inflammatory responses than the proximal part and the RAA. These responses were associated with an increased expression level of SGLT 2 in endothelial cells, cardiomyocytes, and macrophages in areas showing an inflammatory response. Moreover, the pro-oxidant signal was sensitive to inhibitors of the local angiotensin system, NO synthase, SGLT2 and TNF-α. Thus, the AT1R/NADPH oxidases/SGLT2 pathway appears as an interesting target to blunt the stimulatory pro-oxidant signal in cardiac tissues affected by low-grade inflammation that leads to remodeling and fibrosis.

Effect of non-selective NO-synthase inhibitor administered during pregnancy on the development of the cerebral cortex in 20-day-old rat pups

Aim. To study the morphology of neurons in the cerebral cortex of rat pups on day 20 under conditions of administration of a nitric oxide synthase inhibitor (NOS) during placentation.Materials and methods. Outbred white female rats (n = 12) were randomly divided into 2 groups of 6 rats each. On the 11th day of pregnancy, the experimental group received a single intramuscular injection of N(omega)-nitro-L-arginine methyl ester (L NAME) at a dose of 25 mg/kg, in the control group – once intramuscularly 0.9% NaCl solution. Born rat pups were randomly selected one from the mother. On the 20th day, after medical euthanasia, the brain was collected. In the anterior part of the frontal cortex, we studied the density and area of neurons, the size and shape of perikarya and the severity of their staining with toluidine blue. Results. In the experimental group of 20-day-old rat pups, compared to the control group, the density and area of neurons were less by 10% (p > 0.05) and 22% (p > 0.05), respectively, the shape of the perikarya also changed to elongated, the elongation factor increased by 0.3 units. (p < 0.05) and there was a sixfold increase in the proportion of hyperchromic neurons (p < 0.05), hyperchromic wrinkled (p < 0.001) neurons appeared, which were absent in control animals. Conclusion. Morphological changes in neurons of the cerebral cortex in rat pups born from females who received a NOS inhibitor during placentation may be a consequence of a decrease in the formation of nitric oxide in the neurons themselves and in the endothelium of the vessels supplying the brain

Effect of the nitric oxide synthesis inhibitor L-NAME on the isolated rat heart after hypokinesia

The effects of a non-selective nitric oxide synthase inhibitor L-NAME on the functional parameters of the isolated rat heart after a 30-day period of hypokinesia were studied. Electron paramagnetic resonance spectroscopy was employed in the analysis of a role for L-NAME in the intensity of nitric oxide production in rat heart tissues. The intensity of nitric oxide synthesis was assessed by the intensity of the signal belonging to the (DETC)2-Fe2+-NO complex. It was found that L-NAME decreased nitric oxide production on average by 69%. The Langendorff isolated perfused heart was used to evaluate cardiac activity, and the following parameters were measured: pressure generated by the left ventricle, heart rate, and coronary flow. Addition of the nitric oxide synthesis inhibitor L-NAME induced an increase in inotropic function and normalization of heart rate.

Knockdown of miR-19a suppresses gastrointestinal dysmotility diarrhea after TBI by regulating VIP expression.

Traumatic brain injury (TBI) is the main cause of death and can lead to a variety of physiological complications, including gastrointestinal dysfunction. The present study aimed to confirm the miR-19a-mediated suppression of diarrhea after TBI through the regulation of VIP expression. A rat model of TBI induced by controlled cortical injury was used to observe gastrointestinal morphology by opening the abdomen after TBI. After 72h of injury, the fecal water content of the rats was measured. The end ileal segments were removed, and HE staining was used to observe the histopathological changes in the intestine. The levels of serum miR-19a and VIP mRNA were detected by qRT-PCR. ELISA was performed to detect VIP levels in serum. Immunohistochemistry was used to detect the level of VIP in ileal tissues, and immunofluorescence was used to detect c-kit expression in ileal tissue. CCK-8 assay was used to detect the cell viability of interstitial cells of Cajal (ICCs), and TUNEL assay was used to detect apoptosis of ICCs. miR-19a and VIP were highly expressed in the serum of TBI rats, and the knockdown of miR-19a alleviated TBI-induced diarrhea. In addition, the overexpression of miR-19a or VIP inhibited the proliferation of ICCs, promoted apoptosis, and suppressed intracellular Ca2+ levels, whereas miR-19a suppression had the opposite effects. A nonselective nitric oxide synthase inhibitor (L-NA), PKG inhibitors (KT-5823 and RP-8CPT-cGMPS), and a guanylate cyclase inhibitor (ODQ) restored the inhibitory effects of VIP on ICC proliferation, anti-apoptosis effects, and Ca2+ concentrations. Knockdown of miR-19a inhibits activation of the VIP-NO-cGMP-PKG pathway through suppression of VIP expression, which in turn inhibits diarrhea after TBI.

Gastroprotective effect of Artemisia absinthium L.: A medicinal plant used in the treatment of digestive disorders

Ethnopharmacological relevanceWormwood (Artemisia absinthium L.) is traditionally used for stomach pain and gastric relief. However, its possible gastroprotective effect has not yet been experimentally evaluated. Aim of the studyThis study evaluated the gastroprotective effect of aqueous extracts obtained through hot and room temperature maceration of A. absinthium aerial parts in rats. Materials and methodsThe gastroprotective effect of hot aqueous extract (HAE) and room temperature aqueous extract (RTAE) from A. absinthium aerial parts were evaluated in rats using a model of acute gastric ulcer induced by ethanol p.a. The stomachs were collected to measure the gastric lesion area and histological and biochemical analysis. UHPLC-HRMS/MS analysis was used to determine the chemical profile of the extracts. ResultsEight main peaks in the UHPLC chromatogram were identified in both HAE and RTAE extracts: tuberonic acid glycoside (1), rupicolin (2), 2-hydroxyeupatolide (3), yangabin (4), sesartemin (5), artemetin (6), isoalantodiene (7), and dehydroartemorin (8). For RTAE, a higher diversity of sesquiterpene lactones was observed. The groups treated with RTAE at 3%, 10%, and 30% presented a gastroprotective effect, reducing the lesion area by 64.68%, 53.71%, and 90.04%, respectively, when compared with the vehicle (VEH)-treated group. On the other hand, the groups treated with HAE at 3%, 10%, and 30% presented values of lesion areas higher than those of the VEH group. Changes in the submucosa layer, inflammatory process with edema, cellular infiltration, and mucin depletion were detected in the gastric mucosa exposed to ethanol, which was fully prevented by RTAE treatment. Neither HAE nor RTAE could increase the reduced glutathione levels in the injured gastric tissue, but RTAE (30%) reduced the formation of lipid hydroperoxides. When the rats were pre-treated with NEM (a chelator of non-protein thiols) or L-NAME (non-selective nitric oxide synthase inhibitor), the RTAE lost the ability to protect the gastric mucosa. ConclusionsThis study corroborates the ethnopharmacological use of this specie to treat gastric disorders revealing the gastroprotective effect of the room-temperature aqueous extract of A. absinthium aerial parts. Its mode of action may involve the ability of the infusion to maintain the gastric mucosal barrier integrity.

The Acute Effects and Mechanism of Ketamine on Nicotine-Induced Neurogenic Relaxation of the Corpus Cavernosum in Mice.

The present study aimed to investigate the acute effects and the mechanism of ketamine on nicotine-induced relaxation of the corpus cavernosum (CC) in mice. This study measured the intra-cavernosal pressure (ICP) of male C57BL/6 mice and the CC muscle activities using an organ bath wire myograph. Various drugs were used to investigate the mechanism of ketamine on nicotine-induced relaxation. Direct ketamine injection into the major pelvic ganglion (MPG) inhibited MPG-induced increases in ICP. D-serine/L-glutamate-induced relaxation of the CC was inhibited by MK-801 (N-methyl-D-aspartate (NMDA) receptor inhibitor), and nicotine-induced relaxation was enhanced by D-serine/L-glutamate. NMDA had no effect on CC relaxation. Nicotine-induced relaxation of the CC was suppressed by mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist), lidocaine, guanethidine (an adrenergic neuronal blocker), Nw-nitro-L-arginine (a non-selective nitric oxide synthase inhibitor), MK-801, and ketamine. This relaxation was almost completely inhibited in CC strips pretreated with 6-hydroxydopamine (a neurotoxic synthetic organic compound). Ketamine inhibited cavernosal nerve neurotransmission via direct action on the ganglion and impaired nicotine-induced CC relaxation. The relaxation of the CC was dependent on the interaction of the sympathetic and parasympathetic nerves, which may be mediated by the NMDA receptor.

Results of Using the Method of Studying the Degree of Chromatophilia of Brain Neurons in Animals Under Prenatal Administration Of L-NAME

Annotation. Endothelial dysfunction during pregnancy underlies many complications of the gestational process [5]. The main mechanism for the development of endothelial dysfunction is a change in the metabolism of nitric oxide. The introduction of a non-selective NO–synthase inhibitor - L-NAME during placentation caused the occurrence of morphological disorders of neurons in the frontal cortex of the offspring of rats.

Using L-Arginine-No Pathway Modulators in Rats with Subtotal Cerebral Ischemia. Histological Changes

Acute cerebrovascular accident is one of the most urgent problems in modern medicine. The frequency of strokes varies in different regions of the world from 1 to 4 cases per 1000 population per year, increasing significantly with age. Cerebrovascular diseases of ischemic origin tend to grow, rejuvenate, are associated with a severe clinical course, high rates of disability and mortality [1-6]. The relevance of the problem of cerebrovascular diseases can rightfully be defined as extraordinary, requiring the concentration of efforts of specialists of different profiles to solve it. Subtotal cerebral ischemia leads to the development of morphological and functional disorders of the cerebral cortex. The introduction of a non-selective NO–synthase inhibitor - L-NAME aggravated histological disorders of neurons that occur with SCI: an increase in the number of hyperchromic shrunken neurons, a decrease in the size and deformation of their pericaryons. Additional use of L -arginine partially eliminated the negative effect of L – NAME [1-8].

Oxidative Stress Activity in Rats with Subtotal Brain Ischemia Under the Conditions of The Use of Modulators of the L-Arginine-No Pathway

Acute cerebrovascular accident is one of the most urgent problems in modern medicine. The urgency of the problem of cerebrovascular diseases can rightfully be defined as extraordinary, requiring the concentration of efforts of specialists of different profiles to solve it. One of the promising neuroprotective amino acids is L-arginine. Most of the effects caused by this amino acid are associated with its ability to increase the formation of NO, acting as a source for its formation. It has been shown that the use of L-arginine reduces the size of the infarct, reduces vascular tone and causes a hypotensive effect, prevents and corrects ischemic and reperfusion damage to the brain and other organs. The use of "L-arginine" in animals of the group with cerebral ischemia and the introduction of a non-selective NOS inhibitor – L-NAME did not have a significant positive effect. Thus, nitric oxide hyperproduction has been studied by stimulating synthesis by introducing the substrate L-arginine, and inhibition by using the non-selective NO synthase inhibitor N-nitro-L-arginine methyl ester.

The Role of NO Synthase in the Infarct-Limiting Effect of Urgent and Chronic Adaptation to Normobaric Hypoxia.

We studied the role of NO synthase in the infarct-limiting effect of short-term (SNH) and chronic continuous normobaric hypoxia (CNH). In male Wistar rats, SNH (6 sessions of 10-min hypoxia 8% O2 and 10-min reoxygenation) or CNH (12% O2 for 21 days) was modeled. In 30 min after SNH or 24 h after CNH, the rats were subjected to coronary artery occlusion (45 min) and reperfusion (2 h). The following drugs were administered to rats: non-selective NO synthase inhibitor L-NAME (10 mg/kg), inhibitor of inducible NO synthase S-methylthiourea (3 mg/kg), and inhibitor of neuronal NO-synthase 7-nitroindazole (50 mg/kg). NO donor diethylenetriamine was administered intravenously in a dose 2 mg/kg. It was found that L-NAME and S-methylthiourea abolished the infarct-limiting effect of SNH and CNH. Diethylenetriamine increased cardiac tolerance to ischemia/reperfusion. It is believed that inducible NO synthase plays an important role in the cardioprotective effect of normobaric hypoxia.

Oroxylin-A and its phosphonate derivative potentiate eNOS/NO-mediated relaxation and attenuate vasoconstrictor-induced contraction in the mouse aorta

Oroxylin-A (OroA), a flavonoid isolated from Scutellariae baicalensis, alleviates cardiovascular dysfunction. Several procedures for synthesizing OroA have been developed but show low production yield and regioselectivity. We synthesized OroA from baicalin using a one-pot reaction to increase its overall yield. We also determined the chemical properties and mechanism of action of the synthesized OroA and OroA phosphate diethyl ester (OroA-OET) in vascular function. The induction of vascular reactivity by OroA and OroA-OET was evaluated using blood vessel myography and biochemical analysis to assess nitric oxide synthase-mediated nitric oxide production in mouse aortic arteries. OroA and OroA-OET (0.1–30 μM) induced sustained vasorelaxation, which was partly mediated by the endothelium in isolated normal arteries pre-contracted with phenylephrine. OroA and OroA-OET significantly attenuated vasoconstrictors-induced contractile responses. Dilation effects were blocked by the non-selective nitric oxide synthase inhibitor N (omega)-nitro-l-arginine methyl ester but not by tetraethylammonium or 1H-(1,2,4)oxadiazolo [4,3-a]quinoxalin-1-one. Notably, preincubation with OroA and OroA-OET potentiated acetylcholine-induced relaxation and endothelial nitric oxide production in the arteries with the endothelium. OroA and OroA-OET protected against cardiovascular dysfunction. The synthesis and lead compounds used not only improved the yield of OroA from natural sources but also potentially regulated vascular tone.

Redox imaging of dextran sodium sulfate-induced colitis mice treated with nitric oxide synthase inhibitors

Ulcerative colitis (UC) is an inflammatory bowel disease of unknown cause. Redox imbalance, as characterized by excessive production of reactive oxygen species (ROS) and nitric oxide (NO), and deficient levels of endogenous antioxidants, is associated with the pathogenesis of UC. Overhauser-enhanced magnetic resonance imaging (OMRI) combined with a spin-probe method produces high-resolution images of redox imbalance in disease states. We aimed to investigate the effect of NO synthase (NOS) inhibitors on colonic ROS generation in mice with colitis induced by dextran sodium sulfate (DSS) using the OMRI/spin-probe method. Symptoms of DSS-induced colitis were ameliorated by an inducible NOS (iNOS) inhibitor, aminoguanidine (AG), but not by a non-selective NOS inhibitor, NG-Nitro-L-arginine methyl ester (L-NAME). Intracellular ROS generation, observed as the enhancement of OMRI contrast decay rates of a 3-methoxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl probe in the colons of DSS-induced colitis mice, was suppressed by AG, in agreement with the results of an in vivo electron spin resonance study. AG had an inhibitory effect on ROS production at the upper colon and rectum during the initiation stage of colitis, and at the upper and lower colon during the advanced stage. Additionally, there were partial differences in the localization of iNOS protein and nitrite/nitrate levels. L-NAME treatment showed a tendency for increased ROS generation at the upper colon during the advanced stage. The OMRI/spin-probe method provides a novel tool for screening ROS inhibitory activity in vivo in DSS-induced colitis mice.

Leonurine Reduces Oxidative Stress and Provides Neuroprotection against Ischemic Injury via Modulating Oxidative and NO/NOS Pathway.

Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling.

L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through nitric oxide-mediated Drp1 inhibition in mouse C2C12 myoblasts.

Severe heat exposure causes mitochondrial fragmentation and dysfunction, which contribute to the pathogenesis of heat-related illness. L-citrulline is a naturally occurring amino acid and has been suggested to influence heat shock responses. This study aimed to test whether L-citrulline supplementation would preserve mitochondrial integrity and attenuate heat-induced skeletal muscle injury, and elucidate the underlying mechanisms. At 37°C, L-citrulline (2 mM) increased mitochondrial elongation in mouse C2C12 myoblasts, a process associated with a reduction in mitochondrial fission protein Drp1 levels. Mechanistic studies revealed that L-citrulline increased cellular nitric oxide (NO) levels, but not S-nitrosylation of Drp1. L-citrulline caused a decrease in phosphorylation of Drp1 at Ser 616 and an increase in phosphorylation of Drp1 at Ser 637, which resulted in a reduced mitochondrial localization of Drp1. L-NAME, a non-selective NO synthase inhibitor, abolished the increase in L-citrulline-induced NO levels and inhibited Drp1 phosphorylation changes and mitochondrial elongation, which indicates involvement of a NO-dependent pathway. Under 43°C heat stress conditions, L-citrulline prevented translocation of Drp1 to mitochondria, mitochondrial fragmentation and decreased membrane potential. Finally, L-citrulline pretreatment inhibited heat-induced reactive oxygen species (ROS) overproduction, caspase 3/7 activation, apoptotic cell death, and improved cell viability. NO inhibitor L-NAME abolished all the above protective effects of L-citrulline under heat stress. Our results suggest that L-citrulline prevents heat-induced mitochondrial dysfunction and cell injury through NO-mediated Drp1 inhibition in C2C12 myoblasts. L-citrulline may be an effective treatment for heat-related illnesses and other mitochondrial diseases.

Asymmetric Dimethylarginine Protects Neurons from Oxygen Glucose Deprivation Insult by Modulating Connexin-36 Expression.

Background Asymmetric dimethylarginine (ADMA) is a nonselective nitric oxide synthase inhibitor. ADMA is thought to inhibit the production of nitric oxide (NO) by neurons after oxygen-glucose deprivation (OGD). The gap junction protein Connexin-36 (cx-36) is involved in the pathophysiology of stroke. We investigated whether ADMA could protect neurons from OGD insults by regulating the expression of cx-36. Methods Cultured rat cortical neuronal cells were used. Neurons were treated with OGD with or without ADMA pretreatment. The lactate dehydrogenase (LDH) release rate was used to assess neuronal injury. Intracellular NO levels were determined using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Western blotting was performed to detect cx-36 expression. Results The LDH release rate increased in the supernatant of neurons after the OGD insult, whereas ADMA treatment reduced the LDH release rate. Intracellular NO levels increased following OGD treatment, and this increase was not inhibited by ADMA treatment. Expression of cx-36 was upregulated in neurons under OGD conditions, and treatment with ADMA downregulated the expression of cx-36. Conclusions ADMA protects neurons from OGD insult, and cx-36 downregulation may be a possible pathway involved in ADMA-mediated neuronal protection.

N-Nitro-L-Arginine-Methyl ester Exerts Neuroprotection byInhibiting Inflammation in Cerebral Injury Induced by Transient Ischemia/Reperfusion in Rats

Cerebral ischemia-reperfusion injury is a pivotal cause of deaths due to cerebrovascular accidents. Further research efforts are needed to reveal the mechanism underlying its aggravation or alleviation. We previously reported the antioxidant effect of N-Nitro-L-Arginine- Methyl Ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, on rats subjected to transient focal cerebral ischemia-reperfusion (I/R). The aim of this work was to explore further neuroprotective anti-inflammatory effects of L-NAME. This study involved 30 adult male Wistar rats divided into three groups with ten rats in each: sham-operated (control), I/R group of rats subjected to 30 minutes of left common carotid artery (CCA) occlusion followed by 24-hour of reperfusion and test group infused with L-NAME intraperitoneally 15 minutes before the same I/R period. Neurological assessments were evaluated, Western blotting used to estimate Nuclear factor-kappa B (NF-?B), ELISA used to detect Tumor necrosis factor- α (TNF-α), and Nitric oxide metabolites were measured colorimetrically, as well as H&E staining to assess brain damage. Compared with the I/R group, the neurological score, infarction area, and the inflammatory biomarkers NF-?B, TNF-α, and NO were significantly decreased in L-NAME treated rats (P =0.001). As a conclusion from the current study, L-NAME showed potential neuroprotection through it is an anti-inflammatory effect on a rat’s model of transient focal cerebral ischemia-reperfusion.