Reduced Nitric Oxide Production Research Articles

Nitric oxide mediated kisspeptin regulation of steroidogenesis and gametogenesis in the catfish, Clarias batrachus.

Nitric oxide (NO) is a gaseous molecule that regulates various reproductive functions. It is a well-recognized regulator of GnRH-FSH/LH-sex steroid secretion in vertebrates including fish. Kisspeptin is a recently discovered neuropeptide which also regulates GnRH secretion. Nitrergic and kisspeptin neurons are reported in close physical contact in the mammalian brain suggesting their interactive role in the release of GnRH. The existence of kisspeptin and NOS is also demonstrated in vertebrate gonads, but information on their reciprocal relation in gonads, if any, is obscure. Therefore, attempts were made to evaluate the functional reciprocal relation between nitric oxide and kisspeptin in the catfish gonads, if any, by administering the nitric oxide synthase (NOS) inhibitor, L-NAME {N(G)-nitro-L-arginine methyl ester}, which reduces NO production, and kisspeptin agonist (KP-10) and assessing their impacts on the expressions of kisspeptin1, different NOS isoforms, NO and steroid production in the gonadal tissue. The results revealed that L-NAME suppressed the expression of kiss1 in gonads of the catfish establishing the role of NO in kisspeptin expression. However, KP-10 increased the expression of all the isoforms of NOSs (iNOS, eNOS,nNOS) and concurrently NO and steroids in the ovary and testis. In vitro studies also indicate that kisspeptin stimulates the production of NO and estradiol and testosterone levels in the gonadal explants and medium. Thus, in vivo results clearly suggest a reciprocal interaction between kisspeptin and NO to regulate the gonadal activity of the catfish. The in vitro findings further substantiate our contention regarding the interactive role of kisspeptin and NO in gonadal steroidogenesis.

The effcacy of short-term tetrahydrobiopterin administration to improve locomotor muscle microvascular function in heart failure with preserved ejection fraction

Background: Patients with heart failure with preserved ejection fraction (HFpEF) exhibit locomotor muscle microvascular dysfunction due partly to reduced nitric oxide (NO) bioavailability. Tetrahydrobiopterin (BH4) is a requisite cofactor for the enzymatic production of NO by NO synthase (NOS). BH4 insuffciency promotes NOS uncoupling, which reduces NO production and augments cellular oxidative stress. Given the potential of BH4 to improve NO bioavailability, we tested the hypothesis that short-term BH4 administration would improve locomotor muscle microvascular function in patients with HFpEF. Methods: Eight patients with HFpEF (74±7 years; 31.3±4.9 kg/m2) participated in a randomized, double-blind, crossover study, having consumed either BH4 (Sapropterin, 10 mg/kg) or placebo for 10 days, separated by a 14-day washout period. Locomotor muscle microvascular function was assessed as leg blood flow (LBF) and leg vascular conductance (LVC) responses to passive leg movement (PLM) before (pre) and after (post) each treatment. PLM-induced LBF and LVC responses were expressed as peak change (Δpeak) or total hyperemic response (area under the curve above baseline; AUC). Results: LBFΔpeak did not change following BH4 (267 ± 136 mL/min vs. 250 ± 101 mL/min) or placebo (317 ± 119 mL/min vs. 269 ± 97 mL/min) administration (pre vs. post; p>0.05). Similarly, LVCΔpeak was unchanged following either BH4 (396 ± 165 mL/min/100mmHg vs. 465 ± 180 mL/min/100mmHg) or placebo (579 ± 238 mL/min/100mmHg vs. 496 ± 185 mL/min/100mmHg) administration (pre vs. post; p>0.05). The changes in LBFAUC and LVCAUC were not affected by either treatment (LBFAUC; BH4: Δ91 ± 103 mL vs. Δ82 ± 69 mL; placebo: Δ148 ± 119 mL vs. Δ87 ± 81 mL; LVCAUC; BH4: Δ65 ± 55 mL/100mmHg vs. Δ98 ± 78 mL/100mnHg; placebo: Δ182 ± 127 mL/100mmHg vs. Δ105 ± 94 mL/100mmHg) (pre vs. post; p>0.05). Conclusion: Short-term BH4 administration did not improve locomotor muscle microvascular function in patients with HFpEF. This project was funded, in part, by the National Institutes of Health (HL118313, D.W.W.; T32HL139451, K.B.; the U.S. Department of Veterans Affairs (I01RX001311, D.W.W.; IK2RX003670, to K.B.), and the American Heart Association (18POST33960192; K.B.). 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.

IL-6 Contributes to Impaired Nitric Oxide and Increased Mitochondrial ROS Production in Human Aortic Endothelial Cells

Background: Major Depressive disorder (MDD) is associated with chronic low-grade inflammation with elevated proinflammatory cytokines. Notably, elevated inflammation is a known contributor to cardiovascular dysfunction and disease. Interleukin (IL)-6 has been shown to be elevated in rodent models of depression. Additionally, preliminary data from our lab suggests that there is a greater proportion of IL-6 producing CD8+ T cells in adults with MDD. Although IL-6 has been reported to be increased in adults with MDD and known to have a direct effect on vascular endothelial cells, the exact mechanism by which IL-6 interacts with the endothelium is unknown. Therefore, we tested the hypothesis that IL-6 treatment will decrease nitric oxide (NO) production and increase mitochondrial ROS (mitoROS) in endothelial cells. Methods: Human aortic endothelial cells (HAECs) were used to study the effects of IL-6 on NO production. The HAECs were treated with vehicle or 10 ng/mL IL-6 for 16 hours, and then stimulated with Acetylcholine to induce NO production and the real time NO production was measured using a commercially available assay. We also measured mitoROS in HAECs treated with or without IL-6. Results are expressed as mean ± SEM (n=3-6 replicates) and group differences were assessed by one-way ANOVA followed by Tukey’s post-hoc test. Results: L-6 was found to significantly lower NO production in the cells (Control: 276.24 ± 7.01; IL-6: 215.19 ± 6.07 MFI, p=0.022). Acetylcholine stimulation resulted in greater NO production in the control cells (Control: 276.24 ± 7.01; Control + Ach: 354.28 ± 6.26 MFI, p=0.0005) whereas this increase was abolished in the IL-6 treated cells (IL-6: 215.19 ± 6.07; IL-6 + Ach: 225.21 ± 5.16 MFI, p=0.955). Additionally, we also found greater mitoROS in the IL-6 treated cells (Control: 683.02 ± 32.98; IL-6: 840.86 ± 38.63 MFI, p=0.002). Conclusion: These preliminary results suggest that IL-6 reduces NO production in endothelial cells, which may be in part due to elevated mitoROS. This work is supported by grants from NIH K01AG061271, R01AG060395 and AHA940023 (DWT). 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.

The potential role of nitric oxide in the anticonvulsant effects of betulin in pentylenetetrazole (PTZ)-induced seizures in mice

Epilepsy poses a significant challenge, especially for drug-resistant cases, necessitating novel treatment avenues. This study explores the potential interplay between nitric oxide (NO) and the anticonvulsant effects of betulin, a triterpene with promising neuroprotective properties. While betulin exhibits anticonvulsant effects, the specific involvement of NO remains inadequately understood, constituting a pivotal gap in current knowledge. One hundred NMRI mice were randomly assigned to diverse treatment groups, with seizures induced by pentylenetetrazol (PTZ). Parameters such as seizure threshold, nitrite levels, total antioxidant capacity (TAC), malondialdehyde (MDA) levels, and iNOS/nNOS gene expressions were assessed. Betulin significantly increased seizure thresholds and mitigated PTZ-induced NO levels. These findings suggest a potential modulation of NO-related pathways, emphasizing betulin's anti-inflammatory and antioxidant attributes. The study sheds light on betulin's multifaceted impact on oxidative stress, NO regulation, and iNOS/nNOS gene expressions. The ability of betulin to suppress iNOS/nNOS gene expressions, leading to reduce NO production, underscores its potential as an anticonvulsant.

Anti-inflammatory effect of gallic acid-conjugated chitooligosaccharides in lipopolysaccharide-stimulated RAW 264.7 macrophages

The aim of the present study is to investigate the anti-inflammatory effect of gallic acid grafted onto COS chains (GA-COS), focusing on the reduction of nitric oxide production, downregulation of inflammatory signals such as inducible nitric oxide synthase (iNOS), gene expression of cytokines such as TNF-α, IL-1β, IL-6, and nuclear factor kappa B (NF-κB) signalling, including the p50 and p65 subunits. The anti-inflammatory effect is mediated through the reduction of nitric oxide production and downregulation of inflammatory proteins such as inducible nitric oxide synthase (iNOS), gene expression of cytokines like TNF-α, IL-1β, IL-6, and nuclear factor kappa B (NF-κB) signalling, including the p50 and p65 subunits. Target proteins were identified by western blot analysis with specific monoclonal antibodies. The levels of gene expression were determined by the RT-PCR method. The results demonstrate that GA-COS effectively reduces nitric oxide generation and downregulates iNOS protein and cytokine expression and NF-κB signalling in lipopolysaccharide (LPS) -induced RAW 264.7 cells. GA-COS exhibits significantly enhanced anti-inflammatory activity compared to the free chitooligosaccharide chain. This study lays the groundwork for future research to demonstrate that GA-COS holds significant potential as a novel compound for the prevention of inflammatory diseases.

Anti-Inflammatory Effects of Moringa Oleifera Lam Leaf Extract in Lipopolysaccharide-Activated Microglia

Moringa oleifera Lam has been used as a medicinal plant in many Asian countries including Thailand. The plant has a variety of pharmacological effects such as antioxidant, anti-inflammation, hypolipidemic, anti-infection, anticancer, and antidiabetic. The leaves and young pods, which contain various nutritional compounds including proteins, fatty acids, and vitamins, are widely consumed. In this study, we investigated the effects of hexane, ethyl acetate, methanol and ethanol crude extracts of M. oleifera Lam leaves on the viability of microglial cells and on the lipopolysaccharide-induced microglial activation. which is widely used as a model for neuroinflammation. Our findings suggested that all of the crude extracts at concentrations ranging from 10–9 to 10–5 g/mL did not significantly affect the viability of microglial cells and exhibited similar effects. Subsequently, we focused on ethanol extract for investigation of the molecular mechanism involving neuroinflammation. We found that the ethanol extract of M. oleifera Lam leaves at the concentrations of 10–9 to 10–5 g/mL significantly reduced inducible nitric oxide synthase (iNOS) protein expression and nitric oxide (NO) production, accompanied by the reduction of phospho-NF-κB and phospho-IκBα expressions. The results suggested that the ethanol extract of M. oleifera Lam leaves reduced NO production through reduction of iNOS protein expression following the inhibition of NF-κB/IκBα signaling pathways. Our study underscored the therapeutic potential of M. oleifera Lam leaves extract in neurodegenerative diseases associated with neuroinflammation. HIGHLIGHTS The ethanol extract of M. oleifera Lam leaves dose-dependently reduced NO production in LPS-activated microglia The reduction in NO production correlated with the reduction in iNOS protein expression The extract inhibited the expression of NF-kB/I-kBα protein expression in a dose-dependent manner The ethanol extract of oleifera Lam leaves exhibited anti-inflammatory effect via the inhibition of NF-kB/I-kBα signaling pathway GRAPHICAL ABSTRACT

Synaptic mechanisms underlying the elevated sympathetic outflow in fructose-induced hypertension.

Metabolic syndrome is associated with cardiovascular dysfunction, including elevated sympathetic outflow. However, the underlying brain mechanisms are unclear. The nucleus tractus solitarius (NTS) critically regulates autonomic reflexes related to cardiovascular function and contains neurons projecting to the caudal ventrolateral medulla (CVLM). Nitric oxide (NO) is a diffusible free-radical messenger in the vascular, immune, and nervous systems. In this study, we determine if NO in the NTS is involved in the synaptic plasticity underlying the elevated sympathetic outflow in fructose-induced hypertension. We retrogradely labeled CVLM-projecting NTS neurons through the injection of FluoSpheres into the CVLM in a fructose-fed rat model to determine the cellular mechanism involved in increased sympathetic outflow. Fructose feeding increased the blood pressure and glucose levels, which represent metabolic syndrome. We found that fructose feeding reduces the NO precursor L-arginine-induced increase in the firing activity of CVLM-projecting NTS neurons. Furthermore, fructose feeding reduces the L-arginine-induced increase in presynaptic spontaneous glutamatergic synaptic inputs to NTS neurons, while NO donor DEA/NO produces an increase in glutamatergic synaptic inputs in fructose-fed rats similar to that in vehicle-treated rats. In addition, fructose feeding reduces the NO-induced depressor response and sympathoinhibition. These data suggested that fructose feeding reduced NO production and, thus, the subsequent NO-induced glutamate releases in the NTS and depressor response. The findings of this study provide new insights into the central mechanisms involved in the neural control of cardiovascular and autonomic functions in the NTS in metabolic syndrome.

Effect of Latanoprostene Bunod on the Permeability of Trabecular Meshwork Cells

Purpose: To investigate the effects of latanoprostene bunod (LBN) on nitric oxide (NO) production and permeability in human trabecular meshwork cells (HTMC).Methods: HTMC were treated with 50 and 100 µM LBN and latanoprost free acid (LAT) for 30 minutes. Additionally, 100 µM LBN was co-exposed to 0.5 mM L-NAME (N-Nitroarginine methyl ester). Cellular viability and NO production were measured using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) and Griess assays. The permeability and resistance of the HTMC monolayer were evaluated by trans-endothelial electrical resistance (TEER) and carboxyfluorescein permeability.Results: Exposure to 100 µM LBN led to increased NO production, whereas co-exposure to L-NAME reduced NO production. Treatment with 100 µM LBN decreased the TEER of the HTMC monolayer. LBN exposure heightened carboxyfluorescein permeability, but co-exposure to 100 µM LBN and L-NAME reduced permeability. LAT treatment did not affect NO production or permeability.Conclusions: LBN increased the permeability of the HTMC monolayer and increased NO production. Therefore, LBN might increase trabecular outflow in addition to promoting uveoscleral outflow.

Primary Role of the Kidney in Pathogenesis of Hypertension.

Previous transplantation studies and the concept of 'nephron underdosing' support the idea that the kidney plays a crucial role in the development of essential hypertension. This suggests that there are genetic factors in the kidney that can either elevate or decrease blood pressure. The kidney normally maintains arterial pressure within a narrow range by employing the mechanism of pressure-natriuresis. Hypertension is induced when the pressure-natriuresis mechanism fails due to both subtle and overt kidney abnormalities. The inheritance of hypertension is believed to be polygenic, and essential hypertension may result from a combination of genetic variants that code for renal tubular sodium transporters or proteins involved in regulatory pathways. The renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS) are the major regulators of renal sodium reabsorption. Hyperactivity of either the RAAS or SNS leads to a rightward shift in the pressure-natriuresis curve. In other words, hypertension is induced when the activity of RAAS and SNS is not suppressed despite increased salt intake. Sodium overload, caused by increased intake and/or reduced renal excretion, not only leads to an expansion of plasma volume but also to an increase in systemic vascular resistance. Endothelial dysfunction is caused by an increased intracellular Na+ concentration, which inhibits endothelial nitric oxide (NO) synthase and reduces NO production. The stiffness of vascular smooth muscle cells is increased by the accumulation of intracellular Na+ and subsequent elevation of cytoplasmic Ca++ concentration. In contrast to the hemodynamic effects of osmotically active Na+, osmotically inactive Na+ stimulates immune cells and produces proinflammatory cytokines, which contribute to hypertension. When this occurs in the gut, the microbiota may become imbalanced, leading to intestinal inflammation and systemic hypertension. In conclusion, the primary cause of hypertension is sodium overload resulting from kidney dysregulation.

The short-chain fatty acid propionate prevents ox-LDL-induced coronary microvascular dysfunction by alleviating endoplasmic reticulum stress in HCMECs.

Coronary microvascular dysfunction (CMD) is a critical pathogenesis of cardiovascular diseases. Lower endothelial nitric oxide synthase (eNOS) phosphorylation leads to reduced endothelium-derived relaxing factor nitric oxide (NO) generation, causing and accelerating CMD. Endoplasmic reticulum stress (ER stress) has been shown to reduce NO production in umbilical vein endothelial cells. Oxidized low-density lipoprotein (ox-LDL) damages endothelial cell function. However, the relationship between ox-LDL and coronary microcirculation has yet to be assessed. Short-chain fatty acid (SCFA), a fermentation product of the gut microbiome, could improve endothelial-dependent vasodilation in human adipose arterioles, but the effect of SCFA on coronary microcirculation is unclear. In this study, we found ox-LDL stimulated expression of ER chaperone GRP78. Further, we activated downstream PERK/eIF2a, IRE1/JNK, and ATF6 signaling pathways, decreasing eNOS phosphorylation and NO production in human cardiac microvascular endothelial. Furthermore, SCFA-propionate can inhibit ox-LDL-induced eNOS phosphorylation reduction and raise NO production; the mechanism is related to the inhibition of ER stress and downstream signaling pathways PERK/eIF2a, IRE1/JNK, and ATF6. In summary, we demonstrate that ox-LDL induced CMD by activating ER stress, propionate can effectively counteract the adverse effects of ox-LDL and protect coronary microcirculation function via inhibiting ER stress.

Anti-inflammatory Effect of Novel 2-Phenylphthalazin-2-ium Bromides on LPS-induced RAW264.7 Cells and their Mechanism

aims: On this foundation, in this work, we further investigate the therapeutic effects of 2-phenylphthalazin-2-ium bromides on the macrophage-mediated LPS-mediated inflammatory response as well as underlining its molecular mechanisms. background: Quaternary benzo[c]phenanthridine alkaloids (QBAs) have extensively been studied in functions for growth promotion effect and the immune function of the body enhancement, so they have been developed as animal feed additive and attracted the interests of pharmacologists for their very low toxicity to mammals. Meanwhile, quaternary benzo[c]phenanthridine alkaloids such as sanguinarine (SA) and chelerythrine (CH) had been proven to have excellent anti-inflammatory activity. Nevertheless, as potential pant-based antitumor drugs, most of QBAs didn't satisfy our demand for their low content in nature. Thus, it is necessary to develop an effective method to synthesize novel chemical entities with similar structure. Our previous study demonstrated that a novel antifungal 2-phenylphthalazin-2-ium scaffold as a simple analogue was designed and characterized, indicating that they exhibited excellent activity. Most of the compounds showed excellent inhibition activity against almost all eight phytopathogenic fungi, far superior to sanguinarine and chelerythrine. objective: Inspired by quaternary benzo[C]phenanthridine alkaloids, novel 2-phenylphthalazin-2-ium bromides were previously designed and synthesized. The anti-inflammatory effect of 2-phenylphthalazin-2-ium bromides were evaluated based on inflammatory cytokines, and their possible mechanism was explored through NF-κB, TLR4 and MAPK signaling pathways. method: The safe dose range of 2-phenylphthalazin-2-ium bromides was tested by using MTT assay. Griess assay was used to determine the changes of nitric oxide (NO) in the cell culture supernatant. qRT-PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS. The secretion level of TNF-α and IL-1ß was detected by ELISA. Western blot was used to detect the protein expression of IL-6, IL-10, TLR4, iNOS, NF-κB, p-P38/P38, p-ERK/ERK and p-JNK/JNK. result: 2-(3,5-Dichlorophenyl)phthalazin-2-ium Bromide (2) with a concentration below 1 μg/mL showed no significant effect on the growth inhibition of RAW264.7 cells, so the concentrations of compound 2 used for experiments were set to 0, 0.25, 0.5 and 1 μg/mL. Compared with the blank control group, the model group showed increased release of NO, transcription levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4 and iNOS (P<0.05), and ratios of p-P38/P38, p-ERK/ERK, p-JNK/JNK (P<0.05). Compared with model group, sample groups displayed decreased NO release and reduced transcriptional levels of IL-6, IL-1ß, IL-10, TNF-α, TLR4, iNOS and reducing protein expression ratios of IL-6, IL-1ß, IL-10, TNF-α, NF-κB, TLR4, iNOS, p-P38/P38, p-ERK/ERK and p-JNK/JNK (P<0.05). conclusion: This study showed that 2-phenylphthalazin-2-ium bromides partially protected macrophage from LPS-induced inflammatory response via suppressing the TLR4-NF-κB/MAPK and reducing NO production. other: No

High-fat stimulation induces atrial neural remodeling by reducing NO production via the CRIF1/eNOS/P21 axi

BackgroundAutonomic remodeling of the atria plays a pivotal role in the development of atrial fibrillation (AF) and exerts a substantial influence on the progression of this condition. Hyperlipidemia is a predisposing factor for AF, but its effect on atrial nerve remodeling is unclear. The primary goal of this study was to explore the possible mechanisms through which the consumption of a high-fat diet (HFD) induces remodeling of atrial nerves, and to identify novel targets for clinical intervention.MethodsCell models were created in vitro by subjecting cells to palmitic acid (PA), while rat models were established by feeding them a high-fat diet. To investigate the interplay between cardiomyocytes and nerve cells in a co-culture system, we utilized Transwell cell culture plates featuring a pore size of 0.4 μm. The CCK-8 assay was employed to determine cell viability, fluorescent probe DCFH-DA and flow cytometry were utilized for measuring ROS levels, JC-1 was used to assess the mitochondrial membrane potential, the Griess method was employed to measure the nitric oxide (NO) level in the supernatant, a fluorescence-based method was used to measure ATP levels, and MitoTracker was utilized for assessing mitochondrial morphology. The expression of pertinent proteins was evaluated using western blotting (WB) and immunohistochemistry techniques. SNAP was used to treat nerve cells in order to replicate a high-NO atmosphere, and the level of nitroso was assessed using the iodoTMT reagent labeling method.ResultsThe study found that cardiomyocytes’ mitochondrial morphology and function were impaired under high-fat stimulation, affecting nitric oxide (NO) production through the CRIF1/SIRT1/eNOS axis. In a coculture model, overexpression of eNOS in cardiomyocytes increased NO expression. Moreover, the increased Keap1 nitrosylation within neuronal cells facilitated the entry of Nrf2 into the nucleus, resulting in an augmentation of P21 transcription and a suppression of proliferation. Atrial neural remodeling occurred in the HFD rat model and was ameliorated by increasing myocardial tissue eNOS protein expression with trimetazidine (TMZ).ConclusionsNeural remodeling is triggered by high-fat stimulation, which decreases the production of NO through the CRIF1/eNOS/P21 axis. Additionally, TMZ prevents neural remodeling and reduces the occurrence of AF by enhancing eNOS expression.

The Effects of Thymosin Alpha-1 on Macrophages: A Cytological and Anti-Inflammatory Study

Thymosin alpha 1 (Tα-1) is a naturally occurring substance synthesized by the thymus tissue, known to activate various immune system cells. It has been reported to increase the production of Natural Killer cells, CD4 and CD8 cells, and cytokines such as IL-2, IL-3, and IFNγ. Furthermore, it plays a vital role in regulating immunity, inflammation, and tolerance. Its effect on the immune system is exerted via its modulatory action on innate immune system cells, thereby functioning as an endogenous regulator for both inflammatory and adaptive immune systems. In this study, we sought to evaluate the cytological and anti-inflammatory effects of Tα-1 on RAW 264.7 cells. The cytological effects of Tα-1 were assessed using the MTT assay, with the IC50 value against RAW 264.7 cells determined to be 368.105 ug/ml. The morphological observations made at various concentrations of Tα-1 showed increased cytotoxicity and decreased cell density of RAW 264.7 cells with increasing test concentration. Furthermore, the anti-inflammatory effects of Tα-1 were evaluated by analyzing the nitric oxide (NO) production in RAW 264.7 cells. Treatment with the test items at concentrations ranging from 7.813 to 31.25 ug/ml showed a dose-dependent reduction in NO production compared with the control group. These findings suggest that Tα-1 may have the potential as an anti-inflammatory agent in the treatment of various diseases associated with inflammation.

Protection of Pacific white shrimp (Penaeus vannamei) against white spot syndrome virus infection by nitric oxide-generating compound S-nitrosoglutathione

Nitric oxide (NO), a highly reactive free radical generated endogenously by nitric oxide synthase (NOS), plays crucial roles in immune defense against a broad range of pathogens including viruses, bacteria, fungi, and protozoans. However, the regulation and effect of NO during viral infection in invertebrates remain largely unexplored. White spot syndrome virus (WSSV) is one of the most severe pathogens affecting the global shrimp farming industry. Our study shows that NO production in Pacific white shrimp Penaeus vannamei decreases during WSSV infection. Reducing NO production using the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly promotes WSSV replication in primary culture of hemocytes isolated from P. vannamei. This effect is partically reversed by L-arginine, a native substrate for NOS, indicating that L-NAME's immunity-reducing action is NO-dependent. In vitro studies also reveal inhibition of WSSV replication in the presence of three NO donors: S-nitrosoglutathione (GSNO), S-nitro-N-acetylpenicillamine (SNAP), and diethylenetriamine nitric oxide adduct (DETA/NO). The involvement of NO in anti-WSSV protection is further demonstrated by an in vivo study where dietary supplementation with GSNO at non-toxic doses increases NO levels and reduces viral load and mortality in WSSV-challenged shrimp. Our results contribute to understanding the antiviral activity of NO in invertebrates and provide a theoretical basis for developing NO-based therapeutic strategies to control WSSV infection.

Taraxacum coreanum Nakai extract attenuates lipopolysaccharide-induced inflammatory responses and intestinal barrier dysfunction in Caco-2 cells

Ethnopharmacological relevanceTaraxacum coreanum Nakai (TC) is a dandelion native to Korea that has long been used as a medicinal herb with antioxidant and anti-inflammatory properties. Intestinal inflammation is closely associated with intestinal epithelial barrier disruption, which leads to the progression of various intestinal diseases. Aim of the studyThe aim of this study was to investigate the protective effects of TC extract on inflammatory responses and intestinal barrier dysfunction in lipopolysaccharide (LPS)-stimulated Caco-2 cells. Materials and methodsThe inhibitory effect of TC on nitric oxide (NO) and pro-inflammatory cytokines production were determined by Griess reagent and enzyme-linked immunosorbent assay, respectively. The epithelial permeability was evaluated by transepithelial electrical resistance (TEER) assay, and inflammation- and tight junction (TJ)-related protein expression were analyzed by Western blotting. In addition, the presence of ten active compounds was identified and quantified using UHPLC-ESI-MS and HPLC-DAD analyses. ResultsTreatment with TC significantly reduced NO production and pro-inflammatory cytokines production [interleukin (IL)-6 and tumor necrosis factor (TNF)-α] compared to the group treated with LPS only, particularly at 100 μg/mL. TC significantly decreased monolayer permeability as detected by TEER. In addition, the transmission of fluorescein isothiocyanate-dextran 4 across the barrier was decreased after treatment with TC. Inflammation-related proteins (inducible NO synthase, cyclooxygenase-2, TNF-α, IL-6, and IL-1β) were down-regulated after treatment with TC. In contrast, TC significantly increased the protein levels of the TJ-related protein, claudin-5. Ten phytochemicals (protocatechuic acid, chlorogenic acid, caffeic acid, scopoletin, chicoric acid, hyperoside, nicotiflorin, luteoloside, sophoricoside, and luteolin) were identified by UHPLC-ESI-MS and HPLC-DAD analysis. ConclusionOur findings suggest that ethanolic extract of TC could attenuate the LPS-induced intestinal barrier dysfunction by increasing the TJ protein and suppressing inflammatory responses.

Heme Oxygenase 1-Mediated Anti-Inflammatory Effect of Extract from the Aerial Part of Heracleum moellendorffii Hance.

In this study, the anti-inflammatory effects of a methanolic extract from the aerial part of Heracleum moellendorffii Hance (HmAPE) and its underlying mechanisms were investigated. HmAPE demonstrated a significant reduction in nitric oxide production in lipopolysaccharide (LPS)-treated murine macrophage RAW264.7 cells, and HmAPE decreased the protein and mRNA expression of inducible nitric oxide synthase. Further mechanistic studies on inflammatory signaling pathways revealed that HmAPE-mediated downregulation of inflammatory gene expressions was not associated with mitogen-activated protein kinases or nuclear factor-κB signaling pathways. However, HmAPE treatment activated nuclear factor E2-related factor 2 (Nrf2) and upregulated heme oxygenase-1 (HO-1) expression, which is known to suppress pro-inflammatory cytokine production. Additionally, treatment with a selective HO-1 inhibitor, tin protoporphyrin IX, partially reversed the effects of HmAPE in LPS-treated RAW264.7 cells, indicating that HmAPE inhibited LPS-induced NO production, at least in part, through induction of Nrf2-mediated HO-1 expression. These findings suggest that HmAPE could serve as a potential edible source with anti-inflammatory properties, and further studies are required to ascertain its anti-inflammatory efficacy in vivo.

NAD+-boosting compounds enhance nitric oxide production and prevent oxidative stress in endothelial cells exposed to plasma from patients with COVID-19

SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), induces vascular endothelial dysfunction, but the mechanisms are unknown. We tested the hypothesis that the “circulating milieu” (plasma) of patients with COVID-19 would cause endothelial cell dysfunction (characterized by lower nitric oxide (NO) production), which would be linked to greater reactive oxygen species (ROS) bioactivity and depletion of the critical metabolic co-substrate, nicotinamide adenine dinucleotide (NAD+). We also investigated if treatment with NAD+-boosting compounds would prevent COVID-19-induced reductions in endothelial cell NO bioavailability and oxidative stress. Human aortic endothelial cells (HAECs) were exposed to plasma from men and women (age 18–85 years) who were hospitalized and tested positive (n = 34; 20 M) or negative (n = 13; 10 M) for COVID-19. HAECs exposed to plasma from patients with COVID-19 also were co-incubated with NAD+ precursors nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN). Acetylcholine-stimulated NO production was 27% lower and ROS bioactivity was 54% higher in HAECs exposed to plasma from patients with COVID-19 (both p < 0.001 vs. control); these responses were independent of age and sex. NAD+ concentrations were 30% lower in HAECs exposed to plasma from patients with COVID-19 (p = 0.001 vs. control). Co-incubation with NR abolished COVID-19-induced reductions in NO production and oxidative stress (both p > 0.05 vs. control). Co-treatment with NMN produced similar results. Our findings suggest the circulating milieu of patients with COVID-19 promotes endothelial cell dysfunction, characterized by lower NO bioavailability, greater ROS bioactivity, and NAD+ depletion. Supplementation with NAD+ precursors may exert a protective effect against COVID-19-evoked endothelial cell dysfunction and oxidative stress.

Investigation of the Effects of Opioids on Microglial Nitrite and Nitric Oxide Synthase (iNOS) Production and Phagocytosis during Inflammation.

This study aimed to investigate how opioids affect phagocytosis and microglial nitrite and nitric oxide synthase (iNOS) production during inflammation. Opioids are a group of chemicals that are naturally found in the opium poppy plant and exert a variety of effects on the brain, including pain alleviation in some cases. They are commonly used in surgery and perioperative analgesia. However, research on the impact of opioids on microglial inflammatory factor production and phagocytosis is limited. This study was designed to investigate the effects of opioids on inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) generation. Moreover, the influence of opioids on the engulfment of C8-B4 microglial cells after stimulation with LPS was also examined. C8-B4 mouse microglial cells were exposed to various concentrations of opioids after stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Nitrite production was assayed. The iNOS and Cox-2 were determined by Western blotting, and fluorescent immunostaining was performed to assess the percentage of microglia that engulfed fluorescent microspheres in total microglia cultivating with opioids after being activated by LPS. After LPS and IFN-γ stimulation, microglia produced lower amounts of nitric oxide (NO) production with buprenorphine, salvinorin A, and naloxone (P<0.05). When combined with naloxone, no significant differences were found than buprenorphine. It was observed that buprenorphine and salvinorin A could suppress iNOS expression activated by LPS and IFN-γ. Phagocytosis was greatly increased after LPS stimulation, and a significant increase was observed after adding salvinorin A. Buprenorphine and salvinorin A were found to reduce NO production and iNOS induction in microglial cells activated by LPS and IFN-γ. Salvinorin A promoted the phagocytosis of microglia cells treated by LPS.

TAK1 Deficiency in Macrophages Increases Host Susceptibility to Leishmania Infection

Abstract Leishmania parasites mainly infect macrophages and may cause severe immunopathologies in their host, which are called leishmaniases. In the current work, we infected human and mouse macrophages in vitro with Leishmania major, an etiological agent of cutaneous leishmaniasis, and found that inhibition or deletion of the transforming growth factor β–activated kinase 1 (TAK1) gene resulted in increased parasite loads. In vivo, following a challenge with L. major, mice with a macrophage-specific deletion of TAK1 showed increased clinical signs and higher parasite loads compared with wild-type controls. TAK1 deficiency in mouse macrophages led to biased Th2 cell responses during the acute stage of infection, characterized by a decrease in interferon-γ expression, and increased expression of IL-4, IL-5 and IL-10. Finally, we found that, in the late stage of L. major infection, excessive Th2-related cytokines led to high arginase 1 expression in mouse tissues and a significant reduction of NO production both locally and systemically, resulting in compromised control of Leishmania. These findings suggest that TAK1 plays a vital role in host resistance to Leishmania infection.

Exercise and vascular function in sedentary lifestyles in humans.

People with sedentary lifestyles engage in minimal or no physical activity. A sedentary lifestyle promotes dysregulation of cellular redox balance, diminishes mitochondrial function, and increases NADPH oxidase activity. These changes collectively increase cellular oxidative stress, which alters endothelial function by oxidizing LDL-C, reducing NO production, and causing eNOS uncoupling. Reduced levels of nitric oxide (NO) leads to vasoconstriction, vascular remodeling, and vascular inflammation. Exercise modulates reactive oxygen species (ROS) to modify NRF2-KEAP signaling, leading to the activation of NRF2 to alleviate oxidative stress. While regular moderate exercise activates NRF2 through ROS production, high-intensity intermittent exercise stimulates NRF2 activation to a greater degree by reducing KEAP levels, which can be more beneficial for sedentary individuals. We review the damaging effects of a sedentary lifestyle on the vascular system and the health benefits of regular and intermittent exercise.