Increased NADPH Oxidase Activity Research Articles

Oxidative Stress Induced by Cortisol in Human Platelets.

Hypercortisolism is known to affect platelet function. However, few studies have approached the effect of exogenous cortisol on human platelets, and the results obtained are conflicting and unconvincing. In this study, the effect of exogenous cortisol on several parameters indicative of oxidative status in human platelets has been analysed. We have found that cortisol stimulates ROS production, superoxide anion formation, and lipid peroxidation, with these parameters being in strict correlation. In addition, cortisol decreases GSH and membrane SH-group content, evidencing that the hormone potentiates oxidative stress, depleting platelet antioxidant defence. The involvement of src, syk, PI3K, and AKT enzymes in oxidative mechanisms induced by cortisol is shown. The main sources of ROS in cells can include uncontrolled increase of NADPH oxidase activity and uncoupled aerobic respiration during oxidative phosphorylation. Both mechanisms seem to be involved in ROS formation induced by cortisol, as the NADPH oxidase 1 inhibitor 2(trifluoromethyl)phenothiazine, and rotenone and antimycin A, complex I and III inhibitor, respectively, significantly reduce oxidative stress. On the contrary, the NADPH oxidase inhibitor gp91ds-tat, malate and NaCN, complex II and IV inhibitor, respectively, have a minor effect. It is likely that, in human platelets, oxidative stress induced by cortisol can be associated with venous and arterial thrombosis, greatly contributing to cardiovascular diseases.

Upregulation of NADPH-oxidase, inducible nitric oxide synthase and apoptosis in the hippocampus following impaired insulin signaling in the rats: Development of sporadic Alzheimer’s disease

NADPH-oxidase (NOX) is a multi-subunit enzyme complex. The upregulation of NOX causes massive production of superoxide (O2¯), which avidly reacts with nitric oxide (NO) and increases cellular reactive oxygen/nitrogen species (ROS/RNS). Increased ROS/RNS plays pivotal role in the sporadic Alzheimer’s disease (sAD) development and brain damage following impaired insulin signaling. Hence, this study aimed to examine early-time course of changes in NOX and NOS expression, and apoptotic proteins in the rats hippocampi following insulin signaling impairment [induced by STZ injection; intraperitoneal (IP) or in cerebral ventricles (ICV)]. Early effects (1, 3, or 6 weeks) on the NOX activity, translocation of NOX subunits from cytosol to the membrane, NO-synthases [neuronal-, inducible- and endothelial-NOS; nNOS, iNOS and eNOS], The Rac-1 protein expression, levels of NO and O2¯, cytochrome c release, caspase-3 and 9 activations (cleavage) were studied. STZ injection (in both models) increased NOX activity, O2¯ production, and enhanced cytosolic subunits translocation into membrane. The iNOS but not nNOS and eNOS expression and NO levels were increased in STZ treated rats. Finally, STZ injection increased cytochrome c release, caspase-3 and 9 activations in a manner that was significantly associated with levels of O2¯ and NO in the hippocampus. ICV-STZ administration resulted in significant profound changes over the IP route. In conclusion, impairment in insulin function induces early changes in ROS/RNS contents through NOX and iNOS upregulation and neuronal apoptosis in the hippocampus. Our results could mechanistically explain the role of impaired insulin function in the development of sAD.

Toxicity of mercuric oxide nanoparticles on freshwater microalgae: Comprehensive analysis on their interactive effects and detoxification pathways

So far, there is limited research on the potential risks and environmental consequences of mercuric oxide nanoparticles (HgO-NPs) on phytoplankton. To this end, we investigated the moderate and acute concentrations of HgO-NPs (0.8 and 1.6 mg/L, respectively) on the growth, photosynthesis, and detoxification processes in two microalgae, namely Scenedesmus obliquus and Nostoc muscorum. Compared to N. muscorum, S. obliquus had greater accumulation and removal efficiencies of HgO-NPs (92.8 %, 91.4 %) at moderate and acute levels. HgO-NPs accumulation inhibited the growth and decreased photosynthesis, RuBisCo activity and chlorophyll a content in a dose-dependent manner. At oxidative damage level, acute level of HgO-NPs evoked severe lipid and protein peroxidation, and increased NADPH oxidase activity particularly in N. muscorum. Exposure to HgO-NPs also resulted in distinct metabolic perturbations in both microalgal species. Notably, there was a dose-dependent increase in the activity of antioxidant enzymes (e.g., peroxidase and superoxide dismutase), as well as metabolites like tocopherols and flavonoids in S. obliquus. However, the activity of catalase, ascorbate peroxidase and dehydroascorbate reductase enzymes, and ascorbate levels were significantly increased in N. muscorum. At HgO-NPs detoxification level, S. obliquus increased glutathione level and glutathione-S-transferases activity. However, N. muscorum showed high levels of metal chelator (phytochelatins and metalothionine). Investigating the HgO-NPs bioremediation capacity and stress tolerance mechanisms opens possibilities to introduce S. obliquus as a promising bioremediator to relieve ecological risk of HgO-NPs.

Dynamics of reactive oxygen species and lignin biosynthesis during leaf spot disease of Withania somnifera (L.) Dunal.

Withania somnifera is an important medicinal plant, however, its cultivation and quality are compromised through infestation by leaf spot disease caused by the fungus, Alternaria alternata. To find suitable strategies against this disease, studies on post-infectional changes are important. ROS are critical as they interact with other defence signalling pathways. We analyzed ROS-generating and scavenging systems in healthy and diseased leaf samples of W. somnifera and ROS-driven downstream defence pathways. We used DAB and NBT assays for ROS detection, spectrophotometry and in-gel assays for ROS scavenging enzymes, a thioglycolic acid (TGA) based assay, histochemical staining for lignin, and qRT-PCR for transcript-level expression. Leaf spot infection in W. somnifera increased NADPH oxidase activity and ROS accumulation in infected leaves, together with enhanced antioxidant enzyme activity. Leaf spot-infected leaves had increased lignin content and higher expression of lignin biosynthesis genes. In addition, transcript levels of defence-related genes, NPR1 and PR, were also upregulated. The present work provides insights into responses to leaf spot disease through defence-related signalling in W. somnifera. It demonstrates crosstalk between ROS and lignin biosynthesis. This work identified potential targets for developing strategies to confer disease resistance against A. alternata in W. somnifera.

Exercise and vascular function in sedentary lifestyles in humans.

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

Interactive effects of salicylic acid and jasmonic acid on secondary metabolite production in Echinacea purpurea

Secondary metabolites are highly beneficial to human health and have commercial and industrial values. So, this research aimed to study the effects of exogenous salicylic acid (SA) and jasmonic acid (JA) on some secondary metabolites in purple coneflower. A field experiment as a randomized complete block design with three replications was conducted in Shahrood, Iran. Treatments were the factorial arrangement of 3 SA (0, 0.5, and 1 millimole) and 4 JA concentrations (0, 5, 20, and 50 micromole). The non-linear regression procedure was employed to quantify the relation of these materials with each other. The results indicated that the SA effect on all ten measured secondary metabolites changed with changing the JA levels as there was the interaction between these elicitors. On average, most (7 out of 11) of the combined SA_JA levels up-regulated the production of secondary metabolites as compared to the plants not sprayed with SA and JA. In terms of average response to elicitation with 11 combined SA_JA levels, they ranked from higher to lower as the guaiacol peroxidase, hydrogen proxide (H2O2), polyphenol oxidase, glutathione S-transferase, superoxide dismutase, NADPH oxidase, total phenolic content, phenylalanine ammonia-lyase, anthocyanin, and flavonoid. A few secondary metabolites appeared to have a biphasic relationship with each other. For instance, over lower and medium values of NADPH oxidase activity, anthocyanin content increased linearly with increasing NADPH oxidase activity; over higher values of NADPH oxidase activity, it showed a plateau state.

Oxidative Stress Response and Metal Transport in Roots of Macleaya cordata Exposed to Lead and Zinc.

Heavy metal pollution possesses potential hazards to plant, animal and human health, which has become the focus of recent attention. Hence, phytoremediation has been regarded as one of the most important remediation technologies for heavy-metal-contaminated soils. In this research, a dominant mine tailing plant, Macleaya cordata, was used as the experimental material to compare the metal transport and oxidative stress response in its roots under lead (Pb) and zinc (Zn) treatments. The result showed that Pb was mainly accumulated in the roots of M. cordata under the Pb treatment; less than 1% Pb was transported to the parts above. An analysis of the Zn content demonstrated a 39% accumulation in the shoots. The production of reactive oxygen species was detected using the in situ histological staining of roots, which showed that hydrogen peroxide in the root tips was observed to increase with the increase in both Pb and Zn concentrations. No significant superoxide anion changes were noted in the root tips under the Pb treatment. An analysis of the root enzyme activity showed that increase in NADPH oxidase activity can be responsible for the production of superoxide anions, subsequent the inhibition of root growth and decrease in antioxidant enzyme activities in the roots of M. cordata exposed to excess Zn. In total, this research provides evidence that the root of M. cordata has a high antioxidant capacity for Pb stress, so it can accumulate more Pb without oxidative damage. On the other hand, the Zn accumulated in the roots of M. cordata causes oxidative damage to the root tips, which can stimulate more Zn transport to the shoots to reduce the damage to the roots. This result will provide a basis for the application of M. cordata in the phytoremediation of soil polluted by Pb-Zn compounds.

Chemerin-9 in paraventricular nucleus increases sympathetic outflow and blood pressure via glutamate receptor-mediated ROS generation

Chemerin is an adipokine involved in regulating energy homeostasis and reproductive function. Excessive sympathetic activity contributes to hypertension, chronic heart failure and chronic renal disease. Hypothalamic paraventricular nucleus (PVN) is crucial in regulating sympathetic activity and blood pressure. The present study is designed to investigate the roles of chemerin in the PVN in regulating sympathetic activity and blood pressure and underlying mechanisms. Microinjections were performed in the bilateral PVN in male adult rats under anesthesia. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded. The PVN microinjection of chemerin-9, an active fragment of chemerin, increased RSNA and MAP, which were abolished by chemokine-like receptor 1 (CMKLR1) antagonist α-NETA, a superoxide scavenger tempol, antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and apocynin. Immunofluorescence analyses showed that N-methyl-D-aspartate (NMDA) receptors existed in most of cells of the PVN, and some of them co-existed with chemerin. The effects of chemerin-9 on RSNA and MAP were prevented by glutamate-binding site antagonist L-phenylalanine, NMDA receptor antagonist MK-801, and calcium channel blocker verapamil or nifedipine, but only attenuated by non-NMDA receptor antagonist CNQX. Moreover, chemerin-9 increased NADPH oxidase activity and superoxide production, which were prevented by α-NETA, MK-801, or verapamil. These results indicate that chemerin-9 in the PVN increases sympathetic activity and blood pressure via CMKLR1-dependent calcium influx, and glutamate receptor-mediated NADPH oxidase activation and subsequent superoxide production.

Asprosin in the Paraventricular Nucleus Induces Sympathetic Activation and Pressor Responses via cAMP-Dependent ROS Production.

Asprosin is a newly discovered adipokine that is involved in regulating metabolism. Sympathetic overactivity contributes to the pathogenesis of several cardiovascular diseases. The paraventricular nucleus (PVN) of the hypothalamus plays a crucial role in the regulation of sympathetic outflow and blood pressure. This study was designed to determine the roles and underlying mechanisms of asprosin in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male adult SD rats under anesthesia. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) were recorded, and PVN microinjections were performed bilaterally. Asprosin mRNA and protein expressions were high in the PVN. The high asprosin expression in the PVN was involved in both the parvocellular and magnocellular regions according to immunohistochemical analysis. Microinjection of asprosin into the PVN produced dose-related increases in RSNA, MAP, and HR, which were abolished by superoxide scavenger tempol, antioxidant N-acetylcysteine (NAC), and NADPH oxidase inhibitor apocynin. The asprosin promoted superoxide production and increased NADPH oxidase activity in the PVN. Furthermore, it increased the cAMP level, adenylyl cyclase (AC) activity, and protein kinase A (PKA) activity in the PVN. The roles of asprosin in increasing RSNA, MAP, and HR were prevented by pretreatment with AC inhibitor SQ22536 or PKA inhibitor H89 in the PVN. Microinjection of cAMP analog db-cAMP into the PVN played similar roles with asprosin in increasing the RSNA, MAP, and HR, but failed to further augment the effects of asprosin. Pretreatment with PVN microinjection of SQ22536 or H89 abolished the roles of asprosin in increasing superoxide production and NADPH oxidase activity in the PVN. These results indicated that asprosin in the PVN increased the sympathetic outflow, blood pressure, and heart rate via cAMP–PKA signaling-mediated NADPH oxidase activation and the subsequent superoxide production.

Hypertension secondary to nitric oxide depletion produces oxidative imbalance and inflammatory/fibrotic outcomes in the cornea of C57BL/6 mice

Arterial hypertension (AH) leads to oxidative and inflammatory imbalance that contribute to fibrosis development in many target organs. Here, we aimed to highlight the harmful effects of severe AH in the cornea. Our experimental model was established by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) to C57BL/6 mice, which were monitored weekly for arterial blood pressure and intraocular pressure (IOP). Morphological studies of ocular tissues were accompanied by analyses of reactive oxygen species generation, and localization/expression of NAPDH oxidase isoforms (NOX1, NOX2, NOX4) and inflammatory biomarkers (PPARα, PPARγ, IL-1β, IL-6, IL-10, TNF-α, and COX-2). Masson’s trichrome and Sirius Red staining were used to explore the fibrotic status of the cornea. The expression of collagen isoforms (COL1α1, COL1α2, COL3α1, COL4α1, COL4α2) and relevant metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were also quantified to evaluate the participation of collagen metabolism in AH-related corneal damage. Hypertensive animals showed an increase in IOP values, and a thinner cornea compared with normotensive controls. Moreover, AH increased NADPH oxidase activity and reactive oxygen species generation in the cornea, which was accompanied by transcriptional upregulation of NOX isoforms and inflammatory biomarkers, while reducing PPAR expression. L-NAME-treated animals also developed corneal fibrosis with overexpression of collagen isoforms and reduction of factors responsible for collagen degradation. This is the first study reporting structural changes in the cornea and elevated IOP in L-NAME-treated mice. Overexpression of the NADPH oxidase system and collagen deposition might play a substantial role in the pathogenic mechanisms contributing to ocular disturbances in a context of severe hypertension.

Diet-induced obesity impairs spermatogenesis: the critical role of NLRP3 in Sertoli cells

BackgroundAccumulating evidence indicates a key role of Sertoli cell (SC) malfunction in spermatogenesis impairment induced by obesity. Nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) is expressed in SCs, but the role of NLRP3 in the pathological process of obesity-induced male infertility remains unclear.MethodsNLRP3-deficient mice were fed a high-fat diet for 24 weeks to establish obesity-related spermatogenesis impairment. In another set of experiments, a lentiviral vector containing a microRNA (miR)-451 inhibitor was injected into AMP-activated protein kinase α (AMPKα)-deficient mouse seminiferous tubules. Human testis samples were obtained by testicular puncture from men with obstructive azoospermia whose samples exhibited histologically normal spermatogenesis. Isolated human SCs were treated with palmitic acid (PA) to mimic obesity model in vitro.ResultsIncreased NLRP3 expression was observed in the testes of obese rodents. NLRP3 was also upregulated in PA-treated human SCs. NLRP3 deficiency attenuated obesity-related male infertility. SC-derived NLRP3 promoted interleukin-1β (IL-1β) secretion to impair testosterone synthesis and sperm performance and increased matrix metalloproteinase-8 (MMP-8) expression to degrade occludin via activation of nuclear factor-kappa B (NF-κB). Increased miR-451 caused by obesity, decreased AMPKα expression and sequentially increased NADPH oxidase activity were responsible for the activation of NLRP3. miR-451 inhibition protected against obesity-related male infertility, and these protective effects were abolished by AMPKα deficiency in mice.ConclusionsNLRP3 promoted obesity-related spermatogenesis impairment. Increased miR-451 expression, impaired AMPKα pathway and the subsequent ROS production were responsible for NLRP3 activation. Our study provides new insight into the mechanisms underlying obesity-associated male infertility.

Trimetazidine Reduces Cardiac Fibrosis in Rats by Inhibiting NOX2-Mediated Endothelial-to-Mesenchymal Transition.

PurposeEndothelial-to-mesenchymal transition (EndMT) is an important mechanism underlying cardiac fibrosis. The anti-ischemic drug trimetazidine (TMZ) is reportedly useful in ventricular remodeling and associated with NADPH oxidase (NOX) 2. This study aimed to investigate the possible effect of TMZ on cardiac fibrosis exerted via the inhibition of NOX2-mediated EndMT.MethodsA cardiac fibrosis model was established in Sprague-Dawley rats through a subcutaneous injection of isoproterenol (ISO, 5 mg/kg/d). Echocardiographic parameters, myocardial fibrosis, NOX2 expression and EndMT were assessed. An in vitro model of EndMT was developed using human umbilical vein endothelial cells (HUVECs) via treatment with transforming growth factor-β (TGF-β) at 10 ng/mL for 24 h. HUVECs were administrated with TMZ or TMZ and lentivirus, the expression of EndMT and related proteins was observed by wound healing assay, immunoblotting, and immunofluorescence.ResultsRats injected with ISO exhibited severe interstitial cardiac fibrosis and perivascular fibrosis, decreased left ventricular ejection fraction, and increased NOX activity. TMZ treatment mitigated cardiac fibrosis, ameliorated left ventricular dysfunction, and reduced NOX activity. In addition, TMZ effectively inhibited EndMT in ISO-treated rat hearts and TGF-β-treated HUVECs, as manifested by increased CD31 expression, decreased α-SMA expression, and suppressed cell migration. Compared with the control group, the expression of NOX2, nuclear factor-κB (NF-κB), and Snail was increased in vivo and in vitro but decreased with TMZ treatment. Furthermore, the overexpression of NOX2 by lentivirus abolished the protective effects of TMZ on TGF-β-induced EndMT.ConclusionTMZ may ameliorate EndMT and ISO-induced cardiac fibrosis through the NOX2/NF-κB/Snail pathway. The findings of the study may provide new insights into the potential role of TMZ in the pathophysiology of cardiac fibrosis.

Delay of endothelial cell senescence protects cerebral barrier against age-related dysfunction: role of senolytics and senomorphics

ABSTRACT Accumulation of senescent cells in cerebrovasculature is thought to play an important role in age-related disruption of blood–brain barrier (BBB). Using an in vitro model of human BBB, composed of brain microvascular endothelial cells (BMECs), astrocytes and pericytes, this study explored the so-called correlative link between BMEC senescence and the BBB dysfunction in the absence or presence of functionally distinct senotherapeutics. Replicative senescence was deemed present at passage ≥19 where BMECs displayed shortened telomere length, reduced proliferative and tubulogenic potentials and increased NADPH oxidase activity, superoxide anion production (markers of oxidative stress), S-β-galactosidase activity and γ-H2AX staining. Significant impairments observed in integrity and function of a model of BBB established with senescent BMECs, ascertained successively by decreases in transendothelial electrical resistance and increases in paracellular flux, revealed a close correlation between endothelial cell senescence and BBB dysfunction. Disruptions in the localization or expression of tight junction proteins, zonula occludens-1, occludin, and claudin-5 in senescent BMECs somewhat explained this dysfunction. Indeed, treatment of relatively old BMEC (passage 16) with a cocktail of senolytics (dasatinib and quercetin) or senomorphics targeting transcription factor NF-κB (QNZ), p38MAPK signaling pathway (BIRB-796) or pro-oxidant enzyme NADPH oxidase (VAS2870) until passage 20 rendered these cells more resistant to senescence and totally preserved BBB characteristics by restoring subcellular localization and expression of tight junction proteins. In conclusion, attempts that effectively mitigate accumulation of senescent endothelial cells in cerebrovasculature may prevent age-related BBB dysfunction and may be of prophylactic or therapeutic value to extend lifelong health and wellbeing.

Trimethylamine N-Oxide Promotes Autoimmunity and a Loss of Vascular Function in Toll-like Receptor 7-Driven Lupus Mice.

Plasma levels of trimethylamine N-oxide (TMAO) are elevated in lupus patients. We analyzed the implication of TMAO in autoimmunity and vascular dysfunction of the murine model of systemic lupus erythematosus (SLE) induced by the activation of the Toll-like receptor (TLR)7 with imiquimod (IMQ). Female BALB/c mice were randomly divided into four groups: untreated control mice, control mice treated with the trimethylamine lyase inhibitor 3,3-dimethyl-1-butanol (DMB), IMQ mice, and IMQ mice treated with DMB. The DMB-treated groups were administered the substance in their drinking water for 8 weeks. Treatment with DMB reduced plasma levels of TMAO in mice with IMQ-induced lupus. DMB prevents the development of hypertension, reduces disease progression (plasma levels of anti-dsDNA autoantibodies, splenomegaly, and proteinuria), reduces polarization of T lymphocytes towards Th17/Th1 in secondary lymph organs, and improves endothelial function in mice with IMQ-induced lupus. The deleterious vascular effects caused by TMAO appear to be associated with an increase in vascular oxidative stress generated by increased NADPH oxidase activity, derived in part from the vascular infiltration of Th17/Th1 lymphocytes, and reduced nrf2-driven antioxidant defense. In conclusion, our findings identified the bacterial-derived TMAO as a regulator of immune system, allowing for the development of autoimmunity and endothelial dysfunction in SLE mice.

Improving astaxanthin production of Haematococcus pluvialis by an efficient fed-batch strategy in a photobioreactor

Astaxanthin has attracted extensive attention due to its multiple physiological functions, but its production from Haematococcus pluvialis remains challenging due to low biomass concentration. In this study, an efficient fed-batch strategy was designed to improve the astaxanthin productivity at a stable pH level during mixotrophic growth of H. pluvialis. The acid feeding position was equipped with feeding medium to provide sufficient nutrients based on pH changes. Results suggested that pH level was increasing during acetate cultivation, and was a limiting factor for H. pluvialis growth. The biomass concentration was enhanced by 25.7% through stabilizing pH levels in batch culture. Based on linear relationship between nutrient consumption and pH change, a fed-batch strategy was designed and tested, increasing the biomass concentration by 90.6% to 1.62 g L−1 in the photobioreactor. At the molecular level, the photosynthetic efficiency was limited by reducing Rubisco activity and increasing NADPH oxidase activity, while carbon metabolism was enhanced by elevating pyruvate and ace-CoA contents. Furthermore, astaxanthin productivity was increased to 4.5 mg L−1 d−1 within pH range from 5 to 6, which was accompanied by the elevated pyruvate, ace-CoA, and lipid contents. The fed-batch strategy was promising for microalgal production due to its easy operation and high astaxanthin productivity.

Astaxanthin induces NADPH oxidase activation and receptor‑interacting protein kinase 1‑mediated necroptosis in gastric cancer AGS cells.

Astaxanthin (ASX), a red-colored xanthophyll carotenoid, functions as an antioxidant or pro-oxidant. ASX displays anticancer effects by reducing or increasing oxidative stress. Reactive oxygen species (ROS) promote cancer cell death by necroptosis mediated by receptor-interacting protein kinase 1 (RIP1) and RIP3. NADPH oxidase is a major source of ROS that may promote necroptosis in some cancer cells. The present study aimed to investigate whether ASX induces necroptosis by increasing NADPH oxidase activity and ROS levels in gastric cancer AGS cells. AGS cells were treated with ASX with or without ML171 (NADPH oxidase 1 specific inhibitor), N-acetyl cysteine (NAC; antioxidant), z-VAD (pan-caspase inhibitor) or Necrostatin-1 (Nec-1; a specific inhibitor of RIP1). As a result, ASX increased NADPH oxidase activity, ROS levels and cell death, and these effects were suppressed by ML171 and NAC. Furthermore, ASX induced RIP1 and RIP3 activation, ultimately inducing mixed lineage kinase domain-like protein (MLKL) activation, lactate dehydrogenase (LDH) release and cell death. Moreover, the ASX-induced decrease in cell viability was reversed by Nec-1 treatment and RIP1 siRNA transfection, but not by z-VAD. ASX did not increase the ratio of apoptotic Bax/anti-apoptotic Bcl-2, the number of Annexin V-positive cells, or caspase-9 activation, which are apoptosis indices. In conclusion, ASX induced necroptotic cell death by increasing NADPH oxidase activity, ROS levels, LDH release and the number of propidium iodide-positive cells, as well as activating necroptosis-regulating proteins, RIP1/RIP3/MLKL, in gastric cancer AGS cells. The results of this study demonstrated the necroptotic effect of ASX on gastric cancer AGS cells, which required NADPH oxidase activation and RIP1/RIP3/MLKL signaling in vitro.

The atypical small GTPase GEM/Kir is a negative regulator of the NADPH oxidase and NETs production through macroautophagy.

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.

Inhibitory Effect of Korean Red Ginseng Extract on Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells

ObjectivesInfection of Helicobacter pylori (H. pylori), a gram-negative bacterium, leads to various gastric diseases, such as gastritis, peptic ulcer and gastric cancer. H. pylori increases cytokine release and activates inflammatory mediators in gastric mucosa. Particularly, H. pylori upregulates the inflammatory chemokine interleukin-8 (IL-8), which are activated by oxidative stress. IL-8 can cause severe inflammation of the stomach and gastric cancer. Korean red ginseng is the steamed root of 6-year-old Korean ginseng (Panax ginseng Meyer). Ginsenosides, triterpene glycosides, are the active components of Korean red ginseng. Ginsenosides have antioxidant, anti-inflammatory, and antitumor activities. The present study is aimed at determining whether Korean red ginseng extract inhibits H. pylori-induced IL-8 expression in gastric epithelial cells. MethodsThe human gastric epithelial cell line AGS was used. Gastric epithelial AGS cells were treated with Korean red ginseng extract, and infected with H. pylori (NCTC 11,637). Reactive oxygen species (ROS) levels were determined using dichlorofluorescein fluorescence. NADPH oxidase activity was measured using lucigenin chemiluminescence. IL-8 mRNA expression was measured by using real-time PCR. NADPH oxidase subunits were determined in cytosolic extract and membrane extract by using Western blotting. ResultsH. pylori increased NADPH oxidase activity, ROS levels, and upregulated IL-8 expression in gastric epithelial cells. Korean red ginseng extract inhibited IL-8 expression by suppressing NADPH oxidase activity and reducing ROS levels in gastric epithelial cells. H. pylori induced translocation of NADPH oxidase cytosolic subunits to membrane, which is a marker of NADPH oxidase activation, in AGS cells. Korean red ginseng extract inhibited translocation of cytosolic subunits of NADPH oxidase to membrane in AGS cells. ConclusionsKorean red ginseng may be beneficial for preventing H. pylori-associated gastric inflammation by inhibiting oxidative stress and IL-8 expression. Funding SourcesThis study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Urban air pollution induces alterations in redox metabolism and mitochondrial dysfunction in mice brain cortex

Previous reports indicate that the central nervous system (CNS) is a target of air pollution, causing tissue damage and functional alterations. Oxidative stress and neuroinflammation have been pointed out as possible mechanisms mediating these effects. The aim of this work was to study the chronic effects of urban air pollution on mice brain cortex, focusing on oxidative stress markers, and mitochondrial function. Male 8-week-old BALB/c mice were exposed to filtered air (FA, control) or urban air (UA) inside whole-body exposure chambers, located in a highly polluted area of Buenos Aires city, for up to 4 weeks. Glutathione levels, assessed as GSH/GSSG ratio, were decreased after 1 and 2 weeks of exposure to UA (45% and 25% respectively vs. FA; p < 0.05). A 38% increase in lipid peroxidation was found after 1 week of UA exposure (p < 0.05). Regarding protein oxidation, carbonyl content was significantly increased at week 2 in UA-exposed mice, compared to FA-group, and an even higher increment was found after 4 weeks of exposure (week 2: 40% p < 0.05, week 4: 54% p < 0.001). NADPH oxidase (NOX) and glutathione peroxidase (GPx) activities were augmented at all the studied time points, while superoxide dismutase (Cu,Zn-SOD cytosolic isoform) and glutathione reductase (GR) activities were increased only after 4 weeks of UA exposure (p < 0.05). The increased NOX activity was accompanied with higher expression levels of NOX2 regulatory subunit p47phox, and NOX4 (p < 0.05). Also, UA mice showed impaired mitochondrial function due to a 50% reduction in O2 consumption in active state respiration (p < 0.05), a 29% decrease in mitochondrial inner membrane potential (p < 0.05), a 65% decrease in ATP production rate (p < 0.01) and a 30% increase in H2O2 production (p < 0.01). Moreover, respiratory complexes I-III and II-III activities were decreased in UA group (30% and 36% respectively vs. FA; p < 0.05). UA exposed mice showed alterations in mitochondrial function, increased oxidant production evidenced by NOX activation, macromolecules damage and the onset of the enzymatic antioxidant system. These data indicate that oxidative stress and impaired mitochondrial function may play a key role in CNS damage mechanisms triggered by air pollution.

Renal Tubular Epithelial TRPA1 Acts as An Oxidative Stress Sensor to Mediate Ischemia-Reperfusion-Induced Kidney Injury through MAPKs/NF-κB Signaling.

Oxidative stress and inflammation play important roles in the pathophysiology of acute kidney injury (AKI). Transient receptor potential ankyrin 1 (TRPA1) is a Ca2+-permeable ion channel that is sensitive to reactive oxygen species (ROS). The role of TRPA1 in AKI remains unclear. In this study, we used human and animal studies to assess the role of renal TRPA1 in AKI and to explore the regulatory mechanism of renal TRPA1 in inflammation via in vitro experiments. TRPA1 expression increased in the renal tubular epithelia of patients with AKI. The severity of tubular injury correlated well with tubular TRPA1 or 8-hydroxy-2′-deoxyguanosine expression. In an animal model, renal ischemia-reperfusion injury (IR) increased tubular TRPA1 expression in wild-type (WT) mice. Trpa1−/− mice displayed less IR-induced tubular injury, oxidative stress, inflammation, and dysfunction in kidneys compared with WT mice. In the in vitro model, TRPA1 expression increased in renal tubular cells under hypoxia-reoxygenation injury (H/R) conditions. We demonstrated that H/R evoked a ROS-dependent TRPA1 activation, which elevated intracellular Ca2+ level, increased NADPH oxidase activity, activated MAPK/NF-κB signaling, and increased IL-8. Renal tubular TRPA1 may serve as an oxidative stress sensor and a crucial regulator in the activation of signaling pathways and promote the subsequent transcriptional regulation of IL-8. These actions might be evident in mice with IR or patients with AKI.