Inhibition Of Superoxide Production Research Articles

Piceatannol reduces radiation-induced DNA double-strand breaks by suppressing superoxide production and enhancing ATM-dependent repair efficiency

In contemporary society, humans are susceptible to various radiation-borne hazards, including exposure to therapeutic modalities using low-linear energy transfer (low-LET) radiations (X-rays and γ-rays), natural high-LET radiation sourced from cosmic rays, as well as nuclear accidents such as the Fukushima Daiichi Nuclear Power Plant incident. Therefore, this threat incites an imminent necessity to develop novel radioprotective agents against a wide range of LET radiation and elucidate the underlying molecular mechanisms. This study aimed at assessing the radioprotectivity of Piceatannol (PIC), a potent antioxidant polyphenol present in abundance in passion fruit, by investigating its effects on radiation-induced reactive oxygen species (ROS) production and the consequent DNA double-strand break (DSB) capacity and cellular senescence. Specifically, total ROS was evaluated by DCFDA staining, mitochondrial superoxide by MitoSOX staining, DSB by γ-H2AX immunostaining, and cellular senescence by Senescence-associated-β-galactosidase staining.The results demonstrated that PIC administration prior to exposure to both X-ray and high-LET radiation impeded radiation-induced DSB by suppressing ROS production. Interestingly, post-irradiation PIC treatment did not alter ROS levels but enhanced the efficiency of Ataxia Telangiectasia Mutated (ATM)-mediated DSB repair. Additionally, post-irradiation PIC treatment diminished senescence-associated beta-galactosidase levels, indicating that it hinders cellular senescence.Conclusively, PIC exerts radioprotective effects against a wide range of LET radiation. The study findings validate the potential application of PIC not only as a radical scavenger but also as a novel DSB repair-activating radioprotective agent.

Hypoxia decreases mitochondrial ROS production in cells

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site IQ in complex I and site IIIQo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells’ ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site IIIQo decreases HIF1α expression, and found similar effects of suppressing site IQ or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H2O2 derived from site IQ, site IIIQo and NOX in cells is necessary to permit HIF1α stabilization by other signals.

The water extract and the lectin WSMoL from the seeds of Moringa oleifera prevent the hypertension onset by decreasing renal oxidative stress.

Maternal endotoxemia disturbs the intrauterine environment, impairs nephrogenesis, and increases the risk of hypertension and kidney disease in adulthood. Here, it was investigated whether maternal treatment with the water extract of Moringa oleifera seeds (WEMoS) or the water-soluble M. oleifera seed lectin (WSMoL) prevents the oxidative stress induced by lipopolysaccharide (LPS) in pregnant rats, and the renal injury and hypertension in the adult offspring. The administration of WEMoS or WSMoL prevented the stimulatory effects of LPS on lipid peroxidation in the maternal-placenta-fetuses environment. The impact of WEMoS was linked to decreased superoxide anions production in the placenta. The effects of WSMoL were parallel to the inhibition of superoxide anion production and NADPH oxidase activity. The WSMoL also prevented increased NADPH oxidase activity in the fetal kidney. The LPS offspring presented higher systolic blood pressure (SBP) and increased lipid peroxidation, reactive oxygen species (ROS), NADPH oxidase activity, and nitrate/nitrite in the kidney; the maternal treatment with WEMoS and WSMoL prevented these changes. In conclusion, the present study demonstrates that WEMoS and WSMoL have protective effects on maternal endotoxemia, which involve antioxidant and anti-inflammatory actions that prevent the programming of hypertension.

Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1β production in air pollution fine particulate matter (PM2.5)-primed macrophages

Exposure to air pollution fine particulate matter (PM2.5) aggravates respiratory and cardiovascular diseases. It has been proposed that PM2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1β. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM2.5 surrogates, significant IL-1β release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1β release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1β production in ROFA-exposed NLRP3-deficient (Nlrp3−/−) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1β release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K+ efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1β release. Interestingly, specific inhibition of superoxide anion production (O2•-) from mitochondrial respiratory Complex I, but not III, blunted IL-1β release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM2.5 promotes IL-1β release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM2.5.

Telomerase reverse transcriptase restores pancreatic microcirculation profiles and attenuates endothelial dysfunction by inhibiting mitochondrial superoxide production: A potential target for acute pancreatitis therapy

BackgroundAcute pancreatitis (AP) is a potentially lethal disease related to prominent microcirculation dysfunction. Pancreatic microvascular endothelial dysfunction enhances oxidative stress with tissue damage. Increased superoxide production disrupts endothelial junction integrity and increases endothelial permeability. Endothelial mitochondrial ROS (mtROS) represent a major intracellular source of superoxide anions. The non-canonical function of telomerase reverse transcriptase (TERT) involves the maintenance of cellular redox homeostasis in somatic tissues. MethodsWe investigated whether TERT restores microcirculation dysfunction and attenuates the endothelium injury by inhibiting superoxide production during AP progression. We established TERT transgenic and TERT knock-down mice and used cerulein (CER) and lipopolysaccharide (LPS) injections to induce AP models. In addition, we exposed HUVECs to LPS following TERT overexpression or silencing to explore the role of TERT in endothelial dysfunction. We also performed flow cytometry and confocal microscopy assays by using HUVECs. And a mtROS inhibitor, MitoTempo, was used to scavenge mitochondria superoxide and alkyl. ResultsTERT transgenic mice were found to have restored pancreatic microcirculation profiles and microvascular endothelial morphology compared with wild-type mice under cerulein injection. In contrast, TERT silencing displayed the opposite effect in response to cerulein. Subsequently, we showed that TERT overexpression attenuates mtROS production and mitochondrial dysfunction during LPS-stimulated endothelial dysfunction. Furthermore, we found that TERT overexpression maintains the balance between mitochondrial contents and ATP level during endothelial dysfunction. In addition, the protective trend of MitoTempo is impeded after TERT silencing. ConclusionTERT restores pancreatic microcirculation dysfunction and attenuates microvascular endothelium lesions by inhibiting the increase of superoxide production and mitochondrial dysfunction.

Apelin/APJ system protects placental trophoblasts from hypoxia-induced oxidative stress through activating PI3K/Akt signaling pathway in preeclampsia

BackgroundPreeclampsia is a placentally induced syndrome with diverse clinical presentation that currently has no cure. Oxidative stress is a potent inducer of placental dysfunction. The apelin receptor (APJ) system is a pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. This study examines the alteration of circulating apelin levels and placental APJ expression in preeclampsia and investigates whether apelin/APJ system can protect placental trophoblast from hypoxia-induced oxidative stress injury through PI3K/AKT signaling pathway. ResultsOur results confirmed that maternal apelin concentration was increased in women with preeclampsia, but APJ expression was reduced in the preeclamptic placentas. Apelin-13 treatment not only specifically attenuated CoCl2-induced superoxide production, but also prevented CoCl2-induced reduction of SOD activity and SOD1 expression. In addition, apelin-13 suppressed CoCl2-induced apoptosis by increasing the expression of bcl-2/bax ratio and by decreasing the expression of active caspase-3 in placental trophoblasts. Furthermore, we found that apelin-13 binding APJ activated the PI3K and AKT kinases and inhibition of PI3K kinase significantly blocked the anti-oxidative effects of apelin-13 in placental trophoblasts. ConclusionsDecrease of placental APJ expression is associated with oxidative stress-induced placental dysfunction in preeclampsia, and increased circulating apelin could be a moderately successful marker to differentiate subjects with preeclampsia from healthy pregnant women. Inhibition of superoxide production and caspase-3 cleavage, together with upregulation of SOD activity/expression and bcl-2/bax ratio, could be the potential molecular mechanisms by which apelin-13/APJ protects placental trophoblasts from oxidative stress injury.

Reactivity of renal and mesenteric resistance vessels to angiotensin II is mediated by NOXA1/NOX1 and superoxide signaling.

Activation of NADPH oxidase (NOX) enzymes and the generation of reactive oxygen species and oxidative stress regulate vascular and renal function and contribute to the pathogenesis of hypertension. The present study examined the role of NOXA1/NOX1 function in vascular reactivity of renal and mesenteric resistance arteries/arterioles of wild-type and Noxa1-/- mice. A major finding was that renal blood flow is less sensitive to acute stimulation by angiotensin II (ANG II) in Noxa1-/- mice compared with wild-type mice, with a direct action on resistance arterioles independent of nitric oxide (NO) bioavailability. These functional results were reinforced by immunofluorescence evidence of NOXA1/NOX1 protein presence in renal arteries, afferent arterioles, and glomeruli as well as their upregulation by ANG II. In contrast, the renal vascular response to the thromboxane mimetic U46619 was effectively blunted by NO and was similar in both mouse genotypes and thus independent of NOXA1/NOX1 signaling. However, phenylephrine- and ANG II-induced contraction of isolated mesenteric arteries was less pronounced and buffering of vasoconstriction after acetylcholine and nitroprusside stimulation was reduced in Noxa1-/- mice, suggesting endothelial NO-dependent mechanisms. An involvement of NOXA1/NOX1/O2•- signaling in response to ANG II was demonstrated with the specific NOXA1/NOX1 assembly inhibitor C25 and the nonspecific NOX inhibitor diphenyleneiodonium chloride in cultured vascular smooth muscle cells and isolated mesenteric resistance arteries. Collectively, our data indicate that the NOX1/NOXA1/O2•- pathway contributes to acute vasoconstriction induced by ANG II in renal and mesenteric vascular beds and may contribute to ANG II-induced hypertension.NEW & NOTEWORTHY Renal reactivity to angiotensin II (ANG II) is mediated by superoxide signaling produced by NADPH oxidase (NOX)A1/NOX1. Acute vasoconstriction of renal arteries by ANG was blunted in Noxa1-/- compared with wild-type mice. NOXA1/NOX1/O2•- signaling was also observed in ANG II stimulation of vascular smooth muscle cells and isolated mesenteric resistance arteries, indicating that it contributes to ANG II-induced hypertension. A NOXA1/NOX1 assembly inhibitor (C25) has been characterized that inhibits superoxide production and ameliorates the effects of ANG II.

Inside the Mechanism of Action of Three Pyrazole Derivatives in Human Platelets and Endothelial Cells

In the effort to obtain multitarget compound interfering with inflammation, oxidative stress, and tumorigenesis, we synthesized a small library of pyrazole compounds, selecting 4a, 4f, and 4g as the most noteworthy being IC50 against platelet ROS production induced by thrombin of about 10 µM. The in vitro antioxidant potential of the three molecules was evaluated, and since they show a remarkable antioxidative activity, their effect on several parameter indicative of oxidative status and on the efficiency of the aerobic metabolism was tested. The three molecules strongly inhibit superoxide anion production, lipid peroxidation, NADPH oxidase activity and almost restore the oxidative phosphorylation efficiency in thrombin-stimulated platelet, demonstrating a protective effect against oxidative stress. This effect was confirmed in endothelial cell in which 4a, 4f, and 4g show an interesting inhibition activity on H2O2-stimulated EA.hy926 cells. At last, antiproliferative activity of 4a, 4f, and 4g was submitted to a large screening at the NCI. The molecules show interesting anticancer activity, among them the most remarkable is 4g able to strongly inhibit the proliferation of both solid tumor and leukemia cells lines. In conclusion, all the three newly synthetized pyrazoles show remarkable antioxidant and antiproliferative effect worthy of further study.

Mito-TEMPO protects preimplantation porcine embryos against mitochondrial fission-driven apoptosis through DRP1/PINK1-mediated mitophagy

AimsMdivi-1 (Md-1) is a well-known inhibitor of mitochondrial fission and mitophagy. The mitochondrial superoxide scavenger Mito-TEMPO (MT) exerts positive effects on the developmental competence of pig embryos. This study aimed to explore the adverse effects of Md-1 on developmental capacity in porcine embryos and the protective effects of MT against Md-1-induced injury. Main methodsWe exposed porcine embryos to Md-1 (10 and 50 μM) for 2 days after in vitro fertilization (IVF). MT (0.1 μM) treatment was applied for 4 days after exposing embryos to Md-1. We assessed blastocyst development, DNA damage, mitochondrial superoxide production, and mitochondrial distribution using TUNEL assay, Mito-SOX, and Mito-tracker, respectively. Subsequently, the expression of PINK1, DRP1, and p-DRP1Ser616 was evaluated via immunofluorescence staining and Western blot analysis. Key findingsMd-1 compromised the developmental competence of blastocysts. Apoptosis and mitochondrial superoxide production were significantly upregulated in 50 μM Md-1-treated embryos, accompanied by a downregulation of p-DRP1Ser616, PINK1, and LC3B levels and lower mitophagy activity at the blastocyst stage. We confirmed the protective effects of MT against the detrimental effect of Md-1 on blastocyst developmental competence, mitochondrial fission, and DRP1/PINK1-mediated mitophagy activation. Eventually, MT recovered DRP1/PINK1-mediated mitophagy and mitochondrial fission by inhibiting superoxide production in Md-1-treated embryos. SignificanceMT protects against detrimental effects of Md-1 on porcine embryos by suppressing superoxide production. These findings expand available scientific knowledge on improving outcomes of IVF.

Characterization of the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages.

Previous studies have demonstrated that cytosolic phospholipase A2α (cPLA2α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47phox in human phagocytes. Intravenous administration of antisense against cPLA2α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA2α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA2α-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2α in oxidase regulation also in mouse phagocytes. A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47phox-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47phox-PX domain (present in the human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain CONCLUSIONS: The binding between cPLA2α-C2 and p47phox-PX domains exists in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice, probably due to the absence of amino acids Cys98, Ser 99, and Thr100in the p47phox-PX domain that facilitate the binding to cPLA2α.

Astragalus mongholicus Bunge Water Extract Exhibits Anti-inflammatory Effects in Human Neutrophils and Alleviates Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice.

Neutrophils are the primary immune cells in innate immunity, which are related to various inflammatory diseases. Astragalus mongholicus Bunge is a Chinese medicinal herb used to treat various oxidative stress-related inflammatory diseases. However, there are limited studies that elucidate the effects of Astragalus mongholicus Bunge in human neutrophils. In this study, we used isolated human neutrophils activated by various stimulants to investigate the anti-inflammatory effects of Astragalus mongholicus Bunge water extract (AWE). Cell-free assays were used to examine free radicals scavenging capabilities on superoxide anion, reactive oxygen species (ROS), and nitrogen-centered radicals. Imiquimod (IMQ) induced psoriasis-like skin inflammation mouse model was used for investigating anti-psoriatic effects. We found that AWE inhibited superoxide anion production, ROS generation, and elastase release in human neutrophils, which exhibiting a direct anti-neutrophil effect. Moreover, AWE exerted a ROS scavenging ability in the 2,2’-Azobis (2-amidinopropane) dihydrochloride assay, but not superoxide anion in the xanthine/xanthine oxidase assay, suggesting that AWE exhibited anti-oxidation and anti-inflammatory capabilities by both scavenging ROS and by directly inhibiting neutrophil activation. AWE also reduced CD11b expression and adhesion to endothelial cells in activated human neutrophils. Meanwhile, in mice with psoriasis-like skin inflammation, administration of topical AWE reduced both the affected area and the severity index score. It inhibited neutrophil infiltration, myeloperoxidase release, ROS-induced damage, and skin proliferation. In summary, AWE exhibited direct anti-inflammatory effects by inhibiting neutrophil activation and anti-psoriatic effects in mice with IMQ-induced psoriasis-like skin inflammation. Therefore, AWE could potentially be a pharmaceutical Chinese herbal medicine to inhibit neutrophilic inflammation for anti-psoriasis.

The role of transient receptor potential ankyrin 1 in age-related endothelial dysfunction.

Oxidative stress plays a key role in age-related vascular disease. The present study aimed to investigate the role of an antioxidant channel, transient receptor potential ankyrin 1 (TRPA1), in age-related endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) were grown to induce replicative senescence, and 6-month-old young, 12-month-old middle-aged, and 24-month-old aged mice were used. TRPA1 was downregulated in senescent HUVECs, so were endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), and uncoupling protein 2 (UCP2). Activating TRPA1 with cinnamaldehyde prevented downregulation of eNOS, Nrf2, and UCP2, inhibited superoxide production and apoptosis, and preserved nitric oxide bioavailability in senescent HUVECs. TRPA1, phosphorylated eNOS, Nrf2 and UCP2 were significantly downregulated in aged aortas compared with young aortas after a compensatory upregulation in middle-aged aortas. Dietary administration of cinnamaldehyde for 12months prevented mitochondrial dysfunction, improved endothelium-dependent relaxation, and increased expression of eNOS, Nrf2, and UCP2 in aged aortas. Importantly, the effects of cinnamaldehyde can be blocked by a TRPA1 antagonist HC-030031. These findings suggest that TRPA1 may play a critical role in age-related endothelial dysfunction and may become a therapeutic target for the treatment and prevention of age-related vascular disease.

Synthesis, Structure and Impact of 5-Aminoorotic Acid and Its Complexes with Lanthanum(III) and Gallium(III) on the Activity of Xanthine Oxidase.

The superoxide radical ion is involved in numerous physiological processes, associated with both health and pathology. Its participation in cancer onset and progression is well documented. Lanthanum(III) and gallium(III) are cations that are known to possess anticancer properties. Their coordination complexes are being investigated by the scientific community in the search for novel oncological disease remedies. Their complexes with 5-aminoorotic acid suppress superoxide, derived enzymatically from xanthine/xanthine oxidase (X/XO). It seems that they, to differing extents, impact the enzyme, or the substrate, or both. The present study closely examines their chemical structure by way of modern methods—IR, Raman, and 1H NMR spectroscopy. Their superoxide-scavenging behavior in the presence of a non-enzymatic source (potassium superoxide) is compared to that in the presence of an enzymatic source (X/XO). Enzymatic activity of XO, defined in terms of the production of uric acid, seems to be impacted by both complexes and the pure ligand in a concentration-dependent manner. In order to better relate the compounds’ chemical characteristics to XO inhibition, they were docked in silico to XO. A molecular docking assay provided further proof that 5-aminoorotic acid and its complexes with lanthanum(III) and gallium(III) very probably suppress superoxide production via XO inhibition.

Ex Vivo Antioxidant Capacities of Fruit and Vegetable Juices. Potential In Vivo Extrapolation.

Background: In support of claims that their products have antioxidant properties, the food industry and dietary supplement manufacturers rely solely on the in vitro determination of the ORAC (oxygen radical antioxidant capacity) value, despite its acknowledged lack of any in vivo relevance. It thus appears necessary to use tests exploiting biological materials (blood, white blood cells) capable of producing physiological free radicals, in order to evaluate more adequately the antioxidant capacities of foods such as fruit and vegetable juices. Materials: Two approaches to assessing the antioxidant capacities of 21 commercial fruit and vegetable juices were compared: the ORAC assay and the “PMA–whole blood assay,” which uses whole blood stimulated by phorbol myristate acetate to produce the superoxide anion. We described in another paper the total polyphenol contents (TPCs) and individual phenolic compound contents of all the juices were investigated. Results: Ranking of the juices from highest to lowest antioxidant capacity differed considerably according to the test used, so there was no correlation (r = 0.33, p = 0.13) between the two assays when considering all juices. Although the results of the ORAC assay correlated positively with TPC (r = 0.50, p = 0.02), a much stronger correlation (r = 0.70, p = 0.004) emerged between TPC and % superoxide anion inhibition. In the PMA–whole blood assay, peonidin-3-O-glucoside, epigallocatechin gallate, catechin, and quercetin present in juices were found to inhibit superoxide anion production at concentrations below 1 µM, with a strong positive correlation. Conclusions: Associated with the determination of total and individual phenolic compounds contained in fruit and vegetable juices, the PMA–whole blood assay appears better than the ORAC assay for evaluating juice antioxidant capacity.

Leishmania donovani Metacyclic Promastigotes Impair Phagosome Properties in Inflammatory Monocytes.

Leishmaniasis, a debilitating disease with clinical manifestations ranging from self-healing ulcers to life-threatening visceral pathologies, is caused by protozoan parasites of the Leishmania genus. These professional vacuolar pathogens are transmitted by infected sand flies to mammalian hosts as metacyclic promastigotes and are rapidly internalized by various phagocyte populations. Classical monocytes are among the first myeloid cells to migrate to infection sites. Recent evidence shows that recruitment of these cells contributes to parasite burden and the establishment of chronic disease. However, the nature of Leishmania-inflammatory monocyte interactions during the early stages of host infection has not been well investigated. Here, we aimed to assess the impact of Leishmania donovani metacyclic promastigotes on antimicrobial responses within these cells. Our data showed that inflammatory monocytes are readily colonized by L. donovani metacyclic promastigotes, while infection with Escherichia coli is efficiently cleared. Upon internalization, metacyclic promastigotes inhibited superoxide production at the parasitophorous vacuole (PV) through a mechanism involving exclusion of NADPH oxidase subunits gp91phox and p47phox from the PV membrane. Moreover, we observed that unlike phagosomes enclosing zymosan particles, vacuoles containing parasites acidify poorly. Interestingly, whereas the parasite surface coat virulence glycolipid lipophosphoglycan (LPG) was responsible for the inhibition of PV acidification, impairment of the NADPH oxidase assembly was independent of LPG and GP63. Collectively, these observations indicate that permissiveness of inflammatory monocytes to L. donovani may thus be related to the ability of this parasite to impair the microbicidal properties of phagosomes.

Exogenous ketone salts inhibit superoxide production in the rat caudal solitary complex during exposure to normobaric and hyperbaric hyperoxia.

The use of hyperbaric oxygen (HBO2) in hyperbaric and undersea medicine is limited by the risk of seizures [i.e., central nervous system (CNS) oxygen toxicity, CNS-OT] resulting from increased production of reactive oxygen species (ROS) in the CNS. Importantly, ketone supplementation has been shown to delay onset of CNS-OT in rats by ∼600% in comparison with control groups (D'Agostino DP, Pilla R, Held HE, Landon CS, Puchowicz M, Brunengraber H, Ari C, Arnold P, Dean JB. Am J Physiol Regu Integr Comp Physiol 304: R829-R836, 2013). We have tested the hypothesis that ketone body supplementation inhibits ROS production during exposure to hyperoxygenation in rat brainstem cells. We measured the rate of cellular superoxide ([Formula: see text]) production in the caudal solitary complex (cSC) in rat brain slices using a fluorogenic dye, dihydroethidium (DHE), during exposure to control O2 (0.4 ATA) followed by 1-2 h of normobaric oxygen (NBO2) (0.95 ATA) and HBO2 (1.95, and 4.95 ATA) hyperoxia, with and without a 50:50 mixture of ketone salts (KS) dl-β-hydroxybutyrate + acetoacetate. All levels of hyperoxia tested stimulated [Formula: see text] production similarly in cSC cells and coexposure to 5 mM KS during hyperoxia significantly blunted the rate of increase in DHE fluorescence intensity during exposure to hyperoxia. Not all cells tested produced [Formula: see text] at the same rate during exposure to control O2 and hyperoxygenation; cells that increased [Formula: see text] production by >25% during hyperoxia in comparison with baseline were inhibited by KS, whereas cells that did not reach that threshold during hyperoxia were unaffected by KS. These findings support the hypothesis that ketone supplementation decreases the steady-state concentrations of superoxide produced during exposure to NBO2 and HBO2 hyperoxia.NEW & NOTEWORTHY Exposure of rat medullary tissue slices to levels of O2 that mimic those that cause seizures in rats stimulates cellular superoxide ([Formula: see text]) production to varying degrees. Cellular [Formula: see text] generation in the caudal solitary complex is variable during exposure to control O2 and hyperoxia and significantly decreases during ketone supplementation. Our findings support the theory that ketone supplementation delays onset of central nervous system oxygen toxicity in mammals, in part, by decreasing [Formula: see text] production in O2-sensitive neurons.

Beta3-Adrenergic Receptor Activation Alleviates Cardiac Dysfunction in Cardiac Hypertrophy by Regulating Oxidative Stress.

Background Excessive myocardial oxidative stress could lead to the congestive heart failure. NADPH oxidase is involved in the pathological process of left ventricular (LV) remodeling and dysfunction. β3-Adrenergic receptor (AR) could regulate cardiac dysfunction proved by recent researches. The molecular mechanism of β3-AR regulating oxidative stress, especially NADPH oxidase, remains to be determined. Methods Cardiac hypertrophy was constructed by the transverse aortic constriction (TAC) model. ROS and NADPH oxidase subunits expression were assessed after β3-AR agonist (BRL) or inhibitor (SR) administration in cardiac hypertrophy. Moreover, the cardiac function, fibrosis, heart size, oxidative stress, and cardiomyocytes apoptosis were also detected. Results β3-AR activation significantly alleviated cardiac hypertrophy and remodeling in pressure-overloaded mice. β3-AR stimulation also improved heart function and reduced cardiomyocytes apoptosis, oxidative stress, and fibrosis. Meanwhile, β3-AR stimulation inhibited superoxide anion production and decreased NADPH oxidase activity. Furthermore, BRL treatment increased the neuronal NOS (nNOS) expression in cardiac hypertrophy. Conclusion β3-AR stimulation alleviated cardiac dysfunction and reduced cardiomyocytes apoptosis, oxidative stress, and fibrosis by inhibiting NADPH oxidases. In addition, the protective effect of β3-AR is largely attributed to nNOS activation in cardiac hypertrophy.

MIF as a biomarker and therapeutic target for overcoming resistance to proteasome inhibitors in human myeloma

AbstractMultiple myeloma (MM) remains largely incurable despite significant advances in biotherapy and chemotherapy. The development of drug resistance is a major problem in MM management. Macrophage migration inhibitory factor (MIF) expression was significantly higher in purified MM cells from relapsed patients than those with sustained response, and MM patients with high MIF had significantly shorter progression-free survival (PFS) and overall survival (OS). MM cell lines also express high levels of MIF, and knocking out MIF made them more sensitive to proteasome inhibitor (PI)-induced apoptosis not observed with other chemotherapy drugs. Mechanistic studies showed that MIF protects MM cells from PI-induced apoptosis by maintaining mitochondrial function via suppression of superoxide production in response to PIs. Specifically, MIF, in the form of a homotrimer, acts as a chaperone for superoxide dismutase 1 (SOD1) to suppress PI-induced SOD1 misfolding and to maintain SOD1 activity. MIF inhibitor 4-iodo-6-phenylpyrimidine and homotrimer disrupter ebselen, which do not kill MM cells, enhanced PI-induced SOD1 misfolding and loss of function, resulting in significantly more cell death in both cell lines and primary MM cells. More importantly, inhibiting MIF activity in vivo displayed synergistic antitumor activity with PIs and resensitized PI-resistant MM cells to treatment. In support of these findings, gene-profiling data showed a significantly negative correlation between MIF and SOD1 expression and response to PI treatment in patients with MM. This study shows that MIF plays a crucial role in MM sensitivity to PIs and suggests that targeting MIF may be a promising strategy to (re)sensitize MM to the treatment.

High-content screening assay for compounds that enhance or inhibit leukocyte superoxide production to identify drugs for treating immune-mediated diseases

Abstract Reactive oxygen species (ROS) are produced by cells to play important roles in the effector and regulatory functions of immune cells. Our previous studies revealed that ROS act as negative regulators in IL-1β-mediated inflammation. Enhanced severity of inflammation in immune-mediated arthritis was noted when leukocyte NADPH oxidase (NOX2), which mediates the production of ROS, was defective. We hence hypothesized that drugs that modulate the leukocyte ROS production may regulate the pathogenicity of immune-mediated arthritis. Here, we devised a high-content image assay using the dye H2DCF-DA for identifying LOPAC 1280 library compounds that may facilitate or suppress superoxide production in differentiated HL-60 (dHL-60) cells. ROS levels were measured after the cells were treated with phorbol 12-myristate 13-acetate (PMA) or menadione which may induce ROS production from NOX2 or mitochondria. From a screening of 640 compounds in a 3-point interpolate titration in cell-based imaging analysis, we identified 74 drugs that may enhance the ROS production and 72 compounds that may inhibit ROS production by PMA-stimulated-dHL-60 cells. The drugs that we identified included the agonist/antagonist of neurotransmitters and their receptors, ion channel blockers, serotonin antagonists, cell stress modulators and cell cycle inhibitors. To further clarified our hypothesis, we treated NOX2-deficient mice with menadione, which tended to increase the oxidant stress in the joint tissue. The severity of the immune-mediated arthritic joint was significantly suppressed after the treatment. This novel screening method hence may be used to identify potential drugs for treating redox-related inflammatory diseases.

P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK

Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression.