Increase In Nitric Oxide Levels Research Articles

H2S contributed from CSE during cellular senescence suppresses inflammation and nitrosative stress

Aging involves the time-dependent deterioration of physiological functions attributed to various intracellular and extracellular factors. Cellular senescence is akin to aging and involves alteration in redox homeostasis. This is primarily marked by increased reactive oxygen/nitrogen species (ROS/RNS), inflammatory gene expression, and senescence-associated beta-galactosidase activity, all hallmarks of aging. It is proposed that gasotransmitters which include hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO), may affect redox homeostasis during senescence. H2S has been independently shown to induce DNA damage and suppress oxidative stress. While an increase in NO levels during aging is well established, the role of H2S has remained controversial. To understand the role of H2S during aging, we evaluated H2S homeostasis in non-senescent and senescent cells, using a combination of direct measurements with a fluorescent reporter dye (WSP-5) and protein sulfhydration analysis. The free intracellular H2S and total protein sulfhydration levels are high during senescence, concomitant to cystathionine gamma-lyase (CSE) expression induction. Using lentiviral shRNA-mediated expression knockdown, we identified that H2S contributed by CSE alters global gene expression, which regulates key inflammatory processes during cellular senescence. We propose that H2S decreases inflammation during cellular senescence by reducing phosphorylation of IκBα and the p65 subunit of nuclear factor kappa B (NF-κB). H2S was also found to reduce NO levels, a significant source of nitrosative stress during cellular senescence. Overall, we establish H2S as a key gasotransmitter molecule that regulates inflammatory phenotype and nitrosative stress during cellular senescence.

Markers of Endothelial Dysfunction Are Attenuated by Resveratrol in Preeclampsia.

Preeclampsia (PE) is characterized by great endothelial dysfunction, decreased nitric oxide (NO) bioavailability, and higher levels of arginase activity. In the present study, we investigated the potential modulatory effects of trans-resveratrol (RSV) on arginase and endothelial dysfunction biomarkers in endothelial cells exposed to plasma from patients with PE and healthy pregnant (HP) women, and umbilical arteries from patients with PE. Human umbilical vein endothelial cells (HUVECs) were incubated with pooled plasma from 10 HP or 10 PE pregnant women and RSV; umbilical arteries from patients with PE were incubated with RSV; intracellular NO and total reactive oxygen species (ROS) levels were assessed using a probe that interacted with these radicals; total arginase activity was evaluated measuring the urea produced; total antioxidant capacity was measured using the ferric reduction ability power (FRAP) assay; and endothelial dysfunction biomarkers were assessed using qPCR in endothelial cells and umbilical arteries. RSV increased NO levels and decreased total arginase activity in endothelial cells incubated with plasma from patients with PE. In addition, RSV increased total antioxidant capacity and downregulated endothelial dysfunction biomarkers, such as intercellular adhesion molecule-1 (ICAM-1), von Willebrand factor (vWF), and Caspase-3, (CASP-3), in endothelial cells and umbilical arteries from PE patients. RSV treatment positively modulated the L-arginine-NO pathway, decreased arginase activity, and increased antioxidant capacity, in addition to downregulating endothelial dysfunction biomarkers.

Effect of D4 Dopamine Receptor on Na+-K+-ATPase Activity in Renal Proximal Tubule Cells.

NKA activity, nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels were measured in RPT cells treated with the D4 receptor agonist PD168077 and/or the D4 receptor antagonist L745870, the NO synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME) or the soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (ODQ). Total D4 receptor expression and its expression in the plasma membrane were investigated by immunoblotting in RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Activation of D4 receptors with PD168077, inhibited NKA activity in RPT cells from WKY rats in a concentration- and time-dependent manner. The inhibitory effect of PD168077 on NKA activity was prevented by the addition of the D4 receptor antagonist L745870, which by itself had no effect. The NO synthase inhibitor L-NAME and the soluble guanylyl cyclase inhibitor ODQ, which by themselves had no effect on NKA activity, eliminated the inhibitory effect of PD168077 on NKA activity. Activation of D4 receptors also increased NO levels in the culture medium and cGMP levels in RPT cells. However, the inhibitory effect of D4 receptors on NKA activity was absent in RPT cells from SHRs, which could be related to decreased plasma membrane expression of D4 receptors in SHR RPT cells. Activation of D4 receptors directly inhibits NKA activity via the NO/cGMP signaling pathway in RPT cells from WKY rats but not SHRs. Aberrant regulation of NKA activity in RPT cells may be involved in the pathogenesis of hypertension.

The Effects of Consuming Amino Acids L-Arginine, L-Citrulline (and Their Combination) as a Beverage or Powder, on Athletic and Physical Performance: A Systematic Review

Consumption of amino acids L-arginine (L-Arg) and L-citrulline (L-Cit) are purported to increase nitric oxide (NO) production and improve physical performance. Clinical trials have shown relatively more favorable outcomes than not after supplementing with L-Cit and combined L-Arg and L-Cit. However, in most studies, other active ingredients such as malate were included in the supplement. Therefore, the aim of this study was to determine the efficacy of consuming standalone L-Arg, L-Cit, and their combination (in the form of powder or beverage) on blood NO level and physical performance markers. A systematic review was undertaken following PRISMA 2020 guidelines (PROSPERO: CRD42021287530). Four electronic databases (PubMed, Ebscohost, Science Direct, and Google scholar) were used. An acute dose of 0.075 g/kg of L-Arg or 6 g L-Arg had no significant increase in NO biomarkers and physical performance markers (p > 0.05). Consumption of 2.4 to 6 g/day of L-Cit over 7 to 16 days significantly increased NO level and physical performance markers (p < 0.05). Combined L-Arg and L-Cit supplementation significantly increased circulating NO, improved performance, and reduced feelings of exertion (p < 0.05). Standalone L-Cit and combined L-Arg with L-Cit consumed over several days effectively increases circulating NO and improves physical performance and feelings of exertion in recreationally active and well-trained athletes.

Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells.

Background:Endothelial dysfunction is a critical component in the pathogenesis of cardiovascular diseases and is closely associated with nitric oxide (NO) levels and oxidative stress. Here, we report on novel findings linking endothelial expression of CD70 (also known as CD27 ligand) with alterations in NO and reactive oxygen species.Methods:CD70 expression was genetically manipulated in human aortic and pulmonary artery endothelial cells. Intracellular NO and hydrogen peroxide (H2O2) were measured using genetically encoded biosensors, and cellular phenotypes were assessed.Results:An unbiased phenome-wide association study demonstrated that polymorphisms in CD70 associate with vascular phenotypes. Endothelial cells treated with CD70-directed short-interfering RNA demonstrated impaired wound closure, decreased agonist-stimulated NO levels, and reduced eNOS (endothelial nitric oxide synthase) protein. These changes were accompanied by reduced NO bioactivity, increased 3-nitrotyrosine levels, and a decrease in the eNOS binding partner heat shock protein 90. Following treatment with the thioredoxin inhibitor auranofin or with agonist histamine, intracellular H2O2 levels increased up to 80% in the cytosol, plasmalemmal caveolae, and mitochondria. There was increased expression of NADPH oxidase 1 complex and gp91phox; expression of copper/zinc and manganese superoxide dismutases was also elevated. CD70 knockdown reduced levels of the H2O2 scavenger catalase; by contrast, glutathione peroxidase 1 expression and activity were increased. CD70 overexpression enhanced endothelial wound closure, increased NO levels, and attenuated the reduction in eNOS mRNA induced by TNFα.Conclusions:Taken together, these data establish CD70 as a novel regulatory protein in endothelial NO and reactive oxygen species homeostasis, with implications for human vascular disease.

Investigation of the Changes in Serum Intercellular and Vascular Adhesion Molecules (ICAM-1 and VCAM-1) and Nitric Oxide Levels in Response to Exercise Training and L-arginine Supplementation in Postmenopausal Hypertensive Women

Introduction: The positive effects of exercise training in modulating cardiovascular risk factors in individuals with hypertension have been reported. The present study aimed to investigate the effects of 8 weeks of combined training and L-arginine supplementation on the serum levels of intercellular and vascular adhesion molecules (ICAM-1 and VCAM-1) and nitric oxide (NO) in postmenopausal hypertensive women. Method: The postmenopausal women with hypertension were assigned into four groups consisting of 12 individuals, including placebo (P), L-arginine (L), training (T), and training+Larginine (TL) groups. The combined training program (resistance-endurance) was conducted 3 sessions per week for 8 weeks. The L-arginine ingestion was 6 g daily. Blood sampling was performed at the beginning and the end of 8 weeks of intervention, and the levels of ICAM-1, VCAM-1, and NO were measured. The collected data were analyzed in SPPSS software (version 24), one-way ANOVA, and Tukey post hoc test. Results: The levels of ICAM-1 and VCAM-1 in the T and TL groups compared to the P and L groups significantly decreased (P<0.05). Significant increases of NO levels in the L (P=0.003), T (P<0.001), and TL (P<0.001) groups compared to the P group, and also a significant decrease in systolic and diastolic blood pressure were observed in all intervention groups in comparison to the P group (P<0.05). Conclusion: Based on the findings of the present study, L-arginine ingestion and combined training alone or combined together are effective in the reduction of blood pressure in hypertensive patients. Moreover, L-arginine was associated with amplifying combined training effectiveness in decreasing systolic blood pressure. Blood pressure reduction in the above-mentioned groups is probably exerted partly by the upregulation of NO levels

P1474: MOLECURAL MECHANISM OF CHEMOTHERAPEUTIC HYDROXYUREA IS MEDIATED BY NOS2

Background:: Hydroxyurea (HU) is a chemotherapeutic agent that reduces ribonucleotide reductase, stops DNA synthesis and repar, and therefore causes cell proliferation inhibition and apoptosis. Due to its cytostatic properties, HU is frequently used for treatment of myeloproliferative neoplasms, ovarian cancer, and sickle cell anemia. Nitric oxide (NO), produced by nitric oxide synthase (NOS) enzymes is a potent signaling molecule involved in blood flow regulation, neutrotransmission, and immunity. Although HU treatment increases NO levels, up to date it is not clear whether it originates from activation of NOS enzymes or HU degradation. Aims: The aim of this study was to determine the involvement of NOS2 enzyme in the cytostatic effect of HU. Methods: To examine the involvement of the NOS2 enzyme in the molecular mechanism of HU, we treated erythroleukemic HEL92.7.1 cells with pan-selective NOS inhibitor L-NAME (200µM, 1mM, and 5mM), NOS2 specific inhibitor 1400W (1, 10, and 100µM), or NOS2/NOS3 inhibitor DPI (1, 5, and 10µM), in combination with hydroxyurea (200µM), and monitored their effect on proliferation and cell cycle. Immunocytochemistry for the proliferation marker Ki67 was performed to assess proliferation, while cell distribution in cell cycle phases was determined by flow cytometry after propidium iodide staining. Colony forming assay have been performed with the bone marrow cells of Nos2 null mice after oral HU treatment to corroborate the data obtained by enzymatic inhibition. Results: In this study, we demonstrated that treatment of HEL92.7.1 cells with HU induces a dose-dependent increase in NOS2 protein levels and two products of the enzyme NOS - NO and citrulline. HU-induced citrulline levels can be reduced by treatment with the NOS inhibitor L-NAME, indicating that NO is produced de novo by the NOS enzyme rather than HU degradation. Inhibition of the NOS2 enzyme by L-NAME, 1400W, or DPI was sufficient to abolish HU-mediated inhibition of proliferation. While HU increased the number of cells in S-phase of the cycle at the expanse of the G0/G1 due to blocked DNA synthesis, combined treatment with HU and L-NAME or DPI inhibitor resulted in decreased G0/G1 phase and increased S and G2/M phases pointing to increased proliferation. These data indicate that the cytostatic properties of HU are mediated by the NOS2 enzyme. A colony formation assay showed that Nos2 deficient bone marrow cells isolated from mice treated orally with HU (200mg/kg) formed significantly more erythroid colonies (BFU-E and CFU-E) and granulocyte/macrophage progenitors (CFU-GM) compared to HU treated wild-type and untreated Nos2 null mice showing the involvement of Nos2 in the molecular mechanism of HU in vivo. Summary/Conclusion: Our results show that HU induces the enzymatic activity of the NOS2 protein which in turn is involved in the HU regulation of proliferation and cell cycle. Comprehensive knowledge of the molecular mechanism of HU might help to improve its beneficial properties and decrease adverse effects.

Nitric Oxide-Induced Calcineurin A Mediates Antimicrobial Peptide Production Through the IMD Pathway.

Nitric oxide (NO) at a high concentration is an effector to kill pathogens during insect immune responses, it also functions as a second messenger at a low concentration to regulate antimicrobial peptide (AMP) production in insects. Drosophila calcineurin subunit CanA1 is a ubiquitous serine/threonine protein phosphatase involved in NO-induced AMP production. However, it is unclear how NO regulates AMP expression. In this study, we used a lepidopteran pest Ostrinia furnacalis and Drosophila S2 cells to investigate how NO signaling affects the AMP production. Bacterial infections upregulated the transcription of nitric oxide synthase 1/2 (NOS1/2), CanA and AMP genes and increased NO concentration in larval hemolymph. Inhibition of NOS or CanA activity reduced the survival of bacteria-infected O. furnacalis. NO donor increased NO level in plasma and upregulated the production of CanA and certain AMPs. In S2 cells, killed Escherichia coli induced NOS transcription and boosted NO production, whereas knockdown of NOS blocked the NO level increase caused by E. coli. As in O. furnacalis larvae, supplementation of the NO donor increased NO level in the culture medium and AMP expression in S2 cells. Suppression of the key pathway genes showed that the IMD (but not Toll) pathway was involved in the upregulation of CecropinA1, Defensin, Diptericin, and Drosomycin by killed E. coli. Knockdown of NOS also reduced the expression of CanA1 and AMPs induced by E. coli, indicative of a role of NO in the AMP expression. Furthermore, CanA1 RNA interference and inhibition of its phosphatase activity significantly reduced NO-induced AMP expression, and knockdown of IMD suppressed NO-induced AMP expression. Together, these results suggest that NO-induced AMP production is mediated by CanA1 via the IMD pathway.

Persea americana seed extract restores defective sperm quality and biochemical parameters relevant to reproduction in male wistar rats treated with cyclosporine A.

Studies have shown that some drugs impair spermatogenesis, thereby causing infertility. Thus, this study aims at investigating the effect of Persea americana seed extract on the male reproductive system in cyclosporine-induced rats. Thirty male albino rats were randomly divided into five groups; all groups were induced with cyclosporine except the control group. Group 3 was treated with 10mg/kg of lisinopril, groups 4 and 5 received 50 and 100 mg/kg of PAE, respectively. The experiment lasted for 7 days. The antioxidant enzymes [superoxide dismutase (SOD) and catalase (CAT)], and arginase activities were evaluated, nitric oxide (NO) and malondialdehyde (MDA) level of the penile tissue homogenate were determined with serum hormonal (follicle stimulating hormone, luteinizing hormone and testosterone) concentration and sperm quality. This study showed that cyclosporine induction caused a significant (p < .05) decrease in the SOD, CAT, sperm quality, NO, and hormonal level as compared to the control group, with a simultaneous increase in arginase activity and MDA level. However, treatment with PAE and lisinopril significantly (p < .05) increase antioxidant enzyme activities, sperm quality, NO, and serum hormonal level, with a decrease MDA level and arginase activity when compared with cyclosporine-induced group. This study showed that P. americana seed extract could be useful in the management of hormonal disruption resulting from oxidative stress in male folks. PRACTICAL APPLICATIONS: Some pharmaco-agents have been reported to alter spermatogenesis, thereby causing infertility. Plants represent natural resources use in the management of several human diseases from time immemorial. Persea americana seed is a part of the fruit that most people do throw away after consuming the edible portion of the fruit. However, the therapeutic and pharmacological activities of P. americana seed have been reported. Therefore, this study sought to investigate the effects of P. americana seed extract on cyclosporine-induced reprotoxicity.

Understanding the Cellular Sources of the Fractional Exhaled Nitric Oxide (FeNO) and Its Role as a Biomarker of Type 2 Inflammation in Asthma.

Fractional exhaled nitric oxide (FeNO) has gained great clinical importance as a biomarker of type 2 inflammation in chronic airway diseases such as asthma. FeNO originates primarily in the bronchial epithelium and is produced in large quantities by the enzyme inducible nitric oxide synthase (iNOS). It should be noted that nitric oxide (NO) produced at femtomolar to picomolar levels is fundamental for respiratory physiology. This basal production is induced in the bronchial epithelium by interferon gamma (IFNγ) via Janus kinases (JAK)/STAT-1 signaling. However, when there is an increase in the expression of type 2 inflammatory cytokines such as IL-4 and IL-13, the STAT-6 pathway is activated, leading to overexpression of iNOS and consequently to an overproduction of airway NO. Increased NO levels contributes to bronchial hyperreactivity and mucus hypersecretion, increases vascular permeability, reduces ciliary heartbeat, and promotes free radical production, airway inflammation, and tissue damage. In asthmatic patients, FeNO levels usually rise above 25 parts per billion (ppb) and its follow-up helps to define asthma phenotype and to monitor the effectiveness of corticosteroid treatment and adherence to treatment. FeNO is also very useful to identify those severe asthma patients that might benefit of personalized therapies with monoclonal antibodies. In this review, we revised the cellular and molecular mechanisms of NO production in the airway and its relevance as a biomarker of type 2 inflammation in asthma.

In Vitro Immunomodulatory Effects of Inonotus obliquus Extracts on Resting M0 Macrophages and LPS-Induced M1 Macrophages.

Background Inonotus obliquus (Chaga) is a parasitic fungus that is distributed mainly in northeast China. Our literature research showed chaga polysaccharides have bilateral effects on tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels when they exert antitumor and antidiabetic activities. The current research tried to explore the influence of chaga extracts on inflammatory factors via macrophage polarization which has bilateral immune-regulation not only on healthy tissue homeostasis but also on pathologies. Methods Chaga was extracted with 100°C water and precipitated with 80% ethanol. The extracts were studied on RAW264.7 macrophage at resting condition (M0) and lipopolysaccharide (LPS)-activated subtype (classic activated macrophage, M1). The IL-1β, TNF-α, nitric oxide (NO) level, and the protein expressions of M1 and alternative activated macrophage (M2) markers including IL-1β, inducible NO synthase (iNOS), mannose receptor (CD206), and arginase (Arg)-1 were compared. Results The 100 g extracts contained 13.7 g polysaccharides and 1.9 g polyphenols. Compared with M0, the 50 μg/mL extracts increased NO level (P < 0.05) and decreased CD206 and Arg-1 expression significantly (P < 0.05). The extracts at 100–200 μg/mL increased NO and TNF-α level (P < 0.05), but increased iNOS and IL-1β expression significantly (P < 0.05). Compared with M1, the extracts decreased NO level at 25, 50, 100, and 200 μg/mL and decreased IL-1β and TNF-α level at 100–200 μg/mL significantly (P < 0.05). At 25–200 μg/mL, the extracts significantly increased CD206 and Arg-1 expression and decreased IL-1β and iNOS expression separately (P < 0.05). Conclusions Our research suggested that the bilateral effects of the chaga extracts on iNOS, IL-1β, and NO level on M0/M1 macrophages might be related with chaga polysaccharides and chaga polyphenols. Some in vivo anticancer and antidiabetic research of purified chaga polysaccharides related to macrophage differentiation should be conducted further.

Effects of the probiotic, Lactobacillus rhamnosus GG, on ulcer pathogenesis, HSP70 stress protein and nitric oxide levels in stress induced ulcer

ABSTRACT Peptic ulcer is a gastric or duodenal mucosal injury; psychological stress may participate in development of the lesions. Heat shock protein-70 (HSP70) is a molecular chaperone that is responsible for cellular healing; it is an early biomarker of cellular damage. Nitric oxide (NO) is an intra- and intercellular messenger in the gastrointestinal system that protects mucosal integrity. Lactobacillus rhamnosus is among the microflora of the intestinal tract; it is resistant to gastric acidity. We investigated the efficacy of L. rhamnosus administration on ulcer pathogenesis, stress protein HSP70 and NO levels in experimental stress induced ulcer. The proton pump inhibitor, pantoprazole, was used for comparison with the gastroprotective effect of the probiotic. We administered 10 mg/kg pantoprazole and L. rhamnosus at doses of 3 × 108 cfu/ml (M1), 15 × 108 cfu/ml (M5), 30 × 108 cfu/ml (M10) to rats according to McFarland-1, McFarland-5, McFarland-10 standards, respectively. Rats were stressed by immobilization at 4 °C, then sacrificed. The pH, amounts of gastric mucus, NO and HSP70 levels were measured and the histological structure of stomach was assessed. We found increased NO levels in the M5 group and increased HSP70 expression in the pantoprazole group. Significant epithelial damage was observed in the stressed groups and minimal epithelial damage was observed in M5 group compared to controls. The probiotic, L. rhamnosus, may be useful for preventing stress induced ulcers.

Nebivolol Increases Nitric Oxide Synthase via β3 Adrenergic Receptor in Endothelial Cells Following Exposure to Plasma from Preeclamptic Patients.

Background: Low bioavailability of nitric oxide (NO) is related to the pathophysiology of preeclampsia (PE). In the present study, we investigated the effect of nebivolol (NEB), a β3-receptor agonist with vasodilator properties, on the NO synthesis in endothelial cells incubated with plasma from preeclamptic patients. Methods and results: Human umbilical vein endothelial cells (HUVECs) were incubated with plasma from healthy pregnant (HP) and PE women; NO quantification was assessed by a fluorescence compound. We found that endothelial cells incubated with plasma from women with PE show lower NO levels compared with the HP group (p < 0.0001). However, NEB treatment increases NO levels, partially, mediated by β3 adrenergic receptors (p < 0.0001) and through eNOS activation (p < 0.0001). Conclusions: Our results suggest that NEB acts in NO synthesis through eNOS activation and β3 adrenergic receptors in the endothelium. However, further studies will be needed to understand this molecule.

Altered transsulfuration pathway enzymes and redox homeostasis in inherited retinal degenerative diseases

Retinal degenerative diseases result from apoptotic photoreceptor cell death. As endogenously produced gaseous molecules such as hydrogen sulfide (H2S) and nitric oxide (NO) play a key role in apoptosis, we compared the expression levels of genes and proteins involved in the production of these molecules in the retina of normal dogs and three canine models (rcd1, crd2, and xlpra2) of human inherited retinal degeneration (IRD). Using qRT-PCR, Western blot, and immunohistochemistry (IHC), we showed that mRNA and protein levels of cystathionine β–synthase (CBS), an enzyme that produces H2S in neurons, are increased in retinal degeneration, but those of cystathionine γ-lyase (CSE), an enzyme involved in the production of glutathione (GSH), an antioxidant, are not. Such findings suggest that increased levels of H2S that are not counterbalanced by increased antioxidant potential may contribute to disease in affected retinas. We also studied the expression of neuronal and inducible nitric oxide synthase (nNOS and iNOS), the enzymes responsible for NO production. Western blot and IHC results revealed increased levels of nNOS and iNOS, resulting in increased NO levels in mutant retinas. Finally, photoreceptors are rich in polyunsaturated fatty acids (PUFAs) that can make these cells vulnerable to oxidative damage through reactive oxygen species (ROS). Our results showed increased levels of acrolein and hydroxynonenal (4HNE), two main toxic products of PUFAs, surrounding the membranes of photoreceptors in affected canines. Increased levels of these toxic products, together with increased NO and ROS, likely render these cells susceptible to an intrinsic apoptotic pathway involving mitochondrial membranes. To assess this possibility, we measured the levels of BCL2, an anti-apoptotic protein in the mitochondrial membrane. Western blot results showed decreased levels of BCL2 protein in affected retinas. Overall, the results of this study identify alterations in the expression of enzymes directly involved in maintaining the normal redox status of the retina during retinal degeneration, thereby supporting future studies to investigate the role of H2S and NO in retinal degeneration and apoptosis.

Nitric Oxide on Pathophysiology of SARS-CoV 19: Toward Possible Role of Acupuncture Treatment

The ongoing outbreak of COVID-19 has quickly become a daunting challenge to global health. In the absence of satisfied therapy, effective treatment interventions are urgently needed. Previous studies have demonstrated that acupuncture is effective at relieving common symptoms of COVID-19 including breathlessness, nausea, insomnia, leukopenia, fatigue, vomiting, and abdominal pain. Experiments have shown that nitric oxide (NO) inhibits the replication cycle of severe acute respiratory syndrome (SARS) coronavirus with similar structures of COVID-19. Increase in level of NO by using NO gas inhalation has been shown to restore lung function by reducing airway resistance and improving virus-induced lung infections in SARS patients. Recent case report showed that a medical acupuncturist with symptoms consistent with severe COVID pneumonia achieved full recovery by self-administered medical acupuncture and cupping therapy at home. Clinical features and pathophysiology demonstrated that NO deficiency and endothelial dysfunction contribute to the development of COVID-19. Several studies from different groups consistently demonstrated that acupuncture increases NO synthase expression and induces an elevation of NO production and release in plasma and the local skin regions in both animals and humans. It is suggested that exogenous NO supplies or interventions that induce increasing levels of NO can play an important role in protective effects against inflammation and acute lung injury. This article reviews the rationale for mechanisms of NO induction induced by acupuncture in the possible treatment of COVID-19 and highlights its potential for contributing to better clinical outcomes and improving future clinical studies of acupuncture on treatment of COVID-19.

The Effect of Administration of Omega-3 and Vitamin E on the Levels of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) in Preeclampsia White Rats (Rattus Norvegicus)

Background: Preeclampsia is a disease with a variety of theories that describe the uncertainty of the pathophysiology. According to the oxidative stress theory, preeclampsia originates from the failure of trophoblast invasion during the implantation process, causing ischemia and placental hypoxia, which in turn causes cell damage, including placental endothelial cell dysfunction. Omega-3 fatty acids and vitamin E have an important role in preventing preeclampsia. Omega-3 fatty acids play an important role in maintaining cell membranes and anti-inflammatory processes. At the same time, vitamin E acts as a fat-soluble antioxidant that can prevent oxidative stress, inhibit proinflammatory cytokines, and protect fatty acids from oxidation. Aim: The purpose of this study was to determine the effect of omega-3 and vitamin E on the level of ROS and NO in pregnant rats with preeclampsia models. Method &amp; Material: This type of research is experimental with Post-Test Only Control Group Design. The sample consisted of 35 pregnant rats, which were divided into five groups. On the 19th day, blood serum was taken to check the levels of ROS and NO. The measuring instrument used is a spectrophotometer with the ELISA method. Data were analyzed using the Shapiro Wilks normality test. After the parametric test was completed, the hypothesis was tested using one-way ANOVA. Results: The average levels of ROS in each group were K- : 121,684 ng/L, K+ : 143,885 ng/L, P1 : 136,250 ng/L, P2 : 132.433 ng/L, and P3 : 122,993 ng/L. The average levels of NO obtained were K-: 29,502 ng/L, K+: 26,053 ng/L, P1: 27,250 ng/L, P2: 27,555 ng/L, and P3: 32,278 ng/L. The results of one-way ANOVA analysis showed that the administration of omega-3 and vitamin E had a significant difference between the control and treatment groups, both at levels of ROS (p=0.001) and levels of NO (p=0.001). Conclusion: The administration of omega-3, vitamin E, and omega-3 plus vitamin E can reduce ROS levels in pregnant rats with preeclampsia models. There is an increase of NO levels only in the administration of omega-3 plus vitamin E. Keywords: [Omega-3, Vitamin E, Oxidative stress, ROS, NO, Preeclampsia].

Evaluating the role of nitric oxide in myogenesis in vitro

Skeletal muscle injury activates satellite cells to proliferate as myoblasts and migrate, differentiate and fuse with existing fibres at the site of injury. Nitric oxide (NO), a free radical produced by NO synthase, is elevated and supports healing after in vivo injury. NOS-independent elevation of NO levels in vitro is possible via donors such as molsidomine (SIN-1). We hypothesized that alterations in NO levels may directly influence myogenic processes critical for skeletal muscle wound healing. This study aimed to clarify the role of NO in myoblast proliferation, migration and differentiation. Baseline NO levels were established in vitro, whereafter NO levels were manipulated during myogenesis using l-NAME (NOS inhibitor) or SIN-1. Baseline NO levels generated by myoblasts in proliferation media did not change 1 h after stimulation. Addition of a pro-proliferative dose of HGF slightly elevated NO levels 1 h post-stimulation, whereas cell numbers assessed 24 h later increased significantly; l-NAME reduced the HGF-driven increase in NO and proliferation, reducing wound closure over 16 h. In differentiation media, NO levels increased significantly within 24 h, returning to baseline over several days. Regular addition of l-NAME to differentiating cells significantly reduced NO levels and fusion. SIN-1 increased NO levels in a dose-dependent manner, reaching maximal levels 16 h post-treatment. SIN-1, added at 0, 2 and 4 days, significantly increased myofiber area (26 ± 1.8% vs 18.6 ± 3.4% in control at 5 day, p < 0.0001), without affecting proliferation or migration. In conclusion, this study demonstrates that, during skeletal muscle regeneration, increased NO specifically stimulates myoblast differentiation.

Temporal dynamics of nitric oxide wave in early vasculogenesis.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10-HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

Abstract P509: Novel Protein Kinase C Epsilon Inhibitor Attenuates Uncoupled Endothelial Nitric Oxide Synthase And Vascular Endothelial Dysfunction

Endothelial nitric oxide synthase (eNOS) uncoupling reduces nitric oxide (NO) production when tetrahydrobiopterin (BH 4 ) is oxidized to dihydrobiopterin (BH 2 ), resulting in endothelial dysfunction and leukocyte adherence. Renovascular damage following shockwave lithotripsy (SWL) may occur via the potentiation of this mechanism. We aim to modulate uncoupled eNOS activity with myristoylated protein kinase C epsilon inhibitor ( myr -PKCε–; N- myr -EAVSLKPT) and activator ( myr -PKCε+; N- myr -HDAPIGYD). We hypothesize that myr -PKCε– with uncoupled eNOS (i.e., bound to BH 2 ) should reduce leukocyte-endothelial interactions in postcapillary venules and H 2 O 2 levels after SWL. Myr -PKCε+ should promote similar effects when eNOS is coupled (i.e., bound to BH 4 ). Mesenteric venules of anesthetized male SD rats (300g) were superfused for 120 min with 100 μM BH 2 and test solutions (Krebs’ buffer, 10 μM myr -PKCε+, or 10 μM myr -PKCε–) with or without 100 μM BH 4 . Leukocyte activity was evaluated via intravital microscopy. In separate SD rats, SWL was performed on left kidneys (16 kV, 500 shocks at 60 shocks/min and at 120 shocks/min). Then, normal saline or test solution cocktail ( myr -PKCε+ [0.9 mg/kg]/ myr -PKCε- [0.8 mg/kg] with BH 2 [2mg/kg] or BH 4 [0.8 mg/kg]) was given i.v. NO or H 2 O 2 levels were measured with 100 μm microsensors in left renal veins at baseline, end of SWL, and 5-min intervals for 30 min post-SWL. Data were analyzed using ANOVA Student-Newman-Keuls test. BH 2 -induced leukocyte rolling, adherence, and transmigration were significantly increased by myr -PKCε+ ( n = 6) and attenuated with myr- PKCε– ( n = 5) compared to control (n=5) at 120 min (p&lt;0.01). Following SWL, all treatments except myr -PKCε+ with BH 2 significantly reduced H 2 O 2 ( n = 5, p &lt; .01) and increased NO levels ( n = 5, p &lt; .01) compared to control. NO and H 2 O 2 levels following administration of myr -PKCε+ with BH 2 were similar to SWL control. Results suggest that myr -PKCε– attenuates uncoupled eNOS activity and H 2 O 2 release in rat renal veins, after SWL. Promoting coupled eNOS activity with BH 4 yields similar results. Promoting eNOS coupling with BH 4 or inhibiting uncoupled eNOS with myr- PKCε– attenuates oxidative stress and endothelial dysfunction following SWL and BH 2 -induced inflammation.

Omega-3 and omega-6 fatty acids have distinct effects on endothelial fatty acid content and nitric oxide bioavailability

Treatment with high dose icosapent ethyl (IPE), an ethyl ester of the omega-3 fatty acid eicosapentaenoic acid (EPA), significantly reduced ischemic events in patients with either cardiovascular disease (CV) or diabetes plus other risk factors (REDUCE-IT) but the mechanism is not well understood. We compared the effects of EPA, docosahexaenoic acid (DHA), and the omega-6 fatty acid arachidonic acid (AA) on bioavailability of nitric oxide (NO) and fatty acid composition. Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA, DHA, or AA (10 µM). Cells were stimulated with calcium ionophore and NO and peroxynitrite (ONOO−) were measured using porphyrinic nanosensors. Levels of EPA, DHA, AA and other fatty acids were measured by gas chromatography (GC). EPA treatment caused the greatest NO release (18%, p < 0.001) and reduction in ONOO− (13%, p < 0.05) compared to control; the [NO]/[ ONOO−] ratio increased by 35% (p < 0.001). DHA treatment increased NO levels by 12% (p < 0.01) but had no effect on ONOO− release. AA did not affect either NO or ONOO− release. Fatty acid treatments increased their respective levels in endothelial cells. EPA levels increased 10-fold to 4.59 mg/g protein (p < 0.001) with EPA treatment and the EPA/AA ratio increased by 10-fold (p < 0.001) compared to vehicle. Only EPA increased docosapentaenoic acid (DPA, omega-3) levels by 2-fold (p < 0.001). AA alone decreased the EPA/AA ratio 4-fold (p<0.001). These findings support a preferential benefit of EPA on endothelial function and omega-3 fatty acid content.