NADPH Oxidase Inhibitor Research Articles

Downregulation of SODD mediates carnosol-induced reduction in cell proliferation in esophageal adenocarcinoma cells

Esophageal adenocarcinoma carries a poor prognosis associated with a 5-year survival rate of 12.5–20%. Therefore, a new therapeutic modality is needed for this lethal tumor. Carnosol is a phenolic diterpene purified from the herbs such as rosemary and Mountain desert sage and has been shown to have anticancer activities in multiple cancers. In this study we examined the effect of carnosol on cell proliferation in esophageal adenocarcinoma cells. We found that carnosol dose-dependently decreased cell proliferation in FLO-1 esophageal adenocarcinoma cells and significantly increased caspase-3 protein, indicating that carnosol decreases cell proliferation and increases cell apoptosis in FLO-1 cells. Carnosol significantly increased H2O2 production and N-acetyl cysteine, a reactive oxygen species (ROS) scavenger, significantly inhibited carnosol-induced decrease in cell proliferation, indicating that ROS may mediate carnosol-induced decrease in cell proliferation. Carnosol-induced decrease in cell proliferation was partially reversed by NADPH oxidase inhibitor apocynin, suggesting that NADPH oxidases may be partially involved in carnosol’s effect. In addition, carnosol significantly downregulated SODD protein and mRNA expression and knockdown of SODD significantly inhibited the carnosol-induced reduction in cell proliferation, suggesting that downregulation of SODD may contribute to carnosol-induced reduction in cell proliferation. We conclude that carnosol dose-dependently decreased cell proliferation and significantly increased caspase-3 protein. Carnosol’s effect may be through the overproduction of ROS and the downregulation of SODD. Carnosol might be useful for the treatment of esophageal adenocarcinoma.

Rapid translocation of intracellular toll-like receptors depends on endosomal NADPH oxidase.

Endosomal toll-like receptors (TLRs) must be translocated from the endoplasmic reticulum (ER) to the endosome and proteolytically cleaved within the endosome before they can induce cellular signals. As ligands for these TLRs are also liberated from apoptotic or necrotic cells, this process is controlled by several mechanisms which shall ensure that there is no inadvertent activation. We have shown previously that antiphospholipid antibodies induce endosomal NADPH-oxidase (NOX) followed by the translocation of TLR7/8 to the endosome. We show now that endosomal NOX is required for the rapid translocation of TLR3, TLR7/8, and TLR9. Deficiency of gp91phox, the catalytic subunit of NOX2, or inhibition of endosomal NOX by the chloride channel blocker niflumic acid both prevent immediate (i.e., within 30min) translocation of these TLRs as shown by confocal laser scanning microscopy. Under these conditions, the induction of mRNA synthesis for TNF-α and secretion of TNF-α is delayed by approx. 6-9h. However, maximal expression of TNF-α mRNA or secretion of TNF-α is not significantly reduced. In conclusion, these data add NOX2 as another component involved in the orchestration of cellular responses to ligands of endosomal TLRs.

Intralesional Chemotherapy for Prostate Cancer: In vivo Proof of Principle

Introduction: Prostate cancer (PCA) is one of the most common cancers in the world, and current therapies are debilitating to patients. To develop a novel modality for the treatment of PCA, we evaluated the efficacy of intralesional administration of the Sirt3 activator Honokiol (HK) and the NADPH oxidase inhibitor Dibenzolium (DIB). Methods: We used a well-established transgenic adenocarcinoma mouse prostate (TRAMP-C2) model of hormone-independent PCA. MTS assay, apoptosis assay, wound healing assay, transwell invasion assay, RT-qPCR, and Western blotting were conducted in vitro, and HK and DIB were intratumorally administered to mice bearing TRAMP-C2 tumors. Tumor size and weight were observed over time. After removing tumors, H-E staining and immunohistochemistry (IHC) staining were conducted. Results: Treatment by HK or DIB showed an inhibitory effect on cell proliferation and migration in PCA cells. Poor ability to induce apoptosis in vitro, insufficient expression of caspase-3 on IHC staining, and increased necrotic areas on H-E staining indicated that necrosis plays an important role in cell death in treating groups by HK or DIB. RT-PCR, Western blotting, and IHC staining for epithelial mesenchymal transition (EMT) markers suggested that EMT was suppressed by HK and DIB individually. In addition, HK induced activation of CD3. Mouse experiments showed safe antitumor effects in vivo. Conclusions: HK and DIB suppressed PCA proliferation and migration. Further research will explore the effects of HK and DIB at the molecular level to reveal new mechanisms that can be exploited as therapeutic modalities.

Toxoplasma gondii triggers heterophil extracellular traps via NADPH oxidase, ERK1/2 and P38 signalling pathways, glycolysis and autophagy in chickens.

Toxoplasma gondii is a zoonotic parasite with a global distribution. Heterophil extracellular traps (HETs) are a novel innate immune mechanism of chickens against pathogens, but whether T. gondii can induce HETs release in chickens has not been reported. The effects of T. gondii on heterophils viability were assessed by using Cell Counting Kit-8. T. gondii-induced HETs were observed and analysed by the immunofluorescence method. T. gondii-induced reactive oxygen species (ROS) was determined by the DCFH-DA method. The mechanisms underlying T. gondii-triggered HETs were investigated by inhibitors and fluorescence microplate reader. T. gondii did not significantly affect heterophils viability at a 1:1 ratio within 1 h. It was demonstrated for the first time that T. gondii could induce HETs release in chicken, and the structure of HETs was comprised of DNA, elastase and citrullinated histone 3 (citH3). T. gondii increased ROS production in a dose-dependent manner. Inhibitors of NADPH oxidase, extracellular signal-regulated kinase 1/2 (ERK1/2 ) and P38 signalling pathways, glycolysis and autophagy significantly decreased the release of T. gondii-induced HETs. Taken together, T. gondii can induce HETs release in chickens, and ROS, NADPH oxidase, ERK1/2 and P38 signalling pathways, glycolysis and autophagy participate in the process of HETs release, which provides new insights into the innate immune mechanism of chickens against T. gondii infection.

Peripheral and central effects of NADPH oxidase inhibitor, mitoapocynin, in a rat model of diisopropylfluorophosphate (DFP) toxicity.

Organophosphates (OP) are highly toxic chemical nerve agents that have been used in chemical warfare. Currently, there are no effective medical countermeasures (MCMs) that mitigate the chronic effects of OP exposure. Oxidative stress is a key mechanism underlying OP-induced cell death and inflammation in the peripheral and central nervous systems and is not mitigated by the available MCMs. NADPH oxidase (NOX) is one of the leading producers of reactive oxygen species (ROS) following status epilepticus (SE). In this study, we tested the efficacy of the mitochondrial-targeted NOX inhibitor, mitoapocynin (MPO) (10 mg/kg, oral), in a rat diisopropylfluorophosphate (DFP) model of OP toxicity. In DFP-exposed animals, MPO decreased oxidative stress markers nitrite, ROS, and GSSG in the serum. Additionally, MPO significantly reduced proinflammatory cytokines IL-1β, IL-6, and TNF-α post-DFP exposure. There was a significant increase in GP91phox, a NOX2 subunit, in the brains of DFP-exposed animals 1-week post-challenge. However, MPO treatment did not affect NOX2 expression in the brain. Neurodegeneration (NeuN and FJB) and gliosis [microglia (IBA1 and CD68), and astroglia (GFAP and C3)] quantification revealed a significant increase in neurodegeneration and gliosis after DFP-exposure. A marginal reduction in microglial cells and C3 colocalization with GFAP in DFP + MPO was observed. The MPO dosing regimen used in this study at 10 mg/kg did not affect microglial CD68 expression, astroglial count, or neurodegeneration. MPO reduced DFP-induced oxidative stress and inflammation markers in the serum but only marginally mitigated the effects in the brain. Dose optimization studies are required to determine the effective dose of MPO to mitigate DFP-induced changes in the brain.

The Effects of Apocynin on Monosodium Glutamate Induced Liver Damage of Rats

Monosodium glutamate (MSG) is found in refined foods. Apocynin (APO) is a selective NADPH oxidase (NOX) inhibitor. The aim of this experimental study was to investigate possible effects of MSG and the curative effects of APO in rats. Twenty-eight male Sprague-Dawley rats were randomly divided into four groups (Normal control, APO, MSG and MSG + APO, n:7 for each group). The MSG and MSG + APO groups received 120 mg/kg MSG solution orally for 28 consecutive days. The APO and MSG + APO groups received 25 mg/kg APO solution orally for 5 days until the end of the experiment. At the end of the experiment, all rats were sacrificed and liver tissue and blood samples were taken for histological, ultrastructural, and biochemical analyses. In the MSG group, vacuolization and loss in glycogen content in the hepatocytes, leukocyte infiltration and fibrosis in the liver parenchyme and portal triads, were observed. Terminal deoxynucleotidyl transferase dUTP (TUNEL)-positivity and NADPH oxidase (NOX)-2-positivity were higher in the MSG group compared with the other experimental groups. The concentrations of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), total bilirubin, malondialdehyde (MDA), and myeloperoxidase (MPO) were higher, whereas albumin, glutathione (GSH), and superoxide (SOD) levels were lower in the MSG group. All these data has been reversed in MSG + APO group. The histological and biochemical criteria indicated the prominent ameliorating effect of APO on MSG -induced liver injury.

Attenuation of LPS-Induced Lung Injury by Benziodarone via Reactive Oxygen Species Reduction.

As overproduction of reactive oxygen species (ROS) causes various diseases, antioxidants that scavenge ROS, or inhibitors that suppress excessive ROS generation, can be used as therapeutic agents. From a library of approved drugs, we screened compounds that reduced superoxide anions produced by pyocyanin-stimulated leukemia cells and identified benzbromarone. Further investigation of several of its analogues showed that benziodarone possessed the highest activity in reducing superoxide anions without causing cytotoxicity. In contrast, in a cell-free assay, benziodarone induced only a minimal decrease in superoxide anion levels generated by xanthine oxidase. These results suggest that benziodarone is an inhibitor of NADPH oxidases in the plasma membrane but is not a superoxide anion scavenger. We investigated the preventive effect of benziodarone on lipopolysaccharide (LPS)-induced murine lung injury as a model of acute respiratory distress syndrome (ARDS). Intratracheal administration of benziodarone attenuated tissue damage and inflammation via its ROS-reducing activity. These results indicate the potential application of benziodarone as a therapeutic agent against diseases caused by ROS overproduction.

Analysing the neuroprotective abilities of Apocynin (APO) & Chlorogenic Acid (CGA) in synergism to treat Alzheimer’s disease (AD)

AbstractBackgroundNeurological illnesses accounted for the greatest percentage (6.2%) of Total DALY index among all diseases, according to the World Health Organization (WHO). Alzheimer’s disease (AD) is the second most common neurological illness in the world, after Cerebrovascular disease (12 percent), with epilepsy and migraine accounting for 8% apiece. The cognitive function of people with AD deteriorates over time. It is an irreversible, dynamically changing condition that will afflict approximately 65.7 million people worldwide by 2030. For the treatment of AD, the Food and Drug Administration (FDA) has approved a number of commercial formulations (e.g., donepezil, rivastigmine, galantamine, memantine, and aducanumab). However, they have serious adverse effects and only provide symptomatic relief in people who have the illness. As a result, natural compounds are being investigated in recent years for the treatments of neurodegenerative diseases (NDDs). Picrorhiza kurroa, isolate Apocynin (APO) is a potent inhibitor of NADPH oxidase (NOX) and Chlorogenic Acid (CGA), a product of green coffee beans, has been shown to reduce ROS and NO levels in the brain, which is considered major trigger in AD.MethodIt was possible to execute successful in‐silico validation utilising computational biology, as well as the fabrication of APO‐CGA loaded polymeric nanoparticles using the ionic gelation process. The optimized nano‐formulation (APO – CGA NPs) was characterised by using various techniques to ensure its particle size and physicochemical behaviour along with release kinetics analysis.ResultThe attained results suggested that designed nano formulation will provide enhanced synergistic therapeutic efficacy to treat AD.ConclusionThe formulation and study of optimal APO‐CGA NPs for effective transport to the target site, their function in synergism, and their anti‐oxidative capabilities to evaluate the neuroprotective efficacy were the emphasis of this research work.

Methyl jasmonate (MeJA) enhances salt tolerance of okra (Abelmoschus esculentus L.) plants by regulating ABA signaling, osmotic adjustment substances, photosynthesis and ROS metabolism

Methyl jasmonate (MeJA), as a plant growth regulator, which is involves in many morphological and physiological processes, plays an important role in defense systems of plants under stress conditions. However, the roles of physiological and biochemical mechanisms of enhancing salt tolerance of okra plants based on application of MeJA is poorly understood. In this study, the application of the investigated 50 μM MeJA significantly improved salt tolerance of okra plants based on the germination rates and culture pot experiment under salt stress. Based on optimal concentration of MeJA inducing salt tolerance of okra plants, application of 5 mM diphenyleneiodonium (NADPH oxidase inhibitor, DPI) and 50 μM MeJA + 5 mM DPI, respectively, significantly increased salt tolerance of okra plants. Assays on the content of component matter indicated the significant increases of JA, ABA, proline, soluble protein, soluble sugar, photosynthetic pigment and relative water, glutathione (GSH) and ascorbic acid (AsA) content and the significant decreases of hydrogen peroxide (H2O2), superoxide radicals (O2−) and malondialdehyde (MDA) content and electrolyte leakage rates were observed in okra plants with 50 μM MeJA under salt stress. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX) and peroxidase (POD) related to ROS scavenging were also enhanced in okra plants by applied MeJA under salt stress. Furthermore, the marked decreases of H2O2, O2- and MDA content, and the significant increases of CAT, GR and APX activities and GSH and ASA content involved in ASA-GSH cycle were observed in MeJA-okra, DPI-okra and MeJA+DPI-okra plants under salt stress. H2O2 acted a toxic accumulation of ROS rather than as a second messenger for signal transduction in okra plants. These results demonstrate that exogenous MeJA increases the accumulation of JA, ABA and proline, which enhances salt tolerance of okra plants, by regulating osmotic balance and protecting membrane integrity and photosynthesis and activating ROS scavenging system. As a conclusion, treating okra plants with 50 μM MeJA could alleviate the harmful effects of abiotic stresses.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

Inhibition of NADPH Oxidase (NOX) 2 Mitigates Colitis in Mice with Impaired Macrophage AMPK Function.

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKβ1-deficient (AMPKβ1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKβ1LysM mice, whereas sodium salicylate (SS; AMPK β1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKβ1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKβ1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis.

Dose and Time Dependent Protective Effects of Apocyanin and Curcumin Against Diclofenac-Induced Cardiotoxicity in Male Rats

This study explored the protective potential of apocyanin and curcumin in diclofenac-induced cardiotoxicity utilizing cardiac enzymes, pro-inflammatory markers, and along with histopathological endpoints. A total of 123 male Wistar rats were used for the study. 43 rats were used for the determination of the median lethal dose (LD50) of apocyanin and curcumin while 80 rats were randomly divided into 8 groups of 10 rats each. Group 1(control) received distilled water while others received orally, per mg/kg body weight of treatments as follows: group 2(1000, apocyanin, group 3(1000, curcumin), group 4(10, diclofenac), group 5(500, apocyanin and 10, diclofenac), group 6(1000, apocyanin and 10, diclofenac), group 7(500, curcumin and 10, diclofenac) and group 8(1000, curcumin and 10, diclofenac). The treatments were administered daily for 14 and 28 days. LD50 up to 5000mg/kg body weight of apocyanin and curcumin did not show any fatality in animals. Administration of diclofenac significantly (p < 0.05) elevated the activities of creatinine kinase-MB, lactate dehydrogenase, levels of troponin-T, and tumor necrosis factor. There was no alteration in the activities of Interleukin- 1β. The histological results also showed cardiac insults such as cardiac muscle having severe fibro collagenous stroma, reduced myocytes density, and thick wall blood vessels. However, pretreatment with 500 and 1000 mg/kg body weight of apocyanin or curcumin attenuated all biochemical alterations and histological lesions induced by diclofenac in a dose-dependent manner. Pretreatments with apocyanin and curcumin inhibitors of NADPH oxidases 2weres effective in ameliorating diclofenac-induced cardiotoxicity suppressing inflammation and restoring normal histological architecture, thus, highlighting the therapeutic potentials of apocyanin and curcumin in the management of diclofenac-mediated cardiotoxicity.

Harpin enhances antioxidant nutrient accumulation and decreases enzymatic browning in stored soybean sprouts

Abstract Enzymatic browning causes quality losses in the soybean sprout industry. Herein, the effects of harpin, a proteinaceous bacterial elicitor isolated from Erwinia amylovora, in regulating enzymatic browning and antioxidant nutrient accumulation in stored soybean sprout were investigated. Enhanced soybean sprout growth (evaluated by sprout length and fresh weight) occurred after spraying three times (0, 2, and 4 days after sowing) with 30 mg L−1 harpin during the growth stage. The decline in vitamin C and total phenolic contents and total antioxidant capacity (evaluated by Fe3+ reducing power) were attenuated by harpin during storage. Harpin increased phenylalanine ammonia-lyase, NADPH oxidase (NOX), superoxide dismutase, and catalase activities and inhibited polyphenol oxidase activity and enzymatic browning in soybean sprouts during storage. However, this harpin-promoted sprout growth, enhanced antioxidant accumulation and enzyme activity and improved sprout quality could be partly, but drastically, abolished using diphenyleneiodonium chloride, a specific inhibitor of NOX. Moreover, the mechanisms by which harpin influences antioxidant accumulation and enzymatic browning in soybean sprouts during storage were discussed from the perspective of NOX-mediated H2O2 signalling.

Investigation of NADPH-Oxidase’s Binding Subunit(s) for Catechin Compounds Induce Inhibition

Catechins are natural polyphenolic compounds with ability to minimize excess free radicals through different mechanisms including inhibition of NADPH oxidase (NOX) activity. NOX is a complex enzyme made-up of several subunits, where molecules including catechins binds to exert their effect. Hence, the attempt to probe the NOX enzyme’s binding subunit of catechins-induce effect. Several in-silico techniques were deployed in probing the NOX enzyme’s binding subunit of the catechins. The catechins were downloaded from PubChem database in SDF files. The five NOX subunits with PDB ID: 3A1F, 1OV3, 1HH4, 1OEY, and 7CFZ were downloaded from the protein databank. Drug-likeness properties and biological activities were predicted using ADMETMESH software. Catechin-NOX subunits’ interactions was performed via molecular docking, and the docked conformations were analyzed using Protein-plus software. The results of the study predicted the catechin compounds; epicatechin (E), epicatechin gallate (EG) and epigallocatehin gallate (EGG) are drug-like in nature and possess enzymes inhibitory properties. Docking result predicted catechins are capable of interacting with the various NADPH oxidase (NOX) subunits but in a varied degree. Their (catechins) strongest affinities was predicted on p40phox and p67phox PB1 subunit (PBD: 1OEY) with binding energies in the ranges of -8.3 to -9.9kcal/mol in this order; Apocynin>EGG>EG>E. While a weak affinity was predicted between the catechin compounds and gp91 (phox) subunit (PDB: 3A1F) with binding energies (-4.9 to -6.5 kcal/mol) in this sequence; E<apocynin<EGG<EG. In conclusion, the study predicted catechin compounds possess drug-likeness properties and has affinities for interaction with NADPH-oxidase subunits particularly the p40phox and p67phox PB1 probably to exert their antioxidant effects. Therefore, in vitro and in vivo study is recommended to verify this claim.

Specific inhibition of NADPH Oxidase 2 modifies chronic epilepsy

Specific inhibition of NADPH Oxidase 2 modifies chronic epilepsy

Activation of neuronal NADPH oxidase NOX2 promotes inflammatory neurodegeneration.

Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N-acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases.

In vitro study of deltamethrin-induced extracellular traps in hemocytes of Ruditapes philippinarum

Deltamethrin (DLM), a broad-spectrum pesticide, has been proven to have toxic effects on aquatic organisms. Here, we detected the formation of extracellular traps (ETosis) formation in Manila clam (Ruditapes philippinarum) hemocytes stimulated by three concentrations of DLM (0.01, 0.1 and 1 μg/mL) in vitro, and explored the underlying mechanisms induced by this pesticide. Extracellular DNA structure observation and quantitative results indicated that DLM exposure could obviously induce hemocytes ETosis, especially under high concentration of DLM induction. Moreover, DLM increased the levels of myeloperoxidase (MPO) and reactive oxygen species (ROS) in a dose-dependent manner, and enhanced the mRNA expression of several ROS-related genes. DPI (NADPH oxidase inhibitor) and ABAH (MPO inhibitor) could substantially inhibit DLM-induced extracellular traps (ETs), suggesting that the induced ETs release was caused by the induction of the ROS burst and MPO production. In addition, three concentrations of DLM-induced ETs were also accompanied by mitochondrial dysfunction, such as increasing the production of mitochondrial ROS, leading to a decrease in mitochondrial membrane potential (MMP) and activation of mitochondrial permeability transition pore (MPTP). Taken together, these results will shed new light on the immunotoxicity of DLM in clams and perhaps lays the foundation for health assessment in bivalves.

Exogenous ATP triggers antioxidant defense system and alleviates Cd toxicity in maize seedlings

The role of exogenous adenosine 5′-triphosphate (ATP) in the regulation of antioxidant response in plants under heavy metal stress is unclear. Here, we investigated the effects of exogenous ATP application on plant growth, antioxidant response, and Cd accumulation in maize seedlings. Treatment with 0.1 mM CdCl2 moderately reduced dry weight, decreased chlorophyll content, impaired photosynthesis, and increased lipid peroxidation in maize seedlings compared with controls. However, toxicity due to Cd was alleviated after 10–200 µM ATP treatment. Subsequently, the activity of Cd-regulated antioxidant enzymes, antioxidant metabolite accumulation, and total antioxidant capacity were drastically enhanced after 50 µM ATP treatment. Similar patterns were observed in the ADP-treated group but not in the AMP-treated group under Cd stress. However, the ATP-induced elevation in antioxidant defense ability was decreased by the inhibition of NADPH oxidase (NOX). ATP-induced elevation in NOX activity and H2O2 production was partly reversed by the inhibition of NOX in maize seedlings under Cd stress. Furthermore, ATP promoted Cd accumulation in the roots and shoots of maize seedlings. However, the ATP-induced increase in Cd accumulation was partly abolished by the inhibition of NOX. To our knowledge, this is the first report on the role and mechanism of exogenous ATP in regulating plant growth, antioxidant response, and heavy metal phytoextraction. The study provides a new method based on exogenous ATP for enhancing heavy metal tolerance in plants.

Nitric oxide and hydrogen peroxide mediated regulation of chromium (VI) toxicity in wheat seedlings involves alterations in antioxidants and high affinity sulfate transporter

Chromium contamination of the soil is a major scientific concern with reference to crop productivity and human health. In recent years, several approaches are being employed in managing metal toxicity in crop plants. Here, we have investigated about potential and probable crosstalk of nitric oxide (NO) and hydrogen peroxide (H2O2) in mitigating hexavalent chromium [Cr(VI)] toxicity in wheat seedlings. Cr(VI) toxicity reduced the fresh mass and overall growth due to accumulation of reactive oxygen species (ROS) and decreased efficiency of AsA-GSH cycle and downregulation of high affinity sulfate transporter. However, exogenous treatment of NO and H2O2 significantly alleviated Cr toxicity. Application of NO and ROS scavengers reversed stress mitigating effects of NO and H2O2, respectively suggesting that endogenous NO and H2O2 are necessary for rendering Cr toxicity tolerance. Furthermore, NO rescued negative effect of diphenylene iodonium (DPI, NADPH oxidase inhibitor) and H2O2 reversed the negative effect of c-PTIO suggesting that they exhibit independent signalling in mitigating Cr stress. Altogether, data indicated that NO and H2O2 rendered mitigation of Cr stress by up-regulating enzymes (activity and relative gene expression) and metabolites of AsA-GSH cycle, high affinity sulfate transporter (relative gene expression) and glutathione biosynthesis which collectively controlled occurrence of oxidative stress.

Targeting benign prostate hyperplasia treatments: AR/TGF-β/NOX4 inhibition by apocynin suppresses inflammation and proliferation

IntroductionApocynin (Apo), an NADPH oxidase (NOX) inhibitor, has been widely used to treat various inflammatory diseases. However, the therapeutic effects of Apo on benign prostatic hyperplasia (BPH), a multifactorial disease associated with chronic inflammation and hormone imbalance, remain unknown. ObjectivesThe link between androgen signaling, reactive oxygen species (ROS), and prostate cell proliferation may contribute to the pathogenesis of BPH; therefore, the aim of this study was to identify the specific signaling pathway involved and to demonstrate whether the anti-oxidant Apo plays a role in the prevention and treatment of BPH. MethodsIngenuity pathway analysis and si-RNA transfection were conducted to demonstrate the androgen receptor (AR) and NOX4 linkage in BPH. Pathological markers of BPH were measured by H&E staining, immunoblotting, ELISA, qRT-PCR, and immunofluorescence to examine the effect of Apo. Rats stimulated with testosterone and BPH-1 cells were used as BPH models. ResultsAR and NOX4 network-mediated oxidative stress was upregulated in the BPH model. Next, we examined the effects of Apo on oxidative stress and chronic prostatic inflammation in BPH mouse models. In a testosterone-induced BPH rat model, Apo alleviated pathological prostate enlargement and suppressed androgen/AR signaling. Apo suppressed the upregulation of proinflammatory markers and promoted the expression of anti-oxidant factors. Furthermore, Apo regulated the TGF-β/Glut9/activin pathway and macrophage programming. In BPH-1 cells, Apo suppressed AR-mediated proliferation and upregulation of TGFB and NOX4 expression by alleviating oxidative stress. Apo activated anti-oxidant and anti-inflammatory systems and regulated macrophage polarization in BPH-1 cells. AR knockdown partially abolished the beneficial effects of Apo in prostate cells, indicating AR-dependent effects of Apo. ConclusionIn contrast with existing BPH therapies, Apo may provide a new application for prostatic disease treatment, especially for BPH, by targeting the AR/TGF-β/NOX4 signaling pathway.