NADPH Oxidase-mediated Reactive Oxygen Species Research Articles

Investigating the role of rotenone on human blood platelets: Molecular insights into abnormal platelet functions in Parkinson's disease.

Parkinson's disease (PD) is a predominant neuromotor disorder characterized by the selective death of dopaminergic neurons in the midbrain. The majority of PD cases are sporadic or idiopathic, with environmental toxins and pollutants potentially contributing to its development or exacerbation. However, clinical PD patients are often associated with a reduced stroke frequency, where circulating blood platelets are indispensable. Although platelet structural impairment is evident in PD, the platelet functional alterations and their underlying molecular mechanisms are still obscure. Therefore, we investigated rotenone (ROT), an environmental neurotoxin that selectively destroys dopaminergic neurons mimicking PD, on human blood platelets to explore its impact on platelet functions, thus replicating PD conditions in vitro. Our study deciphered that ROT decreased thrombin-induced platelet functions, including adhesion, activation, secretion, and aggregation in human blood platelets. As ROT is primarily responsible for generating intracellular reactive oxygen species (ROS), and ROS is a key player regulating the platelet functional parameters, we went on to check the effect of ROT on platelet ROS production. In our investigation, it became evident that ROT treatment resulted in the stimulation of ROS production in human blood platelets. Additionally, we discovered that ROT induced ROS production by augmenting Ca2+ mobilization from inositol 1,4,5-trisphosphate receptor. Apart from this, the treatment of ROT triggers protein kinase C associated NADPH oxidase-mediated ROS production in platelets. In summary, this research, for the first time, highlights ROT-induced abnormal platelet functions and may provide a mechanistic insight into the altered platelet activities observed in PD patients.

NADPH oxidase-mediated reactive oxygen species, antioxidant isozymes, and redox homeostasis regulate salt sensitivity in maize genotypes

The aim of the study is to examine the relationship between oxidative bursts, their regulation with ion homeostasis, and NADPH oxidase (NOX) in different salt-sensitive maize genotypes. For this, in the first study, four differently salt-sensitive maize genotypes (BIL214 × BIL218 as tolerant, BHM-5 as sensitive, and BHM-7 and BHM-9 as moderate-tolerant) were selected on the basis of phenotype, histochemical detection of reactive oxygen species (ROS), malondialdehyde (MDA) content, and specific and in-gel activity of NOX. In the next experiment, these genotypes were further examined in 200 mM NaCl solution in half-strength Hoagland media for nine days to study salt-induced changes in NOX activity, ROS accumulation, ion and redox homeostasis, the activity of antioxidants and their isozyme responses, and to find out potential relationships among the traits. Methylglyoxal (MG) and glyoxalse enzymes (Gly I and II) were also evaluated. Fully expanded leaf samplings were collected at 0 (control), 3, 6, 9-day, and after 7 days of recovery to assay different parameters. Na+/K+, NOX, ROS, and MDA contents increased significantly with the progression of stress duration in all maize genotypes, with a significantly higher value in BHM-5 as compared to tolerant and moderate-tolerant genotypes. A continual induction of Cu/Zn-SOD was observed in BIL214 × BIL218 due to salt stress. Substantial decreases in CAT2 and CAT3 isozymes in BHM-5 might be critical for the highest H2O2 burst in that sensitive genotype under salt stress. The highest intensified POD isozymes were visualized in BHM-5, BHM-7, and BHM-9, whereas BIL214 × BIL218 showed a continual induction of POD isozymes, although GPX activity decreased in all the genotypes at 9 days. Under salt stress, the tolerant genotype BIL214 × BIL218 showed superior ASA- and GSH-redox homeostasis by keeping GR and MDHAR activity high. This genotype also had a stronger MG detoxification system by having higher glyoxalase activity. Correlation, comparative heatmap, and PCA analyses revealed positive correlations among Na+/K+, NOX, O2•−, H2O2, MG, proline, GR, GST, and Gly I activities. Importantly, the relationship depends on the salt sensitivity of the genotypes. The reduced CAT activity as well as redox homeostasis were critical to the survival of the sensitive genotype.

Angiotensin II acts through Rac1 to upregulate pendrin: role of NADPH oxidase.

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.

Glycine induces enhancement of bactericidal activity of neutrophils

Severe bacterial infections are frequently accompanied by depressed neutrophil functions. Thus, agents that increase the microbicidal activity of neutrophils could add to a direct antimicrobial therapy. Lysophosphatidylcholine augments neutrophil bactericidal activity via the glycine (Gly)/glycine receptor (GlyR) α2/TRPM2/p38 mitogen-activated protein kinase (MAPK) pathway. However, the direct effect of glycine on neutrophil bactericidal activity was not reported. In this study, the effect of glycine on neutrophil bactericidal activity was examined. Glycine augmented bactericidal activity of human neutrophils (EC50 = 238 μM) in a strychnine (a GlyR antagonist)-sensitive manner. Glycine augmented bacterial clearance in mice, which was also blocked by strychnine (0.4 mg/kg, s.c.). Glycine enhanced NADPH oxidase-mediated reactive oxygen species (ROS) production and TRPM2-mediated [Ca2+]i increase in neutrophils that had taken up E. coli. Glycine augmented Lucifer yellow uptake (fluid-phase pinocytosis) and azurophil granule-phagosome fusion in neutrophils that had taken up E. coli in an SB203580 (a p38 MAPK inhibitor)-sensitive manner. These findings indicate that glycine augments neutrophil microbicidal activity by enhancing azurophil granule-phagosome fusion via the GlyRα2/ROS/calcium/p38 MAPK pathway. We suggest that glycine could be a useful agent for increasing neutrophil bacterial clearance.

Lutein Induces Apoptosis of Gastric Cancer Cells by Producing Reactive Oxygen Species via NADPH Oxidase Activation

ObjectivesApoptosis is well known for its relationship with cancer. Disruption of apoptosis leads to tumor progression, and many cytotoxic anticancer agents induce apoptosis. As a class of phytochemicals, carotenoids are fat-soluble pigments that are abundant in fruits and vegetables and have health-promoting properties, including cancer prevention effects. The anticancer effects of carotenoids are related to their pro-oxidant activities. Lutein is a type of vitamin called a carotenoid. A recent study showed that lutein inhibits the growth of breast cancer cells. The present study was purposed to determine whether lutein induces apoptosis of gastric cancer cells through reactive oxygen species (ROS)-mediated mechanism. MethodsHuman gastric cancer cell line, AGS was treated with lutein. Cell viability was determined by counting the number of viable cells. The protein levels of Bax, Bcl-2, active caspase-3 were examined by western blot analysis. DNA fragmentation was determined by Cell Death Detection ELISA. DCF-DA fluorescence technique was used to quantify the intracellular ROS level. NADPH oxidase activity was measured by lucigenin assay. Colony formation assay was performed and colonies were counted. ResultsLutein decreased cell viability in a dose-dependent manner. Colony formation was also inhibited by lutein. Lutein increased Bax/Bcl-2 ratio, caspase-3 activity, and DNA fragmentation. The intracellular ROS level and NADPH oxidase activity were increased. The decrease in cell viability by lutein was reversed by ML171 (NADPH oxidase 1 specific inhibitor) and N-acetyl cysteine (NAC; antioxidant). ConclusionsLutein induce apoptosis of gastric cancer cells by increasing NADPH oxidase-mediated ROS production. Funding SourcesNo funding sources.

Rapamycin Suppresses Penile NADPH Oxidase Activity to Preserve Erectile Function in Mice Fed a Western Diet.

The mechanistic target of rapamycin (mTOR) is a nutrient-sensitive cellular signaling kinase that has been implicated in the excess production of reactive oxygen species (ROS). NADPH oxidase-derived ROS have been implicated in erectile dysfunction pathogenesis. The objective of this study was to determine if mTOR is an activator of NADPH oxidase in the penis and to determine the functional relevance of this pathway in a translationally relevant model of diet-induced erectile dysfunction. Male mice were fed a control diet or a high-fat, high-sucrose Western style diet (WD) for 12 weeks and treated with vehicle or rapamycin for the final 4 weeks of the dietary intervention. Following the intervention, erectile function was assessed by cavernous nerve-stimulated intracavernous pressure measurement, in vivo ROS production was measured in the penis using a microdialysis approach, and relative protein contents from the corpus cavernosum were determined by Western blot. Erectile function was impaired in vehicle treated WD-mice and was preserved in rapamycin treated WD-mice. Penile NADPH oxidase-mediated ROS were elevated in WD-mice and suppressed by rapamycin treatment. Western blot analysis suggests mTOR activation with WD by increased active site phosphorylation of mTOR and p70S6K, and increased expression of NADPH oxidase subunits, all of which were suppressed by rapamycin. These data suggest that mTOR is an upstream mediator of NADPH oxidase in the corpus cavernosum in response to a chronic Western diet, which has an adverse effect on erectile function.

Melatonin functions in priming of stomatal immunity in Panax notoginseng and Arabidopsis thaliana.

Melatonin (MT) plays important roles in plant disease response, but the mechanisms are largely unknown. Here, we show that MT functions in stomatal immunity in Panax notoginseng and Arabidopsis thaliana. Biochemical analyses showed that MT-induced stomatal closure plays a prominent role in preventing invasion of bacteria Pseudomonas syringe pv. tomato (Pst) DC3000 via activation of mitogen-activated protein kinase (MAPK) and NADPH oxidase-mediated reactive oxygen species production in P. notoginseng. The first putative phytomelatonin receptor 1 (PMTR1) is a plasma membrane protein required for perceiving MT signaling in stomatal closure and activation of MAPK. Biochemical and genetic tests found PMTR1 is essential for flg22- and MT-induced MAPK activation in a heterotrimeric GTP-binding protein Gα subunit GPA1-independent manner. GPA1 functions in the same genetic pathways of FLS2/BAK1 (Flagellin Sensing 2/Brassinosteroid Insensitive 1-associated kinase 1)- as well as PMTR1-mediated flg22 and MT signaling in stomatal closure. The stomata in pmtr1 are insensitive to MT and flg22, but the application of MT induces stomatal closure and reduces the bacterial growth in fls2 and bak1 plants, indicating that PMTR1 might be a downstream signaling component in FLS2- and BAK1-mediated stomatal immunity. In summary, our results (i) demonstrate that phytomelatonin functions in the priming of stomatal immunity and (ii) provide insights into the phytomelatonin signaling transduction pathway.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells.

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

Impact of small RNA RaoN on nitrosative-oxidative stress resistance and virulence of Salmonella enterica serovar Typhimurium.

RaoN is a Salmonella-specific small RNA that is encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11. We previously reported that RaoN is induced under conditions of acid and oxidative stress combined with nutrient limitation, contributing to the intramacrophage growth of Salmonella enterica serovar Typhimurium. However, the role of RaoN in nitrosative stress response and virulence has not yet been elucidated. Here we show that the raoN mutant strain has increased susceptibility to nitrosative stress by using a nitric oxide generating acidified nitrite. Extending previous research on the role of RaoN in oxidative stress resistance, we found that NADPH oxidase inhibition restores the growth of the raoN mutant in LPS-treated J774A.1 macrophages. Flow cytometry analysis further revealed that the inactivation of raoN leads to an increase in the intracellular level of reactive oxygen species (ROS) in Salmonella-infected macrophages, suggesting that RaoN is involved in the inhibition of NADPH oxidase-mediated ROS production by mechanisms not yet resolved. Moreover, we evaluated the effect of raoN mutation on the virulence in murine systemic infection and determined that the raoN mutant is less virulent than the wild-type strain following oral inoculation. In conclusion, small regulatory RNA RaoN controls nitrosative-oxidative stress resistance and is required for virulence of Salmonella in mice.

The redox metabolic pathways function to limit Anaplasma phagocytophilum infection and multiplication while preserving fitness in tick vector cells

Aerobic organisms evolved conserved mechanisms controlling the generation of reactive oxygen species (ROS) to maintain redox homeostasis signaling and modulate signal transduction, gene expression and cellular functional responses under physiological conditions. The production of ROS by mitochondria is essential in the oxidative stress associated with different pathologies and in response to pathogen infection. Anaplasma phagocytophilum is an intracellular pathogen transmitted by Ixodes scapularis ticks and causing human granulocytic anaplasmosis. Bacteria multiply in vertebrate neutrophils and infect first tick midgut cells and subsequently hemocytes and salivary glands from where transmission occurs. Previous results demonstrated that A. phagocytophilum does not induce the production of ROS as part of its survival strategy in human neutrophils. However, little is known about the role of ROS during pathogen infection in ticks. In this study, the role of tick oxidative stress during A. phagocytophilum infection was characterized through the function of different pathways involved in ROS production. The results showed that tick cells increase mitochondrial ROS production to limit A. phagocytophilum infection, while pathogen inhibits alternative ROS production pathways and apoptosis to preserve cell fitness and facilitate infection. The inhibition of NADPH oxidase-mediated ROS production by pathogen infection appears to occur in both neutrophils and tick cells, thus supporting that A. phagocytophilum uses common mechanisms for infection of ticks and vertebrate hosts. However, differences in ROS response to A. phagocytophilum infection between human and tick cells may reflect host-specific cell tropism that evolved during pathogen life cycle.

Mycobacterium tuberculosis PPE2 Protein Interacts with p67phox and Inhibits Reactive Oxygen Species Production.

Mycobacterium tuberculosis employs defense mechanisms to protect itself from reactive oxygen species (ROS)-mediated cytotoxicity inside macrophages. In the current study, we found that a secretory protein of M. tuberculosis PPE2 disrupted the assembly of NADPH oxidase complex. PPE2 inhibited NADPH oxidase-mediated ROS generation in RAW 264.7 macrophages and peritoneal macrophages from BALB/c mice. PPE2 interacted with the cytosolic subunit of NADPH oxidase, p67phox, and prevented translocation of p67phox and p47phox to the membrane, resulting in decreased NADPH oxidase activity. Trp236 residue present in the SH3-like domain of PPE2 was found to be critical for its interaction with p67phox Trp236Ala mutant of PPE2 did not interact with p67phox and thereby did not affect ROS generation. M. tuberculosis expressing PPE2 and PPE2-null mutants complemented with PPE2 survived better than PPE2-null mutants in infected RAW 264.7 macrophages. Altogether, this study suggests that PPE2 inhibits NADPH oxidase-mediated ROS production to favor M. tuberculosis survival in macrophages. The findings that M. tuberculosis PPE2 protein is involved in the modulation of oxidative response in macrophages will help us in improving our knowledge of host-pathogen interactions and the application of better therapeutics against tuberculosis.

Overexpression of the dominant negative nbexo70d1 mutantionconfers tolerance to salt stress in transgenic tobacco

The vesicle trafficking process, which involves exocytotic and endocytotic pathways, has been reported to play a role in regulating plant responses to different environmental stresses. The Exo70 protein is important for the localization of the exocyst in the plasma membrane; however, its role in the physiology of stress tolerance is currently unclear. In this study, we characterized NbExo70D1, an Exo70 gene from tobacco (Nicotiana benthamiana). It was shown to have a role in the plant response to salt stress. More specifically, tolerance to salt stress is conferred by the overexpression of the dominant negative nbexo70d1 domain D mutation in transgenic tobacco. In addition, a reduced accumulation of reactive oxygen species (ROS) under salt treatment was observed in the transgenic lines compared to the wild type. Treatment with diphenylene iodonium, an NADPH oxidase inhibitor, resulted in a decrease in salt stress-triggered ROS production in the roots of both wild type tobacco and transgenic tobacco. Furthermore, there was a reduction in NADPH oxidase activity in the transgenic plants under salt treatment, which indicates NbExo70D1 is involved in NADPH oxidase-mediated ROS production. We also characterized the tissue-specific expression patterns of NbExo70D1 during salt stress response by expressing the ProNbExo70D1-β-glucuronidase reporter construct in plants. Importantly, the GFP-NbExo70D1 fusion protein was localized in both the plasma membrane and the cytoplasm; expressing the dominant negative mutation disrupted the interaction between NbExo70D1 protein and the plasma membrane. Overall, our study suggests that Exo70 plays an important role in regulating the production and transmission of ROS as part of a salt stress response in plants.

067 Rapamycin Suppresses NADPH Oxidase-mediated Reactive Oxygen Species Production and Reverses Erectile Dysfunction in Western Diet-fed Mice

The mammalian target of rapamycin (mTOR) is a nutrient-sensitive cellular signaling kinase that has been shown to be activated by high-fat and/or high-sugar feeding in highly metabolic tissues such as adipose tissue, liver, and skeletal muscle. mTOR activation has been implicated in the excess production of reactive oxygen species (ROS) resulting from overnutrition, which has been associated with activation of NADPH oxidase (Nox) as a source of excess ROS production. A Western style high-fat, high-sugar diet has recently been used to induce erectile dysfunction (ED) in rodents, in which elevated Nox has been implicated in ED pathogenesis. The objective of this study was to determine if mTOR is an upstream activator of Nox in the penis in response to the Western diet (WD) and to determine if this pathway is relevant in WD-induced ED. Young male C57Bl/6J mice (n = 90) were fed a control diet (CD) or WD ad libitum for 12 weeks. For the final four weeks of the dietary intervention, mice were intraperitoneally injected with either vehicle or the mTOR inhibitor rapamycin (Group 1: 1 mg/kg; Group 2: 2 mg/kg) three days/week. Following the intervention, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation. In separate mice following the same intervention, in vivo ROS production was measured in the penis utilizing a microdialysis approach. Microdialysis probes were inserted into the penis of anesthetized mice and perfused with saline containing 100 μM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase. ROS convert the reagents to a fluorescent byproduct resorufin, which was measured in the outflowing dialysate. Three replicate samples were collected, after which 300 μM apocynin, a Nox inhibitor, was added to the perfusate, and additional samples were collected. Nox-mediated ROS were determined by calculation of the ROS that was inhibited by apocynin. Significant differences between groups were determined by two-way ANOVA with Tukey’s multiple comparisons post-hoc analysis, with an alpha level of 0.05.

Leishmania donovani Induces Autophagy in Human Blood-Derived Neutrophils.

Neutrophils, the essential components of the innate immune system, are recruited in large numbers to the pathogen site of entry. Several pathogens induce neutrophil autophagy; however, function of autophagic events during Leishmania parasite infection remain unknown. In this article, we report a finding that is new, to our knowledge, of how Leishmania-induced human polymorphonuclear neutrophil (hPMN) autophagy regulates the silent mode of parasite transfer to macrophages by influencing the engulfment of infected cells. Leishmania infection induced a time-dependent autophagy increase responsive to block by 3-methyladenine but sensitive to ULK1/2 inhibition only after 3 h. This suggested the prevalence of canonical autophagy during later hours, ULK1/2 inhibition being able to block only canonical autophagy. Interaction of Rubicon and Beclin-1 at 1 h postinfection affirmed the prevalence of noncanonical autophagy during early infection. There was a reduction in macrophage uptake of parasite-exposed hPMNs treated with 3-methyladenine or ULK1/2 inhibitor, suggesting the involvement of both noncanonical and canonical autophagy in neutrophil engulfment. Autophagy inducer rapamycin augmented neutrophil engulfment by macrophages. Redistribution of hPMN surface CD47 encouraged neutrophil uptake. Activation of ERK, phosphoinositide 3-kinase, and NADPH oxidase-mediated reactive oxygen species generation were induced after parasite binding. The lpg1-knockout parasites expressing defective lipophosphoglycan did not induce autophagy, indicating that lipophosphoglycan is necessary for interaction with the neutrophils. Autophagy induction was TLR2/4 independent because the receptor blockade did not interfere with infection-induced autophagy. In summary, the engulfment of neutrophils by the macrophages was influenced by the escalation of hPMN autophagy, which is an important event during Leishmania infection.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/β or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1−/− mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1−/− macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Global metabolomics approach in in vitro and in vivo models reveals hepatic glutathione depletion induced by amorphous silica nanoparticles

The present study aimed to investigate the mechanisms involved in amorphous silica nanoparticles (aSiNPs)-mediated hepatotoxicity through the evaluation of changes in global metabolomics in in vitro and in vivo systems. 1H NMR-based non-targeted global metabolomics and biochemical approaches were conducted in an aSiNPs-treated human hepatoma cell line (HepG2) and in ICR mice liver. The non-targeted NMR-based metabolomic analysis, followed by pathway analysis, revealed the perturbation of glutathione metabolism and the depletion of the glutathione pool after aSiNPs treatment in both in vitro (HepG2 cells) and in vivo systems. The total glutathione level, glutathione-S-transferase enzyme activity, and antioxidant gene expression strongly corroborated the metabolomic analysis results. The in vitro results were further supported by the in vivo data, specifically for metabolites profiling (Pearson Correlation coefficient is 0.462 (p = 0.026)). Furthermore, the depletion of glutathione, the formation of NADPH oxidase-mediated reactive oxygen species, and oxidative stress were evident in aSiNPs-treated HepG2 cells. Overall, the suppression of glutathione metabolism and oxidative stress are among the principal causes of aSiNPs-mediated hepatotoxicity.

Dextromethorphan Attenuates NADPH Oxidase-Regulated Glycogen Synthase Kinase 3β and NF-κB Activation and Reduces Nitric Oxide Production in Group A Streptococcal Infection.

Group A Streptococcus (GAS) is an important human pathogen that causes a wide spectrum of diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), an antitussive drug, has been demonstrated to efficiently reduce inflammatory responses, thereby contributing to an increased survival rate of GAS-infected mice. However, the anti-inflammatory mechanisms underlying DM treatment in GAS infection remain unclear. DM is known to exert neuroprotective effects through an NADPH oxidase-dependent regulated process. In the present study, membrane translocation of NADPH oxidase subunit p47phox and subsequent reactive oxygen species (ROS) generation induced by GAS infection were significantly inhibited via DM treatment in RAW264.7 murine macrophage cells. Further determination of proinflammatory mediators revealed that DM effectively suppressed inducible nitric oxide synthase (iNOS) expression and NO, tumor necrosis factor alpha, and interleukin-6 generation in GAS-infected RAW264.7 cells as well as in air-pouch-infiltrating cells from GAS/DM-treated mice. GAS infection caused AKT dephosphorylation, glycogen synthase kinase-3β (GSK-3β) activation, and subsequent NF-κB nuclear translocation, which were also markedly inhibited by treatment with DM and an NADPH oxidase inhibitor, diphenylene iodonium. These results suggest that DM attenuates GAS infection-induced overactive inflammation by inhibiting NADPH oxidase-mediated ROS production that leads to downregulation of the GSK-3β/NF-κB/NO signaling pathway.

Importance of NADPH oxidase-mediated redox signaling in the detrimental effect of CRP on pancreatic insulin secretion

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation.

Partial Protection by Dietary Antioxidants Against Ethanol-Induced Osteopenia and Changes in Bone Morphology in Female Mice.

Chronic alcohol consumption leads to increased fracture risk and an elevated risk of osteoporosis by decreasing bone accrual through increasing osteoclast activity and decreasing osteoblast activity. We have shown that this mechanism involves the generation of reactive oxygen species (ROS) produced by NADPH oxidases. It was hypothesized that different dietary antioxidants, N-acetyl cysteine (NAC; 1.2mg/kg/d), and α-tocopherol (Vit.E; 60mg/kg/d) would be able to attenuate the NADPH oxidase-mediated ROS effects on bone due to chronic alcohol intake. To study the effects of these antioxidants, female mice received a Lieber-DeCarli liquid diet containing ethanol (EtOH) with or without additional antioxidant for 8weeks. Tibias displayed decreased cortical bone mineral density in both the EtOH and EtOH+antioxidant groups compared to pair-fed (PF) and PF+antioxidant groups (p<0.05). However, there was significant protection from trabecular bone loss in mice fed either antioxidant (p<0.05). Microcomputed tomography analysis demonstrated a significant decrease in bone volume (bone volume/tissue volume) and trabecular number (p<0.05), along with a significant increase in trabecular separation in the EtOH compared to PF (p<0.05). In contrast, the EtOH+NAC and EtOH+Vit.E did not statistically differ from their respective PF controls. Ex vivo histologic sections of tibias were stained for nitrotyrosine, an indicator of intracellular damage by ROS, and tibias from mice fed EtOH exhibited significantly more staining than PF controls. EtOH treatment significantly increased the number of marrow adipocytes per mm as well as mRNA expression of aP2, an adipocyte marker in bone. Only NAC was able to reduce the number of marrow adipocytes to PF levels. EtOH-fed mice exhibited reduced bone length (p<0.05) and had a reduced number of proliferating chondrocytes within the growth plate. NAC and Vit.E prevented this (p<0.05). These data show that alcohol's pathological effects on bone extend beyond decreasing bone mass and suggest a partial protective effect of the dietary antioxidants NAC and Vit.E at these doses with regard to alcohol effects on bone turnover and bone morphology.

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence.

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence.