NADPH Oxidase Activity Research Articles

A transient blood IL-17 increase triggers neuroinflammation in cerebellum and motor incoordination in hyperammonemic rats

Patients with liver cirrhosis may show minimal hepatic encephalopathy (MHE) with motor incoordination which is reproduced in hyperammonemic rats. Hyperammonemia induces peripheral inflammation which triggers neuroinflammation and enhanced GABAergic neurotransmission in cerebellum and motor incoordination. The mechanisms involved remain unknown. The aims were to assess if the early increase of peripheral IL-17 triggers motor incoordination in hyperammonemic rats and to identify some underlying mechanisms. We assessed if blocking peripheral IL-17 with anti-IL-17 at 2–4 days of hyperammonemia prevents motor incoordination and analyzed underlying mechanisms. Hyperammonemia induces a transient blood IL-17 increase at days 3–4. This is associated with increased IL-17 receptor membrane expression and activation in cerebellum, leading to NADPH oxidase activation, increased superoxide production and MLCK that induce blood–brain barrier (BBB) permeabilization by reducing occludin and ZO-1. BBB permeabilization facilitates the entry of IL-17, which increases in cerebellum and activates microglia. This increases TNFα and the TNFR1-S1PR2-CCL2-BDNF-TrkB pathway. This enhances GABAergic neurotransmission which impairs motor coordination. Blocking peripheral IL-17 with anti-IL-17 prevents all the above process and prevents motor incoordination. Early treatment to reduce blood IL-17 may be a useful treatment to reverse motor incoordination in patients with MHE.Graphical abstract

Effects of sucrose and 1-MCP on enzymatic and nonenzymatic antioxidants in postharvest Gynura bicolor DC

After harvesting, Gynura bicolor DC (G. bicolor) undergoes rapid quality deterioration, including decay, nutrient loss, and reactive oxygen species (ROS) burst, greatly limiting its shelf life. This study was performed to evaluate the effects of treatment with sucrose and 1-methylcyclopropene (1-MCP) on indices of quality deterioration, ROS metabolism, and phenylpropanoid metabolism, with the goals of resisting oxidative stress and improving the postharvest quality of G. bicolor. Sucrose treatment activated phenylpropanoid metabolism, increased phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and 4-coumarate:CoA ligase (4CL) activities, and promoted the accumulation of phenolics. The increases in nonenzymatic antioxidants decreased O2·−, ·OH, and H2O2 contents. Conversely, 1-MCP treatment increased NADPH oxidase (NOX) activity and induced transient oxidative stress, which significantly activated enzymatic antioxidants, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), thus maintaining a lower ROS level. Treatment with both sucrose and 1-MCP exhibited a synergistic effect on alleviating oxidative stress, downregulating GbSAG101 and GbATG expression. Compared with sucrose treatment, 1-MCP treatment showed a greater inhibitory effect on ROS burst. Phenolic substance contents were similar in plants treated with sucrose and 1-MCP at the end of the storage period. Therefore, we speculated that higher levels of antioxidant enzyme activity may decrease the consumption of nonenzymatic antioxidants. Our experimental results indicated the involvement of different pathways in the effects of sucrose treatment, 1-MCP treatment, and combined treatment on delaying ROS burst, and suggested potential alternative methods for the preservation of vegetables with stems during the postharvest storage.

Effect of elevated ammonium on biotic and abiotic stress defense responses and expression of related genes in cucumber (Cucumis sativus L.) plants

Ammonium (NH4+) enhances plant defense mechanisms but can be phytotoxic as the sole nitrogen source. To investigate the impact of a balanced NH4+ and NO3− ratio on plant defense parameters without adverse effects, cucumber plants (Cucumis sativus L.) were grown under control (14 mM NO3− + 2 mM NH4+) and elevated level of NH4+ (eNH4+, 8 mM NO3−+ 8 mM NH4+). Plants subjected to eNH4+ showed significantly increased shoot and root biomass by about 41% and 47%, respectively. Among the antioxidant enzymes studied, ascorbate peroxidase (EC 1.11.1.11) activity was increased up to 3.3 fold in eNH4+ compared with control plants, which was associated with enhanced resistance to paraquat. Upregulation of PATHOGENESIS RELATED PROTEIN 4 (PR4) and LIPOXYGENASE 1 (LOX1), accompanied by increased concentrations of salicylic acid and nitric oxide, conferred more excellent resistance of eNH4+ plants to powdery mildew infection. However, the expression levels of ACC OXIDASE 1 (ACO1) and RESPIRATORY BURST OXIDASE HOMOLOGS B (RBOHB) were lower in eNH4+ plants, which was consistent with decreased NADPH oxidase activity and lower leaf H2O2 levels. The biosynthesis of phenolics was enhanced, whereas the activities of polymerizing enzymes and lignin deposition were reduced by half in eNH4+ plants. Besides, a significant effect on plant biomass under salt or drought stress has not been observed between control and eNH4+ plants. These results showed that different defense pathways are distinctively affected by eNH4+ treatment, and the NH4+ to NO3− ratio may play a role in fine-tuning the plant defense response.

Angiotensin type-1 receptor autoantibodies promote alpha-synuclein aggregation in dopaminergic neurons.

Angiotensin, through its type-1 receptor (AT1), is a major inducer of inflammation and oxidative stress, contributing to several diseases. Autoimmune processes have been involved in neurodegeneration, including Parkinson's disease (PD). AT1 autoantibodies (AT1-AA) enhance neurodegeneration and PD, which was related to increased neuronal oxidative stress and neuroinflammation. However, the effect of AT1-AA on α-synuclein aggregation, a major factor in PD progression, has not been studied. In cultures of dopaminergic neurons, we observed that AT1-AA promote aggregation of α-synuclein, as AT1-AA upregulated major mechanisms involved in the α-synuclein aggregation process such as NADPH-oxidase activation and intracellular calcium raising. The results further support the role of AT1 receptors in dopaminergic neuron degeneration, and several recent clinical studies observing the neuroprotective effects of AT1 receptor blockers.

Accumulation of advanced oxidation protein products promotes age-related decline of type H vessels in bone.

Type H vessels have been proven to couple angiogenesis and osteogenesis. The decline of type H vessels contributes to bone loss in the aging process. Aging is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, whether AOPP accumulation is involved in age-related decline of type H vessels is unclear. Here, we show that the increase of AOPP levels in plasma and bone were correlated with the decline of type H vessels and loss of bone mass in old mice. Exposure of microvascular endothelial cells to AOPPs significantly inhibited cell proliferation, migration, and tube formation, increased NADPH oxidase activity and excessive reactive oxygen species generation, upregulated the expression of vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1, and eventually impaired angiogenesis, which was alleviated by redox modulator N-acetylcysteine and NADPH oxidase inhibitor apocynin. Furthermore, reduced AOPP accumulation by NAC treatment was able to alleviate significantly the decline of type H vessels, bone mass loss and deterioration of bone microstructure in old mice. Collectively, these findings suggest that AOPPs accumulation contributes to the decline of type H vessels in the aging process, and illuminate a novel potential mechanism underlying age-related bone loss.

β3-adrenergic agonist counters oxidative stress and Na+-K+ pump inhibitory S-glutathionylation of placental cells: Implications for preeclampsia.

Oxidative stress from placental ischemia/reperfusion and hypoxia/reoxygenation (H/R) in preeclampsia is accompanied by Na+-K+ pump inhibition and S-glutathionylation of its β1 subunit (GSS-β1), a modification that inhibits the pump. β3-adrenergic receptor (β3-AR) agonists can reverse GSS-β1. We examined effects of the agonist CL316,243 on GSS-β1 and sources of H/R-induced oxidative stress in immortalized first trimester human trophoblast (HTR-8/SVneo) and freshly isolated placental explants from normal term pregnancies. H/R increased GSS-β1 and, reflecting compromised α1/β1 subunit interaction, it reduced α1/β1 pump subunit co-immunoprecipitation. H/R increased p47phox/p22phox NADPH oxidase subunit co-immunoprecipitation reflecting membrane translocation of cytosolic p47phox that is needed to activate NADPH oxidase. Fluorescence of O2•--sensitive dihydroethidium increased in parallel. H/R increased S-glutathionylation of endothelial nitric oxide synthase (GSS-eNOS) that uncouples NO synthesis towards synthesis of O2•- and reduced trophoblast migration. Oxidative stress induced by tumor necrosis factor α (TNF-α) increased soluble fms-like tyrosine kinase receptor 1 (sFlt-1) trophoblast release, a marker of preeclampsia, and reduced trophoblast integration into endothelial cellular networks. CL316,243 eliminated H/R-induced GSS-β1 and decreases of α1/β1 subunit coimmunoprecipitation, eliminated NADPH oxidase activation and increases in GSS-eNOS, restored trophoblast migration, eliminated increased sFlt-1 release and restored trophoblast integration in endothelial cell networks. H/R induced GSS-β1, α1/β1 subunit co-immunoprecipitation and NADPH oxidase activation of placental explants reflected effects of H/R for trophoblasts and CL316,243 eliminated these changes. We conclude a β3-AR agonist counters key pathophysiological features of preeclampsia in vitro. β3 agonists already in human use for another purpose are potential candidates for re-purposing to treat preeclampsia.

New insights into oxidative damage to yellow catfish (Pelteobagrus fulvidraco) muscle by acute ammonia stress

Ammonia causes stress, inhibits fish growth, and damages tissues and organs. The muscle proliferation contributes to more than 50 % of the growth of fish. This study aimed to investigate the effects of acute ammonia stress on muscle damage, reactive oxygen species (ROS) production and scavenging, and endoplasmic reticulum stress (ERS) in yellow catfish (Pelteobagrus fulvidraco). In this study, yellow catfish were exposed to three concentrations of ammonia (0, 25, and 125 mg/L) for 96 h. The results showed that acute ammonia stress resulted in the deterioration of plasma biochemical parameters, histopathological injury, and oxidative damage in the muscle. In addition, ammonia stress led to the overproduction of muscle ROS, which may be partially related to the activation of the muscle NADPH oxidase (NOX) family. Ammonia stress reduces the ability to scavenge ROS, which may be partly related to the inhibition of the muscle antioxidant system. Ammonia stress leads to ERS, which may be related in part to the activation of muscle-activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1), and protein kinase RNA-activating-like ER kinase (PERK)/eukaryotic translation initiation factor 2 alpha (eIF2α)/transcription factor 4 (ATF4) pathways. In conclusion, the present study provides evidence for the study of muscle toxicity induced by ammonia stress in freshwater fish and provides a reference for the healthy culture of yellow catfish.

CB1 Receptor Activation Provides Neuroprotection in an Animal Model of Glutamate-Induced Excitotoxicity Through a Reduction of NOX-2 Activity and Oxidative Stress.

Excitotoxicity is a process in which NADPH oxidase-2 (NOX-2) plays a pivotal role in the generation of reactive oxygen species (ROS). Oxidative stress influences the expression of Aquaporin 4 (AQP4), a water channel implicated in blood-brain barrier (BBB) permeability and edema formation. The endocannabinoid system is widely distributed in the brain, particularly through the cannabinoid receptor type 1 (CB1) and type 2 (CB2), which have been shown to have a neuroprotective function in brain injury. Given the significant involvement of NOX-2 in ROS production during excitotoxicity, our research aims to assess the participation of NOX-2 in the neuroprotective effect of the cannabinoid receptor agonist WIN55,212-2 against glutamate-induced excitotoxicity damage in the striatum using invivo model. Wild-type mice (C57BL/6) and NOX-2 KO (gp91Cybbtm1Din/J) were stereotactically injected in the striatum with monosodium glutamate or vehicle. Subsequently, a group of mice was administered an intraperitoneal dose of WIN55,212-2, AM251, or AM251/WIN55,212-2 following the intracerebral injection. Motor activity was assessed, and the lesion was examined through histological sections stained with cresyl violet. Additionally, brain water content and Evans blue assay were conducted. The activity of NOX was quantified, and the protein expression of CB1, gp91phox, AQP4, Iba-1, TNF-α, and NF-κB was analyzed using Western blot. Furthermore, ROS formation was measured through the DHE assay. The activation of the endocannabinoid receptors demonstrated a neuroprotective response during excitotoxicity, meditated by NOX-2. The reduction in ROS production led to a decrease in neuroinflammation, and AQP4 expression, resulting in reduced edema formation, and BBB permeability. During excitotoxic damage, WIN55,212-2 inhibits NOX-2-induced ROS production, reducing brain injury.

Formononetin Induces Ferroptosis in Activated Hepatic Stellate Cells to Attenuate Liver Fibrosis by Targeting NADPH Oxidase 4.

Ferroptosis is a newly discovered type of cell death that exerts a crucial role in hepatic fibrosis. Formononetin (FMN), a natural isoflavone compound mainly isolated from Spatholobus suberectus Dunn, shows multiple biological activities, including antioxidant, anti-inflammatory, and hepatoprotection. This research aims to explore the regulatory mechanism of FMN in liver fibrosis and the relationship between NADPH oxidase 4 (NOX4) and ferroptosis. The effects of FMN on HSC ferroptosis were evaluated in rat model of CCl4-induced hepatic fibrosis. In vitro, N-acetyl-L-cysteine (NAC) and deferoxamine (DFO) were used to block ferroptosis and then explored the anti-fibrotic effect of FMN. The target protein of FMN was identified by bio-orthogonal click chemistry reaction as well as drug affinity responsive target stability (DARTS), cellular thermal shift (CETSA), surface plasmon resonance (SPR) assays, and isothermal titration calorimetry (ITC) analysis. Here, we found that FMN exerted anti-fibrotic effects via inducing ferroptosis in activated HSCs. NAC and DFO prevented FMN-induced ferroptotic cell death and collagen reduction. Furthermore, FMN bound directly to NOX4 through possible active amino acid residues sites, and increased NOX4-based NADPH oxidase activity to enhance levels of NADP+/NADPH, thus promoting ferroptosis of activated HSCs and relieving liver fibrosis. These results demonstrate that the direct target and mechanism by which FMN improves liver fibrosis, suggesting that FMN may be a natural candidate for further development of liver fibrosis therapy.

Arginine alleviates LPS-induced leukocytes inflammation and apoptosis via adjusted NODs signaling

Arginine plays a key role in regulating the immune function of fish. To evaluate the effect of arginine on the immune response of largemouth bass (Micropterus salmoides), the effects of arginine on cell viability, NADPH oxidase activity, respiratory burst activity, and NO production of leukocytes were analyzed both in vitro and in vivo. In this study, we found that arginine could promote the respiratory burst activity of leucocytes both in vivo and in vitro. By incubating leukocytes with the combination of LPS and arginine, we found that arginine supplementation inhibited the expression of inflammatory genes (tumor necrosis factor-alpha, tnfα; interleukin(il) 8 and il10) and apoptotic genes (caspase 3, caspase 8, and caspase 9) induced by LPS, as well as promoted the arginine metabolism. Arginine supplementation significantly induced (cd4-like) cd4 gene expression after LPS challenge. Further studies showed that LPS could significantly increase nucleotide-binding oligomerization domain containing 1 (nod1) gene expression, but decreased the nod2 gene. The arginine supplementation increased nuclear factor kappa-B (NF-κB) protein level. In conclusion, arginine can alleviate LPS-induced inflammatory response and apoptosis as well as induce cd4 gene expression against LPS challenge via adjusting the expression of NODs signaling.

UV-B radiation mitigates oxidative stress damage in postharvest Agaricus bisporus by modulating the antioxidant defense system

Agaricus bisporus (A. bisporus) has fragile tissues and is highly susceptible to post-harvest decay and spoilage, which affecting the development of the industry. Ultraviolet B (UV-B) irradiation, as a typical irradiation preservation technology, is effective in inducing the production of endogenous metabolic substances in organisms and enhancing their level of resistance. The objective of this study was to investigate the mechanism of activation of the antioxidant defence system in A. bisporus by UV-B irradiation, utilising a range of UV-B irradiation doses (0, 25, 50 and 100 kJ m−2). In this study we found that 50 kJ m−2 UV-B irradiation effectively delayed the accumulation of reactive oxygen species (ROS), inhibited NADPH oxidase (NOX) activity and the expression of Rbohf, PXMP2, PXMP4, APO, and MPV17. Moreover, it could increase the accumulation of ascorbate (AsA) and glutathione (GSH), enhance the activities of ascorbate peroxidase (APX) and glutathione peroxidase (GSH-PX), and it also effectively induce catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities and up-regulate the expression levels of related genes. In addition, we found that UV-B irradiation upregulated the expression of UVR8 and suppressed the expression of PEX5, PEX11 and PMD1. These results suggest that 50 kJ m−2 UV-B irradiation could stimulate the UV Resistance Loucs 8 (UVR8) receptor, regulate peroxisome proliferation, and enhance the ability of A. bisporus to resist oxidative stress, thereby maintaining the cellular redox homeostasis, this provides a new strategy for the study of extended postharvest storage stability of A. bisporus.

Deciphering the mechanisms underlying the neuroprotective potential of kaempferol: a comprehensive investigation.

Neurodegenerative disorders are characterized by neuronal degradation, dysfunction, or death within the CNS. Oxidative and inflammatory stress play crucial roles in the pathogenesis of various neurodegenerative diseases. The interplay between these stressors and dysregulated cellular signaling pathways contributes to neurodegeneration. Downregulation of NRF-2 compromises antioxidant defense, exacerbating neuronal damage, while increased TLR-4/MAPK and TLR-4/NF-κB signaling promotes neuroinflammation. Excessive ROS production by NADPH oxidase leads to oxidative damage and neuronal apoptosis. The strategies targeting NRF-2, TLR-4-mediated inflammatory stress, and NADPH oxidase activity promise to mitigate neuronal damage and halt the progression of the disease. Kaempferol is a flavonoid polyphenol antioxidant found abundantly in various fruits and vegetables, including apples, grapes, tomatoes, and broccoli. It is widely found in medicinal plants including Equisetum spp., Sophora japonica, Ginkgo biloba, and Euphorbia pekinensis (Rupr.). A substantial body of in vitro and in vivo evidences have demonstrated the neuroprotective potential of kaempferol against neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Kaempferol demonstrates multifaceted potential in mitigating neuroinflammation, apoptosis, and oxidative stress in different neurodegenerative diseases through the modulation of various pathways including NRF-2, NADPH oxidase, TLR-4/MAPK, and TLR-4/NF-κB. This review article was developed through a comprehensive analysis and interpretation of research published between 2009 and 2024, sourced from multiple scientific databases, including PubMed, Scopus, ScienceDirect, and Web of Science. This review aims to provide an in-depth overview of the neuroprotective effects of kaempferol, focusing on its underlying molecular mechanisms. A total of 24 research evidence were included to elucidate the molecular pathways by which kaempferol exerts its protective effects against neurodegenerative diseases.

Endothelial ENaC-α Restrains Oxidative Stress in Lung Capillaries in Murine Pneumococcal Pneumonia-associated Acute Lung Injury.

Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NADPH oxidase 2 (NOX2), involving the pneumococcal virulence factor pneumolysin (PLY). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of the epithelial sodium channel (ENaC) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function -measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans Blue Dye incorporation in mouse lungs- following infection with pneumococci. PLY-induced hyperpermeability in HL-MVEC monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47phox subunit (Western blotting) in vitro and by co-localization of CD31 and gp91phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α KO (enENaC-α KO) mice develop increased capillary leak upon i.t. instillation with PLY or pneumococci, compared to wild type (wt) animals. TIP peptide diminishes capillary leak in Sp-infected wt mice, without significantly increasing lung bacterial load. Lung slices from Sp-infected enENaC-α KO mice have a significantly increased endothelial NOX2 expression, as compared to infected CRE mice. In conclusion, endothelial ENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in ARDS, without impairing host defense.

Plasma Proteomics of Type 2 Diabetes, Hypertension, and Co-Existing Diabetes/Hypertension in Thai Adults.

The study explored proteomics to better understand the relationship between type 2 diabetes (T2DM) and hypertension (HT) in Thai adults, using shotgun proteomics and bioinformatics analysis. Plasma samples were taken from 61 subjects: 14 healthy subjects (mean age = 40.85 ± 7.12), 13 with T2DM (mean age = 57.38 ± 6.03), 16 with HT (mean age = 66.87 ± 10.09), and 18 with coexisting T2DM/HT (mean age = 58.22 ± 10.65). Proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein-protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 11.5. We identified six unique proteins in T2DM patients, including translationally controlled 1 (TPT1) and nibrin (NBN), which are associated with the DNA damage response. In HT patients, seven unique proteins were identified, among them long-chain fatty acid-CoA ligase (ASCL), which functions in the stimulation of triacylglycerol and cholesterol synthesis, and NADPH oxidase activator 1 (NOXA1), which is involved in high blood pressure via angiotensin II-induced reactive oxygen species (ROS)-generating systems. In coexisting T2DM/HT patients, six unique proteins were identified, of which two-microtubule-associated protein 1A (MAP1A)-might be involved in dementia via RhoB-p53 and diacylglycerol kinase beta (DGKB), associated with lipid metabolism. This study identified new candidate proteins that are possibly involved in the pathology of these diseases.

LRRK2 regulates production of reactive oxygen species in cell and animal models of Parkinson's disease.

Oxidative stress has long been implicated in Parkinson's disease (PD) pathogenesis, although the sources and regulation of reactive oxygen species (ROS) production are poorly defined. Pathogenic mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are associated with increased kinase activity and a greater risk of PD. The substrates and downstream consequences of elevated LRRK2 kinase activity are still being elucidated, but overexpression of mutant LRRK2 has been associated with oxidative stress, and antioxidants reportedly mitigate LRRK2 toxicity. Here, using CRISPR-Cas9 gene-edited HEK293 cells, RAW264.7 macrophages, rat primary ventral midbrain cultures, and PD patient-derived lymphoblastoid cells, we found that elevated LRRK2 kinase activity was associated with increased ROS production and lipid peroxidation and that this was blocked by inhibitors of either LRRK2 kinase or NADPH oxidase 2 (NOX2). Oxidative stress induced by the pesticide rotenone was ameliorated by LRRK2 kinase inhibition and was absent in cells devoid of LRRK2. In a rat model of PD induced by rotenone, a LRRK2 kinase inhibitor prevented the lipid peroxidation and NOX2 activation normally seen in nigral dopaminergic neurons in this model. Mechanistically, LRRK2 kinase activity was shown to regulate phosphorylation of serine-345 in the p47phox subunit of NOX2. This, in turn, led to translocation of p47phox from the cytosol to the membrane-associated gp91phox (NOX2) subunit, activation of the NOX2 enzyme complex, and production of ROS. Thus, LRRK2 kinase activity may drive cellular ROS production in PD through the regulation of NOX2 activity.

Loss of endogenous Nox2-NADPH oxidase does not prevent age-induced platelet activation and arterial thrombosis in mice

BackgroundReactive oxygen species are known to contribute to platelet hyperactivation and thrombosis during aging; however, the mechanistic contribution of the specific oxidative pathway remains elusive. ObjectivesWe hypothesized that during aging, endogenous Nox2-NADPH oxidase contributes to platelet reactive oxygen species accumulation and that loss of Nox2 will protect from platelet activation and thrombosis. MethodsWe studied littermates of Nox2 knockout (Nox2-KO) and -wild-type (Nox2-WT) mice at young (3-4 months) and old (18-20 months) age. Within platelets, we examined the expression of subunits of NADPH oxidase and enzyme activity, oxidant levels, activation markers, aggregation, and secretion. We also assessed susceptibility to in vivo thrombosis in 2 experimental models. ResultsWhile aged Nox2-WT mice displayed increased mRNA levels for Nox2, aged Nox2-KO mice showed an increase in Nox4 mRNA. However, neither the protein levels of several subunits nor the activity of NADPH oxidase were found to be altered by age or genotype. Both aged Nox2-WT and aged Nox2-KO mice exhibited similar enhancement in levels of platelet oxidants, granule release, αIIbβ3 activation, annexin V binding, aggregation and secretion, and a greater susceptibility to platelet-induced pulmonary thrombosis compared with young mice. In a photochemical injury model, adoptive transfer of platelets from aged Nox2-WT or Nox2-KO mice to the aged host mice resulted in a similar time to develop occlusive thrombus in the carotid artery. These findings suggest that loss of endogenous Nox2 does not protect against age-related platelet activation and arterial thrombosis in mice. ConclusionWe conclude that Nox2 is not an essential mediator of prothrombotic effects associated with aging.

Molecular characterization of a phytomelatonin receptor and its overexpression as a ‘one-stop’ solution to nullify the toxic effects of hazardous inorganic agro-pollutants

Inorganic toxicants like arsenic, copper, lead, nickel and fluoride are notorious agro-pollutants, impeding plant-productivity due to high bioaccumulation. Consumption of such contaminated plant-parts causes irreversible health hazards. We identified a G-protein coupled receptor, serving as melatonin receptor (MelR) in Nicotiana tabacum (NtMelR), that relayed downstream-signaling after binding melatonin, a potent growth regulator and antioxidant. Using inhibitors against G-protein-α and NADPH oxidase (NOX), and by supplementing epidermal strips with exogenous melatonin and H2O2, we established that NtMelR acted upstream of reactive oxygen species (ROS) production in guard cells. Transgenic lines of N. benthamiana overexpressing NtMelR maintained constitutive melatonin-signaling via MelR, leading to efficient stomatal closure for preventing desiccation during oxidative stress. Melatonin biosynthesis was stimulated in the transgenic lines, exposed to different agro-pollutant stress, providing a steady-abundance of ligand for NtMelR binding and activating the defence machinery, comprising of enzymatic-antioxidants like superoxide dismutase, catalase, peroxidases and glyoxalases. Due to increased antioxidant capacity, the transgenics exhibited less molecular injuries (electrolyte leakage, methylglyoxal accumulation and NOX activity), generated less ROS and bioaccumulated significantly lower levels of toxicants. Unlike the wild-type counterparts, the transgenics maintained high relative water content, photosynthetic efficiency, could flower abundantly and even produce seeds. Overall, we established that overexpression of NtMelR is a single-window strategy to generate multiple-stress tolerant genotypes.

Hydrogen Peroxide Is Involved in Methane-Alleviated Cadmium Toxicity in Alfalfa (Medicago sativa L.) Seedlings by Enhancing Cadmium Chelation onto Root Cell Walls.

Although previous studies have demonstrated that methane (CH4) can mitigate the toxicity of cadmium (Cd) in alfalfa seedlings, the CH4-rich water used in these studies may create hypoxic conditions, potentially influencing the experimental outcomes. Therefore, this study aimed to investigate whether CH4 can reduce Cd toxicity in alfalfa seedlings without the interference of hypoxia and to analyze its underlying mechanisms. Here, it was observed that supplementing oxygen with saturated CH4-rich water can significantly alleviate the inhibition of 75 μM CdCl2 on the growth of alfalfa (Medicago sativa L.) seedlings. Less Cd accumulation was also observed in both root and shoot parts, which could be explained by the CH4-altered cell wall components in alfalfa seedling roots, including covalent and ionic soluble pectin, and the degree of demethylation in pectin, thus enabling a higher proportion of Cd binding to the cell walls and reducing the entry of Cd into the cells. The above actions of CH4 were accompanied by an increase in hydrogen peroxide (H2O2) content and NADPH oxidase activity, which could be blocked by the addition of the NADPH oxidase inhibitor diphenylene iodonium (DPI). Taken together, these results implied that exogenously applied CH4 could alleviate Cd toxicity in alfalfa seedlings by enhancing Cd chelation onto the root cell walls, which might be closely associated with NADPH oxidase-dependent H2O2 signals. These findings could provide insight into the mechanism through which CH4 alleviates Cd toxicity in alfalfa plants.

The Role of Propionate-Induced Rearrangement of Membrane Proteins in the Formation of the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Adhesive-invasive E. coli has been suggested to be associated with the development of Crohn's disease (CD). It is assumed that they can provoke the onset of the inflammatory process as a result of the invasion of intestinal epithelial cells and then, due to survival inside macrophages and dendritic cells, stimulate chronic inflammation. In previous reports, we have shown that passage of the CD isolate ZvL2 on minimal medium M9 supplemented with sodium propionate (PA) as a carbon source stimulates and inhibits the adherent-invasive properties and the ability to survive in macrophages. This effect was reversible and not observed for the laboratory strain K12 MG1655. We were able to compare the isogenic strain AIEC in two phenotypes-virulent (ZvL2-PA) and non-virulent (ZvL2-GLU). Unlike ZvL2-GLU, ZvL2-PA activates the production of ROS and cytokines when interacting with neutrophils. The laboratory strain does not cause a similar effect. To activate neutrophils, bacterial opsonization is necessary. Differences in neutrophil NADH oxidase activation and ζ-potential for ZvL2-GLU and ZvL2-PA are associated with changes in membrane protein abundance, as demonstrated by differential 2D electrophoresis and LC-MS. The increase in ROS and cytokine production during the interaction of ZvL2-PA with neutrophils is associated with a rearrangement of the abundance of membrane proteins, which leads to the activation of Rcs and PhoP/Q signaling pathways and changes in the composition and/or modification of LPS. Certain isoforms of OmpA may play a role in the formation of the virulent phenotype of ZvL2-PA and participate in the activation of NADPH oxidase in neutrophils.

Gamma-aminobutyric acid elicits H2O2 signalling and promotes wheat seed germination under combined salt and heat stress.

In the realm of wheat seed germination, abiotic stresses such as salinity and high temperature have been shown to hinder the process. These stresses can lead to the production of reactive oxygen species, which, within a certain concentration range, may actually facilitate seed germination. γ-aminobutyric acid (GABA), a non-protein amino acid, serves as a crucial signaling molecule in the promotion of seed germination. Nevertheless, the potential of GABA to regulate seed germination under the simultaneous stress of heat and salinity remains unexplored in current literature. This study employed observational methods to assess seed germination rate (GR), physiological methods to measure H2O2 content, and the activities of glutamate decarboxylase (GAD), NADPH oxidase (NOX), superoxide dismutase (SOD), and catalase (CAT). The levels of ABA and GABA were quantified using high-performance liquid chromatography technology. Furthermore, quantitative real-time PCR technology was utilized to analyze the expression levels of two genes encoding antioxidant enzymes, MnSOD and CAT. The findings indicated that combined stress (30 °C + 50 mM NaCl) decreased the GR of wheat seeds to about 21%, while treatment with 2 mM GABA increased the GR to about 48%. However, the stimulatory effect of GABA was mitigated by the presence of ABA, dimethylthiourea, and NOX inhibitor, but was strengthened by H2O2, antioxidant enzyme inhibitor, fluridone, and gibberellin. In comparison to the control group (20 °C + 0 mM NaCl), this combined stress led to elevated levels of ABA, reduced GAD and NOX activity, and a decrease in H2O2 and GABA content. Further investigation revealed that this combined stress significantly suppressed the activity of superoxide dismutase (SOD) and catalase (CAT), as well as downregulated the gene expression levels of MnSOD and CAT. However, the study demonstrates that exogenous GABA effectively reversed the inhibitory effects of combined stress on wheat seed germination. These findings suggest that GABA-induced NOX-mediated H2O2 signalling plays a crucial role in mitigating the adverse impact of combined stress on wheat seed germination. This research holds significant theoretical and practical implications for the regulation of crop seed germination by GABA under conditions of combined stress.