Nox Activity Research Articles

Deletion of NADPH oxidase 2 in chondrocytes exacerbates ethanol-mediated growth plate disruption in mice without major effects on bone architecture or gene expression.

Excess reactive oxygen species generated by NADPH oxidase 2 (Nox2) in response to ethanol exposure mediate aspects of skeletal toxicity including increased osteoclast differentiation and activity. Because perturbation of chondrocyte differentiation in the growth plate by ethanol could be prevented by dietary antioxidants, we hypothesized that Nox2 in the growth plate was involved in ethanol-associated reductions in longitudinal bone growth. Nox2 conditional knockout mice were generated, where the essential catalytic subunit of Nox2, cytochrome B-245 beta chain (Cybb), is deleted in chondrocytes using a Cre-Lox model with Cre expressed from the collagen 2a1 promoter (Col2a1-Cre). Wild-type and Cre-Lox mice were fed an ethanol Lieber-DeCarli-based diet or pair-fed a control diet for 8 weeks. Ethanol treatment significantly reduced the number of proliferating chondrocytes in the growth plate, enhanced bone marrow adiposity, shortened femurs, reduced body length, reduced cortical bone volume, and decreased mRNA levels of a number of osteoblast and chondrocyte genes. Conditional knockout of Nox2 enzymatic activity in chondrocytes did not consistently prevent any ethanol effects. Rather, knockout mice had fewer proliferating chondrocytes than wild-type mice in both the ethanol- and control-fed animals. Additional analysis of tibia samples from Nox4 knockout mice showed that loss of Nox4 activity also reduced the number of proliferating chondrocytes and altered chondrocyte size in the growth plate. Although Nox enzymatic activity regulates growth plate development, ethanol-associated disruption of the growth plate morphology is independent of ethanol-mediated increases in Nox2 activity.

Neuronal NADPH oxidase is required for neurite regeneration of Aplysia bag cell neurons.

NADPH oxidase (Nox), a major source of reactive oxygen species (ROS), is involved in neurodegeneration after injury and disease. Nox is expressed in both neuronal and non-neuronal cells and contributes to an elevated ROS level after injury. Contrary to the well-known damaging effect of Nox-derived ROS in neurodegeneration, recently a physiological role of Nox in nervous system development including neurogenesis, neuronal polarity, and axonal growth has been revealed. Here, we tested a role for neuronal Nox in neurite regeneration following mechanical transection in cultured Aplysia bag cell neurons. Using a novel hydrogen peroxide (H2 O2 )-sensing dye, 5'-(p-borophenyl)-2'-pyridylthiazole pinacol ester (BPPT), we found that H2 O2 levels are elevated in regenerating growth cones following injury. Redistribution of Nox2 and p40phox in the growth cone central domain suggests Nox2 activation after injury. Inhibiting Nox with the pan-Nox inhibitor celastrol reduced neurite regeneration rate. Pharmacological inhibition of Nox is correlated with reduced activation of Src2 tyrosine kinase and F-actin content in the growth cone. Taken together, these findings suggest that Nox-derived ROS regulate neurite regeneration following injury through Src2-mediated regulation of actin organization in Aplysia growth cones.

Formyl-peptide receptor 2 signalling triggers aerobic metabolism of glucose through Nox2-dependent modulation of pyruvate dehydrogenase activity.

The human formyl-peptide receptor 2 (FPR2) is activated by an array of ligands. By phospho-proteomic analysis we proved that FPR2 stimulation induces redox-regulated phosphorylation of many proteins involved in cellular metabolic processes. In this study, we investigated metabolic pathways activated in FPR2-stimulated CaLu-6 cells. The results showed an increased concentration of metabolites involved in glucose metabolism, and an enhanced uptake of glucose mediated by GLUT4, the insulin-regulated member of GLUT family. Accordingly, we observed that FPR2 transactivated IGF-IRβ/IRβ through a molecular mechanism that requires Nox2 activity. Since cancer cells support their metabolism via glycolysis, we analysed glucose oxidation and proved that FPR2 signalling promoted kinase activity of the bifunctional enzyme PFKFB2 through FGFR1/FRS2- and Akt-dependent phosphorylation. Furthermore, FPR2 stimulation induced IGF-IRβ/IRβ-, PI3K/Akt- and Nox-dependent inhibition of pyruvate dehydrogenase activity, thus preventing the entry of pyruvate in the tricarboxylic acid cycle. Consequently, we observed an enhanced FGFR-dependent lactate dehydrogenase (LDH) activity and lactate production in FPR2-stimulated cells. As LDH expression is transcriptionally regulated by c-Myc and HIF-1, we demonstrated that FPR2 signalling promoted c-Myc phosphorylation and Nox-dependent HIF-1α stabilization. These results strongly indicate that FPR2-dependent signalling can be explored as a new therapeutic target in treatment of human cancers.

Stachydrine Hydrochloride Regulates the NOX2-ROS-Signaling Axis in Pressure-Overload-Induced Heart Failure.

Our previous studies revealed the protection of stachydrine hydrochloride (STA) against cardiopathological remodeling. One of the underlying mechanisms involves the calcium/calmodulin-dependent protein kinase Ⅱ (CaMKII). However, the way STA influences CaMKII needs to be further investigated. The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-coupled reactive oxygen species (ROS) overproduction putatively induces the oxidative activation of CaMKII, resulting in the occurrence of pathological cardiac remodeling and dysfunction in experimental models of mice. Thus, in this study, we assessed the role of the NOX2-ROS signal axis in STA cardioprotection. The transverse aortic constriction (TAC)-induced heart failure model of mice, the phenylephrine-induced hypertrophic model of neonatal rat primary cardiomyocytes, and the H2O2-induced oxidative stress models of adult mouse primary cardiomyocytes and H9c2 cells were employed. The echocardiography and histological staining were applied to assess the cardiac effect of STA (6 mg/kg/d or 12 mg/kg/d), which was given by gavage. NOX2, ROS, and excitation-contraction (EC) coupling were detected by Western blotting, immunofluorescence, and calcium transient-contraction synchronous recordings. ROS and ROS-dependent cardiac fibrosis were alleviated in STA-treated TAC mice, demonstrating improved left ventricular ejection fraction and hypertrophy. In the heart failure model of mice and the hypertrophic model of cardiomyocytes, STA depressed NOX2 protein expression and activation, as shown by inhibited translocation of its phosphorylation, p67phox and p47phox, from the cytoplasm to the cell membrane. Furthermore, in cardiomyocytes under oxidative stress, STA suppressed NOX2-related cytosolic Ca2+ overload, enhanced cell contractility, and decreased Ca2+-dependent regulatory protein expression, including CaMKⅡ and Ryanodine receptor calcium release channels. Cardioprotection of STA against pressure overload-induced pathological cardiac remodeling correlates with the NOX2-coupled ROS signaling cascade.

Neuroprotective potential of formononetin, a naturally occurring isoflavone phytoestrogen.

The increased prevalence of neurological illnesses is a burgeoning challenge to the public healthcare system and presents greater financial pressure. Formononetin, an O-methylated isoflavone, has gained a lot of attention due to its neuroprotective potential explored in several investigations. Formononetin is widely found in legumes and several types of clovers including Trifolium pratense L., Astragalus membranaceus, Sophora tomentosa, etc. Formononetin modulates various endogenous mediators to confer neuroprotection. It prevents RAGE activation that results in the inhibition of neuronal damage via downregulating the level of ROS and proinflammatory cytokines. Furthermore, formononetin also increases the expression of ADAM-10, which affects the pathology of neurodegenerative disease by lowering tau phosphorylation, maintaining synaptic plasticity, and boosting hippocampus neurogenesis. Besides these, formononetin also increases the expression of antioxidants, Nrf-2, PI3K, ApoJ, and LRP1. Whereas, reduces the expression of p65-NF-κB and proinflammatory cytokines. It also inhibits the deposition of Aβ and MAO-B activity. An inhibition of Aβ/RAGE-induced activation of MAPK and NOX governs the protection elicited by formononetin against inflammatory and oxidative stress-induced neuronal damage. Besides this, PI3K/Akt and ER-α-mediated activation of ADAM10, ApoJ/LRP1-mediated clearance of Aβ, and MAO-B inhibition-mediated preservation of dopaminergic neurons integrity are the major modulations produced by formononetin. This review covers the biosynthesis of formononetin and key molecular pathways modulated by formononetin to confer neuroprotection.

NADPH oxidase 4 inhibition is a complementary therapeutic strategy for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neurodegenerative disorder, characterized by motor neuron (MN) degeneration and severe muscular atrophy and caused by Survival of Motor Neuron (SMN) depletion. Therapies aimed at increasing SMN in patients have proven their efficiency in alleviating SMA symptoms but not for all patients. Thus, combinational therapies are warranted. Here, we investigated the involvement of NADPH oxidase 4 (NOX4) in SMA-induced spinal MN death and if the modulation of Nox4 activity could be beneficial for SMA patients. We analysed in the spinal cord of severe type SMA-like mice before and at the disease onset, the level of oxidative stress and Nox4 expression. Then, we tested the effect of Nox4 inhibition by GKT137831/Setanaxib, a drug presently in clinical development, by intrathecal injection on MN survival and motor behaviour. Finally, we tested if GKT137831/Setanaxib could act synergistically with FDA-validated SMN-upregulating treatment (nusinersen). We show that NOX4 is overexpressed in SMA and its inhibition by GKT137831/Setanaxib protected spinal MN from SMA-induced degeneration. These improvements were associated with a significant increase in lifespan and motor behaviour of the mice. At the molecular level, GKT137831 activated the pro-survival AKT/CREB signaling pathway, leading to an increase in SMN expression in SMA MNs. Most importantly, we found that the per os administration of GKT137831 acted synergistically with a FDA-validated SMN-upregulating treatment. The pharmacological inhibition of NOX4 by GKT137831/Setanaxib is neuroprotector and could represent a complementary therapeutic strategy to fight against SMA.

Dysregulated anti-oxidant signalling and compromised mitochondrial integrity negatively influence regulatory T cell function and viability in liver disease

Dysregulated inflammatory responses and oxidative stress are key pathogenic drivers of chronic inflammatory diseases such as liver cirrhosis (LC). Regulatory T cells (Tregs) are essential to prevent excessive immune activation and maintain tissue homeostasis. While inflammatory cues are well known to modulate the function and stability of Tregs, the extent to which Tregs are influenced by oxidative stress has not been fully explored. The phenotypic and functional properties of CD4+CD25+CD127lo/- Tregs isolated from patients with LC were compared to healthy controls (HC). Treg redox state was investigated by characterizing intracellular reactive oxygen species (ROS), NADPH oxidase-2 (Nox2) activity, mitochondrial function, morphology, and nuclear factor-erythroid 2-related factor (Nrf2) antioxidant signalling. The relevance of Nrf2 and its downstream target, Heme-oxygenase-1 (HO-1), in Treg function, stability, and survival, was further assessed using mouse models and CRISPR/Cas9-mediated HO-1 knock-out. Circulating Tregs from LC patients displayed a reduced suppressive function, correlating with liver disease severity, associated with phenotypic abnormalities and increased apoptosis. Mechanistically, this was linked to a dysregulated Nrf2 signalling with resultant lower levels of HO-1, enhanced Nox2 activation, and impaired mitochondrial respiration and integrity. The functional deficit in LC Tregs could be partially recapitulated by culturing control Tregs in patient sera. Our findings reveal that Tregs rely on functional redox homeostasis for their function, stability, and survival. Targeting Treg specific anti-oxidant pathways may have therapeutic potential to reverse the Treg impairment in conditions of oxidative damage such as advanced liver disease. This study was funded by the Wellcome Trust (211113/A/18/Z).

Apocynin, an NADPH Oxidase Enzyme Inhibitor, Prevents Amebic Liver Abscess in Hamster.

Amebiasis is an intestinal infection caused by Entamoeba histolytica. Amebic liver abscess (ALA) is the most common extraintestinal complication of amebiasis. In animal models of ALA, neutrophils have been shown to be the first cells to come into contact with Entamoeba histolytica during the initial phase of ALA. One of the multiple mechanisms by which neutrophils exhibit amebicidal activity is through reactive oxygen species (ROS) and the enzyme NADPH oxidase (NOX2), which generates and transports electrons to subsequently reduce molecular oxygen into superoxide anion. Previous reports have shown that ROS release in the susceptible animal species (hamster) is mainly stimulated by the pathogen, in turn provoking such an exacerbated inflammatory reaction that it is unable to be controlled and results in the death of the animal model. Apocynin is a natural inhibitor of NADPH oxidase. No information is available on the role of NOX in the evolution of ALA in the hamster, a susceptible model. Our study showed that administration of a selective NADPH oxidase 2 (NOX2) enzyme inhibitor significantly decreases the percentage of ALA, the size of inflammatory foci, the number of neutrophils, and NOX activity indicated by the reduction in superoxide anion (O2-) production. Moreover, in vitro, the apocynin damages amoebae. Our results showed that apocynin administration induces a decrease in the activity of NOX that could favor a decrease in ALA progression.

Promoting effect of N2 calcination on FeCeOx for selective catalytic reduction of NOx with NH3

AbstractBACKGROUNDSelective catalytic reduction of NOx with NH3 (NH3‐SCR) technology is effective for NOx removal. Acidity and redox ability are two significant characteristics for NH3‐SCR catalysts. This study investigated the effects of N2 calcination on the NH3‐SCR performance of FeCeOx, inspired by the promotion of active oxygen species with inert atmosphere calcination. The structure‐activity relationship and reaction mechanism of the two catalysts (Fe9CeOx‐N2 and Fe9CeOx‐air) were concluded based on the results of characterization.RESULTSFe9CeOx‐N2 showed better NOx conversion and SO2 resistance than Fe9CeOx‐air, especially in low and medium temperature range. For fresh catalysts, a higher amount of Oα, Fe3+ and Ce3+ promoted the redox ability of Fe9CeOx‐N2, and further improved the NOx adsorption and activation. Additionally, the greater number of acid sites of Fe9CeOx‐N2 was another reason for its competitive performance. For SO2‐poisoned catalysts, the primary reaction pathway was the reaction between pre‐adsorbed NH3 species and NOx. The higher amount of acidity from metal sulfate contributed to its better SO2 tolerance of Fe9CeOx‐N2.CONCLUSIONHigher redox ability and acidity led to better NH3‐SCR activity and SO2 tolerance of Fe9CeOx‐N2. This study provides a feasible preparation method for enhancing the NOx conversion and SO2 tolerance of NH3‐SCR catalysts. © 2023 Society of Chemical Industry.

NOX2 deficiency exacerbates diet-induced obesity and impairs molecular training adaptations in skeletal muscle

The production of reactive oxygen species (ROS) by NADPH oxidase (NOX) 2 has been linked to both insulin resistance and exercise training adaptations in skeletal muscle. This study explores the previously unexamined role of NOX2 in the interplay between diet-induced insulin resistance and exercise training (ET). Using a mouse model that harbors a point mutation in the essential NOX2 regulatory subunit, p47phox (Ncf1*), we investigated the impact of this mutation on various metabolic adaptations. Wild-type (WT) and Ncf1* mice were assigned to three groups: chow diet, 60% energy fat diet (HFD), and HFD with access to running wheels (HFD + E). After a 16-week intervention, a comprehensive phenotypic assessment was performed, including body composition, glucose tolerance, energy intake, muscle insulin signaling, redox-related proteins, and mitochondrial adaptations. The results revealed that NOX2 deficiency exacerbated the impact of HFD on body weight, body composition, and glucose intolerance. Moreover, in Ncf1* mice, ET did not improve glucose tolerance or increase muscle cross-sectional area. ET normalized body fat independently of genotype. The lack of NOX2 activity during ET reduced several metabolic adaptations in skeletal muscle, including insulin signaling and expression of Hexokinase II and oxidative phosphorylation complexes. In conclusion, these findings suggest that NOX2 mediates key beneficial effects of exercise training in the context of diet-induced obesity.

NADPH oxidase 1: A target in the capacity of dimeric ECG and EGCG procyanidins to inhibit colorectal cancer cell invasion

Colorectal cancer (CRC) is prevalent worldwide. Dietary consumption of procyanidins has been linked to a reduced risk of developing CRC. The epidermal growth factor (EGF) receptor (EGFR) signaling pathway is frequently dysregulated in CRC. Our earlier research showed that the procyanidin dimers of epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), through their interaction with lipid rafts, inhibit the EGFR signaling pathway and decrease CRC cell growth. The process of cancer cell invasion and metastasis involves matrix metalloproteinases (MMPs), which are partially EGFR-regulated. This study investigated whether ECG and EGCG dimers can inhibit EGF-induced CRC cell invasion by suppressing the redox-regulated activation of the EGFR/MMPs pathway. Both dimers mitigated EGF-induced cell invasion and the associated increase of MMP-2/9 expression and activity in different CRC cell lines. In Caco-2 cells, both dimers inhibited the activation of the EGFR and downstream of NF-κB, ERK1/2 and Akt, which was associated with decreased MMP-2/9 transcription. EGF induced a rapid NOX1-dependent oxidant increase, which was diminished by both ECG and EGCG dimers and NOX inhibitors (apocynin, Vas-2870, DPI). Both dimers inhibited NOX1 gene expression, as well as NOX1 activity with evidence of direct binding to NOX1. Both dimers, all NOX chemical inhibitors and NOX1 silencing inhibited EGF-mediated activation of the EGFR signaling pathway and the increased MMP-2/9 mRNA levels and activity. Pointing to the relevance of NOX1 on ECG and EGCG dimer effects on CRC invasiveness, silencing of NOX1 also inhibited EGF-stimulated Caco-2 cell invasion. In summary, ECG and EGCG dimers can act inhibiting CRC cell invasion/metastasis both, by downregulating MMP-2 and MMP-9 expression via a NOX1/EGFR-dependent mechanism, and through a direct inhibitory effect on MMPs enzyme activity.

New NADPH Oxidase 2 Inhibitors Display Potent Activity against Oxidative Stress by Targeting p22phox-p47phox Interactions.

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1.

Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

Mechanisms involved in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)-derived reactive oxygen species (ROS) modulation of muscle function in human and dog bladders.

Roles of redox signaling in bladder function is still under investigation. We explored the physiological role of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) in regulating bladder function in humans and dogs. Mucosa-denuded bladder smooth muscle strips obtained from 7 human organ donors and 4 normal dogs were mounted in muscle baths, and trains of electrical field stimulation (EFS) applied for 20 minutes at 90-second intervals. Subsets of strips were incubated with hydrogen peroxide (H2O2), angiotensin II (Ang II; Nox activator), apocynin (inhibitor of Noxs and ROS scavenger), or ZD7155 (specific inhibitor of angiotensin type 1 (AT1) receptor) for 20 minutes in continued EFS trains. Subsets treated with inhibitors were then treated with H2O2 or Ang II. In human and dog bladders, the ROS, H2O2 (100μM), caused contractions and enhanced EFS-induced contractions. Apocynin (100μM) attenuated EFS-induced strip contractions in both species; subsequent treatment with H2O2 restored strip activity. In human bladders, Ang II (1μM) did not enhance EFS-induced contractions yet caused direct strip contractions. In dog bladders, Ang II enhanced both EFS-induced and direct contractions. Ang II also partially restored EFS-induced contractions attenuated by prior apocynin treatment. In both species, treatment with ZD7155 (10μM) inhibited EFS-induced activity; subsequent treatment with Ang II did not restore strip activity. Collectively, these data provide evidence that ROS can modulate bladder function without exogenous stimuli. Since inflammation is associated with oxidative damage, the effects of Ang II on bladder smooth muscle function may have pathologic implications.

Hierarchical TiO2-g-C3N4 photocatalyst with purification effect for NOx oxidation under cyan light

NOx photocatalytic oxidation is a promising approach for practical NOx abatement at sub-ppb levels and under ambient conditions. However, conventional photocatalysts are characterized by their low NOx adsorption capacity and activity under visible light. This results in poor NO (g) conversion and may form more toxic NO2 (g), hampering the utilization of photocatalysis in NOx removal. Herein, we employed a hierarchical macro-mesoporous structure composed of Z-scheme TiO2-g-C3N4 hybrid embedded in parallel TiO2 channels as a high adsorption capacity and low band gap photocatalyst for NOx oxidation. This unique structure provides aligned macroporous channels and mesoporous walls for efficient NOx adsorption and storage of NOx oxidation product (i.e., NO3-(s)) while enabling visible light absorption. NOx oxidation was conducted in a laminar-flow reactor under 50% relative humidity and simultaneous exposure to blue and green lights (cyan light). TiO2-g-C3N4 showed NO conversion (37%) which is four times higher than that of TiO2 (7%) and two times higher than that of g-C3N4 (17%). In addition, TiO2-g-C3N4 hybrids showed high NOx adsorption capacity and converted NO to nontoxic NO3- with minimal formation of NO2, evident by its positive DeNOx index (+74 ppm) in contrast to g-C3N4 with a negative DeNOx (-4 ppm) and TiO2 with a slightly positive DeNOx index (+15 ppm). The study demonstrates the delicate relationship between H2O adsorption on the hybrid structure and NOx photooxidation reaction and provides detailed mechanistic insights on the photocatalytic response. It is expected that the results of this study will benefit ongoing efforts to design visible-light active photocatalysts.

Nicotinamide Antagonizes Lipopolysaccharide-Induced Hypoxic Cell Signals in Human Macrophages.

Mechanisms to control the immune response are important to pathogen evasion and host defense. Gram-negative bacteria are common pathogens that can activate host immune responses through their outer membrane component, LPS. Macrophage activation by LPS induces cell signals that promote hypoxic metabolism, phagocytosis, Ag presentation, and inflammation. Nicotinamide (NAM) is a vitamin B3 derivative and precursor in the formation of NAD, which is a required cofactor in cellular function. In this study, treatment of human monocyte-derived macrophages with NAM promoted posttranslational modifications that antagonized LPS-induced cell signals. Specifically, NAM inhibited AKT and FOXO1 phosphorylation, decreased p65/RelA acetylation, and promoted p65/RelA and hypoxia-inducible transcription factor-1α (HIF-1α) ubiquitination. NAM also increased prolyl hydroxylase domain 2 (PHD2) production, inhibited HIF-1α transcription, and promoted the formation of the proteasome, resulting in reduced HIF-1α stabilization, decreased glycolysis and phagocytosis, and reductions in NOX2 activity and the production of lactate dehydrogenase A. These NAM responses were associated with increased intracellular NAD levels formed through the salvage pathway. NAM and its metabolites may therefore decrease the inflammatory response of macrophages and protect the host against excessive inflammation but potentially increase injury through reduced pathogen clearance. Continued study of NAM cell signals invitro and invivo may provide insight into infection-associated host pathologies and interventions.

LRRC8A anion channels modulate vascular reactivity via association with myosin phosphatase rho interacting protein.

Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.

Zinc(Ⅱ)-induced Neuroimmunological Renal Chronic Kidney Disease Prevention and Suppressive Progression

Zinc(Ⅱ) supplementation with 30 mg/day should become likely therapeutic chronic kidney disease (CKD) prevention implications with immense importance of CKD-associated immunological and neurological complications. Zinc induced neuroimmunological suppressive CKD progression with Stage 2 ～ 3A, 3B, zinc supplementation (30 mg/day) may be beneficial for nutritional status in CKD children and adolescents, and zinc could reduce urinary protein excretion composed of albumin and/or minimize proteinuria excretion in proteinuric CKD. In CKD 4-5 stage, risk of cardiovascular disease (CVD) become higher that zinc supplementation of 25 mg/day in zinc homeostasis has a beneficial effect for CVD in vascular dysfunction. Thus, zinc intake of 30 mg/day and serum zinc level of 60～76 μg/dL could slow down CKD Stage 4 progression. Zinc induced CKD Stage 5 (eGFR < 15) is involved that zinc is actively bound to several proteins that the decrease of circulating zinc levels in CKD is partly caused by renal losses of zinc, becoming zinc-deficiency with advanced stage CKD and ESRD in CKD. Serum zinc and serum albumin levels are 37.4 ± 5.4 and 2.6 ± 0.6 µg/dL, respectively, for as eGFR 6.6 (5.2–8.3). Zn intake of 45-75 mg/day and serum zinc level of ≤78.3 µg/dL are beneficial for HD and maintenance hemodialysis (MHD) patients, in which Zinc 15 mg/day and Selenium 50–70 μg/day that are recommended may be thought to prevent ESRD stage. Zinc induced NAD(P)H oxidase (Nox) activation occurring ROS generation in CKD cell involves assembly that ROS include the superoxide anion (O2•–), hydrogen peroxide (H2O2), and hydroxyl radicals (OH•), and in the kidney. Zinc(Ⅱ) ions-binding protein molecular mechanism is involved that Zn2+ ions-several protein complexes coordinated binding model of ligands of such as albumin, a-macroglobulin and transferrin in several proteins had been found on the binding specificity by Zn2+ ions-centered tetrahedral geometric coordination, in which causing zinc-activated serine, histidine and aspartate hydrogen residues enhance renal function activity and resulting inhibition of CKD progression proceeds.

Synergisticeffect of elevated glucose levels with SARS-CoV-2 spike protein induced NOX-dependent ROS production in endothelial cells.

Diabetic patients infected with coronavirus disease 2019 (COVID-19) often have a higher probability of organ failure and mortality. The potential cellular mechanisms through which blood glucose exacerbates tissue damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still unclear. We cultured endothelial cells within differing glucose mediums with an increasing concentration gradient of SARS-CoV-2 Spike protein (S protein). S protein can cause the reduction of ACE2 and TMPRSS2, and activation of NOX2 and NOX4. A high glucose medium was shown to aggravate the decrease of ACE2 and activation of NOX2 and NOX4 in cultured cells, but had no effect on TMPRSS2. S protein mediated activation of the ACE2-NOX axis induced oxidative stress and apoptosis within endothelial cells, leading to cellular dysfunction via the reduction of NO and tight junction proteins which may collectively be exacerbated by elevated glucose. In addition, the glucose variability model demonstrated activation of the ACE2-NOX axis in a similar manner observed in the high glucose model in vitro. Our present study provides evidence for a mechanism through which hyperglycemia aggravates endothelial cell injury resulting from S protein mediated activation of the ACE2-NOX axis. Our research thus highlights the importance of strict monitoring and control of blood glucose levels within the context of COVID-19 treatment to potentially improve clinical outcomes.

OXYCINNAMIC ACIDS AS NOX4 INHIBITORS IN ALZHEIMER'S DISEASE THERAPY. AN EXPERIMENTAL STUDY

Relevance. Alzheimer's disease is a terminal form of dementia with a complex pathogenesis in which NOX-dependent oxidative stress plays an ex-tremely important role. Aim of the study. To evaluate the influence of oxycinnamic acids on the alteration of NOX4 activity in brain tissue of animals with experimental Alz-heimer's disease. Material and methods. Alzheimer's disease was modeled in Wistar rats by injection of β-amyloid aggregates with amino acid sequence 1-42 into hippocampal tissue (CA1 segment). Oxycinnamic acids: caffeic acid, coumaric acid, ferulic acid and synapic acid were administered at a dose of 100 mg/kg, orally for 60 days after the Alzheimer's disease model. Ethylmethylhydroxypyridine succinate was used as a reference drug in a similar dosage and dosing mode. Changes in the concentration of active NOX4 isophrome, hydrogen peroxide as well as β-amyloid in brain tissue of rats were as-sessed after the indicated time period. Results. This study showed that the analyzed oxycinnamic acids were comparable with each other and the referent