Reduced Reactive Oxygen Species Formation Research Articles

Reactive Oxygen Species Formation in the Brain at Different Oxygen Levels: The Role of Hypoxia Inducible Factors.

Hypoxia inducible factor (HIF) is the master oxygen sensor within cells and is central to the regulation of cell responses to varying oxygen levels. HIF activation during hypoxia ensures optimum ATP production and cell integrity, and is associated both directly and indirectly with reactive oxygen species (ROS) formation. HIF activation can either reduce ROS formation by suppressing the function of mitochondrial tricarboxylic acid cycle (TCA cycle), or increase ROS formation via NADPH oxidase (NOX), a target gene of HIF pathway. ROS is an unavoidable consequence of aerobic metabolism. In normal conditions (i.e., physioxia), ROS is produced at minimal levels and acts as a signaling molecule subject to the dedicated balance between ROS production and scavenging. Changes in oxygen concentrations affect ROS formation. When ROS levels exceed defense mechanisms, ROS causes oxidative stress. Increased ROS levels can also be a contributing factor to HIF stabilization during hypoxia and reoxygenation. In this review, we systemically review HIF activation and ROS formation in the brain during hypoxia and hypoxia/reoxygenation. We will then explore the literature describing how changes in HIF levels might provide pharmacological targets for effective ischaemic stroke treatment. HIF accumulation in the brain via HIF prolyl hydroxylase (PHD) inhibition is proposed as an effective therapy for ischaemia stroke due to its antioxidation and anti-inflammatory properties in addition to HIF pro-survival signaling. PHD is a key regulator of HIF levels in cells. Pharmacological inhibition of PHD increases HIF levels in normoxia (i.e., at 20.9% O2 level). Preconditioning with HIF PHD inhibitors show a neuroprotective effect in both in vitro and in vivo ischaemia stroke models, but post-stroke treatment with PHD inhibitors remains debatable. HIF PHD inhibition during reperfusion can reduce ROS formation and activate a number of cellular survival pathways. Given agents targeting individual molecules in the ischaemic cascade (e.g., antioxidants) fail to be translated in the clinic setting, thus far, HIF pathway targeting and thereby impacting entire physiological networks is a promising drug target for reducing the adverse effects of ischaemic stroke.

293. Preclinical assessment of new generation antiplatelet therapies to prevent preeclampsia

Key pathophysiological steps in preeclampsia include 1) placental oxidative stress, 2) elevated sFlt1 and 3) endothelial dysfunction. While the antiplatelet drug aspirin has shown potential to prevent preeclampsia, its effectiveness remains limited. New generation antiplatelet drugs (Clopidogrel, Prasugrel and Ticagrelor) have distinct properties beyond antiplatelet actions (inflammation and endothelial dysfunction), thus may be more effective to prevent preeclampsia. We examined whether new generation antiplatelet drugs are better than aspirin at countering various pathophysiological steps in models of preeclampsia. Methods Primary human cytotrophoblast, placental explants and endothelial cells (HUVEC and uterine microvascular (UtMVs)) were treated with increasing doses of Clopidogrel, Prasugrel, Ticagrelor or Aspirin (0–100 μM). Endothelial dysfunction was induced and antiplatelet agents were added. Functional assays measured the adherence of primary human monocytes to stimulated endothelial cells. Media and cell lysates were collected to assess 1) ROS production, 2) antioxidant response element signaling pathways, 3) production of vasoactive mediators, 4) production of sFlt1, PlGF and pro-inflammatory mediators and 4) markers of endothelial dysfunction. Results New generation antiplatelet agents induced nuclear Nrf2 translocation (antioxidant transcription factor), increased antioxidant gene expression (HO-1, NQO1 and GCLC). Consistently reactive oxygen species (ROS) production was reduced with new generation antiplatelet treatment. Furthermore the new generation antiplatelets potently reduced sFlt1 secretion from preeclamptic placental explants, and importantly they also increased PlGF mRNA expression. Antiplatelet agents rescued endothelial dysfunction, mitigating monocyte-endothelial adhesion, and reduced VCAM1 and ET-1 expression (both mRNA and protein) and enhanced eNOS activity. Aspirin however had no beneficial effects in our in vitro/ex vivo models of preeclampsia. Conclusions In contrast to aspirin, new generation antiplatelets potently upregulate antioxidant defences, reduce ROS formation, decrease sFlt1 secretion, enhance activity of vasoactive mediators and counter endothelial dysfunction in human models of preeclampsia. Given they are classified as category B/C drugs, they represent exciting candidate therapies to prevent preeclampsia.

Bone Response to Dietary Co-Enrichment with Powdered Whole Grape and Probiotics.

Nutrition is a primary modifiable determinant of chronic noncommunicable disease, including osteoporosis. An etiology of osteoporosis is the stimulation of bone-resorbing osteoclasts by reactive oxygen species (ROS). Dietary polyphenols and probiotics demonstrate protective effects on bone that are associated with reduced ROS formation and suppressed osteoclast activity. This study tested the effect of dietary enrichment with powdered whole grape and probiotics (composed of equal parts Bifidobacterium bifidum, B. breve, Lactobacillus casei, L. plantarum, and L. bulgaricus) on bone microarchitecture in a mouse model of age-related osteoporosis. Groups (n = 7 each) of 10-month-old male mice were fed one of six diets for 6 months: 10% grape powder with sugar corrected to 20%; 20% grape powder; 1% probiotic with sugar corrected to 20%; 10% grape powder + 1% probiotic with sugar corrected to 20%; 20% grape powder + 1% probiotic; 20% sugar control. Femur, tibia and 4th lumbar vertebrae from 10-month-old mice served as comparator baseline samples. Bone microarchitecture was measured by micro-computed tomography and compared across diet groups using analysis of variance. Aging exerted a significant effect on tibia metaphysis trabecular bone, with baseline 10-month-old mice having significantly higher bone volume/total volume (BV/TV) and trabecular number measurements and lower trabecular spacing measurements than all 16-month-old groups (p < 0.001). Neither grape nor probiotic enrichment significantly improved bone microarchitecture during aging compared to control diet. The combination of 20% grape + 1% probiotic exerted detrimental effects on tibia metaphysis BV/TV compared to 10% grape + 1% probiotic, and trabecular number and trabecular spacing compared to 10% grape + 1% probiotic, 1% probiotic and control groups (p < 0.05). Femur metaphysis trabecular bone displayed less pronounced aging effects than tibia bone, but also showed detrimental effects of the 20% grape + 1% probiotic vs. most other diets for BV/TV, trabecular number, trabecular spacing and trabecular pattern factor (p < 0.05). Tibia and femur diaphysis cortical bone (cortical wall thickness and medullary area) displayed neither aging nor diet effects (p > 0.05). Vertebrae bone showed age-related deterioration in trabecular thickness and trabecular spacing and a trend toward preservation of trabecular thickness by grape and/or probiotic enrichment (p < 0.05). These findings demonstrate no benefit to bone of combined compared to independent supplementation with probiotics or whole grape powder and even suggest an interference of co-ingestion.

Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-β-synthase is deficient (cbs−/−), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

In vitro regulation of reactive oxygen species formation in red blood cells of homozygous sickle cell patients using Vitamin C

Background: Sickle cell patients produce more reactive oxygen species (ROS) than healthy individuals, leading to increased cell membrane damage. Theoretically, reducing ROS formation would preserve red cell membranes of sickle cell patients. Vitamin C is a powerful anti-oxidant capable of inhibiting ROS formation in a variety of situations, by functioning as an electron donor to reduce molecular oxygen. This study aimed to determine whether Vitamin C reduced ROS formation in sickle red cells.Methods:27 homozygous (HbSS) patients were recruited from the outpatient clinics of Lagos University Teaching Hospital, Nigeria, and annex at the Sickle Cell Foundation, Lagos, Nigeria. Demographic information and EDTA patient blood samples were collected. The test group were red cells preincubated in 80uM and 100uM Vitamin C concentrations before stressing with tertbutylhydroperoxide. These were compared to stressed matched controls preincubated in phosphate buffered saline. Cell staining was done with CellRox Orange followed by flow cytometry to quantify ROS.Results: ROS count for Vitamin C pre-treated red cells was significantly lower than matched controls (p&lt;0.001). Average ROS count for 80uM test samples was 27.5/ul (95% CI, 17.5 to 72.5) and for 100uM 3.9/ul (95% CI, 1.9 to 5.9). Male gender was significantly associated with elevated baseline ROS count (p=0.03).Conclusion:Vitamin C reduced ROS formation in HbSS cells. Future studies should focus on a role for Vitamin C as a safe, cheap addition to maintenance therapy of sickle cell patients.

Hydrogen sulfide attenuates cardiac injury in takotsubo cardiomyopathy by alleviating oxidative stress

Takotsubo cardiomyopathy (TCM) is characterized by transient left ventricular apical ballooning with the absence of coronary occlusion, which is an acute cardiac syndrome with substantial morbidity and mortality. It was reported that reduced endogenous hydrogen sulfide (H2S) levels may be related to various heart diseases. The present study investigated the mechanism by which H2S administration modulates and protects cardiac function in TCM rats. In order to establish a TCM model, Sprague Dawley (SD) rats were injected with a single dose of β-adrenergic agonist isoprenaline (ISO). We found that ISO induced cardiac dysfunction, which was characterized by a significant decrease in left ventricular systolic pressure (LVSP), maximum contraction velocity (+dp/dtmax), maximum relaxation velocity (−dp/dtmax) and increased left ventricular end-diastolic pressure (LVEDP). Accordingly, we found that plasma and heart tissue H2S levels in TCM rats decreased significantly, and cardiac cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) expression were lower. Moreover, cardiac dysfunction in TCM was associated with oxidative stress response and reactive oxygen species (ROS) formation. NADPH Oxidase 4 (NOX4) and p67 protein expressions significantly increased in TCM cardiac tissues. In addition, Sodium hydrosulfide (NaHS) ameliorated ISO-induced cardiac dysfunction and reversed ISO-induced oxidative stress. This study revealed that H2S exerted cardioprotective effects by reducing NADPH oxidase, which reduced ROS formation and prevented oxidative stress. Our study provided novel evidence that H2S is protective in myocardial dysfunction in TCM rats and could be a therapeutic target for alleviating β-adrenergic system overstimulation-induced cardiovascular dysfunction.

Effect of paroxetine on left ventricular remodeling in an in vivo rat model of myocardial infarction.

Left ventricular (LV) remodeling following a myocardial infarction (MI) involves formation of reactive oxygen species (ROS). Paroxetine, a selective serotonin reuptake inhibitor, has an antioxidant effect in the vascular wall. We investigated whether paroxetine reduces myocardial ROS formation and LV remodeling following a MI. In a total of 32 Wistar rats, MI was induced by a 30-min ligation of the left anterior descending artery followed by 7- or 28-day reperfusion. During the 28days of reperfusion, LV remodeling was evaluated by magnetic resonance imaging (MRI) and echocardiography (n=20). After 28days of reperfusion, the susceptibility to ventricular tachycardia was evaluated prior to sacrifice and histological assessment of myocyte cross-sectional area, fibrosis, and presence of myofibroblasts. Myocardial ROS formation was measured with dihydroethidium after 7days of reperfusion in separate groups (n=12). Diastolic LV volume, evaluated by MRI (417±60 vs. 511±64µL, p<0.05), and echocardiography (515±80 vs. 596±83µL, p<0.05) as well as diastolic LV internal diameter evaluated with echocardiography (7.2±0.6 vs. 8.1±0.7mm, p<0.05) were lower in the paroxetine group than in controls. Furthermore, myocyte cross-sectional area was reduced in the paroxetine group compared with controls (277±26 vs. 354±23mm3, p<0.05) and ROS formation was reduced in the remote myocardium (0.415±0.19 normalized to controls, p<0.05). However, no differences in the presence of fibrosis or myofibroblasts were observed. Finally, paroxetine reduced the susceptibility to ventricular tachycardia (induced in 2/11 vs. 6/8 rats, p<0.05). Paroxetine treatment following MI decreases LV remodeling and susceptibility to arrhythmias, probably by reducing ROS formation.

EFFECT OF PAROXETINE TREATMENT ON LEFT VENTRICULAR REMODELING IN AN IN VIVO RAT MODEL OF MYOCARDIAL INFARCTION

Left ventricular (LV) remodeling following a myocardial infarction (MI) involves formation of reactive oxygen species (ROS). Paroxetine, a selective serotonin reuptake inhibitor, has an antioxidant effect in the vascular wall. We investigated whether paroxetine reduces myocardial ROS formation and LV remodeling following a MI. In a total of 32 Wistar rats, MI was induced by a 30-min ligation of the left anterior descending artery followed by 7- or 28-day reperfusion. During the 28 days of reperfusion, LV remodeling was evaluated by magnetic resonance imaging (MRI) and echocardiography (n = 20). After 28 days of reperfusion, the susceptibility to ventricular tachycardia was evaluated prior to sacrifice and histological assessment of myocyte cross-sectional area, fibrosis, and presence of myofibroblasts. Myocardial ROS formation was measured with dihydroethidium after 7 days of reperfusion in separate groups (n = 12). Diastolic LV volume, evaluated by MRI (417 ± 60 vs. 511 ± 64 µL, p < 0.05), and echocardiography (515 ± 80 vs. 596 ± 83 µL, p < 0.05) as well as diastolic LV internal diameter evaluated with echocardiography (7.2 ± 0.6 vs. 8.1 ± 0.7 mm, p < 0.05) were lower in the paroxetine group than in controls. Furthermore, myocyte cross-sectional area was reduced in the paroxetine group compared with controls (277 ± 26 vs. 354 ± 23 mm3, p < 0.05) and ROS formation was reduced in the remote myocardium (0.415 ± 0.19 normalized to controls, p < 0.05). However, no differences in the presence of fibrosis or myofibroblasts were observed. Finally, paroxetine reduced the susceptibility to ventricular tachycardia (induced in 2/11 vs. 6/8 rats, p < 0.05). Paroxetine treatment following MI decreases LV remodeling and susceptibility to arrhythmias, probably by reducing ROS formation.

The effect of Notch1 on myocardial ischemia reperfusion injury

To investigate the protective effects of Notch1 on myocardial ischemia reperfusion injury and explore underlying molecular mechanism. H9c2 cells were exposed to hypoxia/reoxygenation (H/R), ischemic preconditioning (IPC) and ischemic postconditioning (IPost) treatment following infection with lentiviral vector-Notch1 intracellular domain (Lv-N1ICD) or Lv-N1ICD-shRNA, and divided into control group, H/R group, H/R+ N1ICD group, IPC group, IPC+ N1ICD-shRNA group, IPost group, IPost+ N1ICD-shRNA group, respectively. Apoptosis was detected by Annexin V/propidium iodide (PI), and reactive oxygen species (ROS) was detected by 2', 7'dichlorofluorescin-diacetate (DCFH-DA). The expression of p-glycogen synthase kinase-3β (p-GSK-3β)/GSK-3β were detected by Western blot analysis. The apoptosis rate of cardiomyocytes in control group, H/R group, H/R+ N1ICD group, IPC group, IPC+ N1ICD-shRNA group, IPost group, IPost+ N1ICD-shRNA group was (5.34±1.70)%, (47.03±4.10)%, (33.89±12.25)%, (35.85±3.17)%, (51.12±8.22)%, (37.35±3.82)%, (47.90±3.08)%, respectively, showing significant differences in all group (F=18.47, P<0.05). ROS in control group, H/R group, H/R+ N1ICD group, IPC group, IPC+ N1ICD-shRNA group, IPost group, IPost+ N1ICD-shRNA group was 624.66±79.52, 1 221.87±63.66, 913.12±115.82, 935.85±201.62, 1 204.03±113.82, 967.15±106.11, 1 296.59±222.38, respectively, showing significant differences in all group (F=8.77, P<0.05). The ratio of p-GSK-3β to GSK-3β in control group, H/R group, H/R+ N1ICD group, IPC group, IPC+ N1ICD-shRNA group, IPost group, IPost+ N1ICD-shRNA group was 0.58±0.12, 0.62±0.20, 1.24±0.09, 1.16±0.12, 0.72±0.15, 1.16±0.12, 0.75±0.12, respectively, showing significant differences in all group (F=11.21, P<0.05). As an endogenous cardioprotective factor, Notch1 reduces myocardial ischemia reperfusion injury by promoting GSK-3β phosphorylation, inhibiting cardiomyocyte apoptosis and reducing ROS formation.

Upregulation of Periostin Prevents High Glucose-Induced Mitochondrial Apoptosis in Human Umbilical Vein Endothelial Cells via Activation of Nrf2/HO-1 Signaling

Background/Aims: High glucose-induced oxidative damage to endothelial cells plays a central role in the pathogenesis of diabetic vascular complications. This study was undertaken to explore the role of periostin in high glucose-induced endothelial cell apoptosis and associated molecular mechanisms. Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to high glucose (33.3 mmol/L) and examined for the expression of periostin. The effects of periostin upregulation on high glucose-induced apoptosis, mitochondrial dysfunction, and reactive oxygen species (ROS) production were determined. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) by periostin was checked. HO-1 knockdown experiments were done to confirm its role in the action of periostin in high glucose-exposed HUVECs. Results: High glucose significantly upregulated the expression of periostin in HUVECs. Enforced expression of periostin attenuated high glucose-induced apoptosis in HUVECs, as determined by TUNEL staining and caspase-3 activity assay. Periostin overexpression prevented loss of Δψm, release of mitochondrial cytochrome c, and dysregulation of Bcl-2 and Bax in high glucose-exposed HUVECs. Periostin upregulation suppressed high glucose-induced ROS generation and activated the Nrf2/HO-1 signaling. HO-1 silencing restored high glucose-induced ROS generation and apoptotic response in periostin-overexpressing HUVECs. Conclusion: Periostin mitigates high glucose-induced mitochondrial apoptosis in endothelial cells, via activation of Nrf2/HO-1 signaling and reduction of ROS formation. Further studies are warranted to explore the therapeutic potential of periostin in diabetic vascular complications.

NOX4 in Mitochondria: Yeast Two-Hybrid-Based Interaction with Complex I Without Relevance for Basal Reactive Oxygen Species?

NADPH oxidases (NOXs) represent the only known dedicated source of reactive oxygen species (ROS) and thus a prime therapeutic target. Type 4 NOX is unique as it produces H2O2, is constitutively active, and has been suggested to localize to cardiac mitochondria, thus possibly linking mitochondrial and NOX-derived ROS formation. The aim of this study was to identify NOX4-binding proteins and examine the possible physiological localization of NOX4 to mitochondria and its impact on mitochondrial ROS formation. We here provide evidence that NOX4 can, in principle, enter protein–protein interactions with mitochondrial complex I NADH dehydrogenase subunits, 1 and 4L. However, under physiological conditions, NOX4 protein was neither detectable in the kidney nor in cardiomyocyte mitochondria. The NOX inhibitor, GKT136901, slightly reduced ROS formation in cardiomyocyte mitochondria, but this effect was observed in both wild-type and Nox4−/− mice. NOX4 may thus associate with mitochondrial complex I proteins, but in cardiac and renal mitochondria under basal conditions, expression is beyond our detection limits and does not contribute to ROS formation. Antioxid. Redox Signal. 23, 1106–1112.

Ambient Fine Particulate Matter Suppresses In Vivo Proliferation of Bone Marrow Stem Cells through Reactive Oxygen Species Formation.

AimsSome environmental insults, such as fine particulate matter (PM) exposure, significantly impair the function of stem cells. However, it is unknown if PM exposure could affect the population of bone marrow stem cells (BMSCs). The present study was to investigate the effects of PM on BMSCs population and related mechanism(s).Main MetheodsPM was intranasally distilled into male C57BL/6 mice for one month. Flow cytometry with antibodies for BMSCs, Annexin V and BrdU ware used to determine the number of BMSCs and the levels of their apoptosis and proliferation in vivo. Phosphorylated Akt (P-Akt) level was determined in the BM cells with western blotting. Intracellular reactive oxygen species (ROS) formation was quantified using flow cytometry analysis. To determine the role of PM-induced ROS in BMSCs population, proliferation, and apotosis, experiments were repeated using N-acetylcysteine (NAC)-treated wild type mice or a triple transgenic mouse line with overexpression of antioxidant network (AON) composed of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase-1 with decreased in vivo ROS production.Key FindingsPM treatment significantly reduced BMSCs population in association with increased ROS formation, decreased P-Akt level, and inhibition of proliferation of BMSCs without induction of apoptosis. NAC treatment or AON overexpression with reduced ROS formation effectively prevented PM-induced reduction of BMSCs population and proliferation with partial recovery of P-Akt level.SignificancePM exposure significantly decreased the population of BMSCs due to diminished proliferation via ROS-mediated mechanism (could be partially via inhibition of Akt signaling).

Structural apelin analogues: mitochondrial ROS inhibition and cardiometabolic protection in myocardial ischaemia reperfusion injury.

Background and PurposeMitochondria-derived oxidative stress is believed to be crucially involved in cardiac ischaemia reperfusion (I/R) injury, although currently no therapies exist that specifically target mitochondrial reactive oxygen species (ROS) production. The present study was designed to evaluate the potential effects of the structural analogues of apelin-12, an adipocyte-derived peptide, on mitochondrial ROS generation, cardiomyocyte apoptosis, and metabolic and functional recovery to myocardial I/R injury.Experimental ApproachIn cultured H9C2 cardiomyoblasts and adult cardiomyocytes, oxidative stress was induced by hypoxia reoxygenation. Isolated rat hearts were subjected to 35 min of global ischaemia and 30 min of reperfusion. Apelin-12, apelin-13 and structural apelin-12 analogues, AI and AII, were infused during 5 min prior to ischaemia.Key ResultsIn cardiac cells, mitochondrial ROS production was inhibited by the structural analogues of apelin, AI and AII, in comparison with the natural peptides, apelin-12 and apelin-13. Treatment of cardiomyocytes with AI and AII decreased cell apoptosis concentration-dependently. In a rat model of I/R injury, pre-ischaemic infusion of AI and AII markedly reduced ROS formation in the myocardial effluent and attenuated cell membrane damage. Prevention of oxidative damage by AI and AII was associated with the improvement of functional and metabolic recovery after I/R in the heart.Conclusions and ImplicationsThese data provide the evidence for the potential of the structural apelin analogues in selective reduction of mitochondrial ROS generation and myocardial apoptosis and form the basis for a promising therapeutic strategy in the treatment of oxidative stress-related heart disease.

Oxidized low-density lipoprotein alters endothelial progenitor cell populations

Oxidized low-density lipoprotein (ox-LDL) is critical to atherosclerosis in hyperlipidemia. Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are important to preventing atherosclerosis, and significantly decreased in hyperlipidemia. This study was to demonstrate ox-LDL and hyperlipidemia could exhibit similar effect on EPC population and the role of reactive oxygen species (ROS). ROS production in BM and blood was significantly increased in male C57BL/6 mice with intravenous ox-LDL treatment, and in hyperlipidemic LDL receptor knockout mice with 4-month high-fat diet. ROS formation was effectively blocked with overexpression of antioxidant enzymes or N-acetylcysteine treatment. In hyperlipidemic and ox-LDL-treated mice, c-Kit(+)/CD31(+) cell number in BM and blood, and Sca-1(+)/Flk-1(+) cell number in blood, not in BM, were significantly decreased, which were not affected by inhibiting ROS production, while blood CD34(+)/Flk-1(+) cell number was significantly increased that was prevented with reduced ROS formation. However, blood CD34(+)/CD133(+) cell number increased in ox-LDL-treated mice, while decreased in hyperlipidemic mice. These data suggested that ox-LDL produced significant changes in BM and blood EPC populations similar (but not identical) to chronic hyperlipidemia with predominantly ROS-independent mechanism(s).

Neuroprotective Effects of Alpha-Mangostin on MPP(+)-Induced Apoptotic Cell Death in Neuroblastoma SH-SY5Y Cells.

In vitro studies have shown that extracts from mangosteen (Garcinia mangostana Linn.) act as antioxidants and cytoprotective agents against oxidative damage. The protective effect of alpha-mangostin, the major xanthone found in the pericarp of the mangosteen, in cellular models of Parkinson's disease (PD), has not been investigated. This study aims to investigate whether alpha-mangostin could protect SH-SY5Y neuroblastoma cells from MPP+-induced apoptosis. The effects of alpha-mangostin on MPP+-induced cell death were evaluated with a cell viability assay, staining for nuclear DNA morphology, flow cytometry for apoptotic cells and reactive oxygen species (ROS) production, quantitative real-time PCR for the expression of p53, Bax, and Bcl-2, and western blot analysis for cleaved caspase-3. Concomitant treatment with alpha-mangostin attenuated the effect of MPP+ on cell viability and apoptotic cell death. Alpha-mangostin reduced ROS formation induced by MPP+. Bax/Bcl-2 expression ratio and expression of p53 were significantly lower in cells cocultured with alpha-mangostin and MPP+. The cotreated cells showed a significant decrease in activated caspase-3 compared with MPP+ treatment alone. Our data suggest that cytoprotection of alpha-mangostin against MPP+-induced apoptosis may be associated with the reduction of ROS production, modulating the balance of pro- and antiapoptotic genes, and suppression of caspase-3 activation.

Effects of Glucose Concentration on Propofol Cardioprotection against Myocardial Ischemia Reperfusion Injury in Isolated Rat Hearts.

The anesthetic propofol confers cardioprotection against myocardial ischemia-reperfusion injury (IRI) by reducing reactive oxygen species (ROS). However, its cardioprotection on patients is inconsistent. Similarly, the beneficial effect of tight glycemic control during cardiac surgery in patients has recently been questioned. We postulated that low glucose (LG) may promote ROS formation through enhancing fatty acid (FA) oxidation and unmask propofol cardioprotection during IRI. Rat hearts were isolated and randomly assigned to be perfused with Krebs-Henseleit solution with glucose at 5.5 mM (LG) or 8 mM (G) in the absence or presence of propofol (5 μg/mL) or propofol plus trimetazidine (TMZ). Hearts were subjected to 35 minutes of ischemia followed by 60 minutes of reperfusion. Myocardial infarct size (IS) and cardiac CK-MB were significantly higher in LG than in G group (P < 0.05), associated with reduced left ventricular developed pressure and increases in postischemic cardiac contracture. Cardiac 15-F2t-isoprostane was higher, accompanied with higher cardiac lipid transporter CD36 protein expression in LG. Propofol reduced IS, improved cardiac function, and reduced CD36 in G but not in LG. TMZ facilitated propofol cardioprotection in LG. Therefore, isolated heart with low glucose lost sensitivity to propofol treatment through enhancing FA oxidation and TMZ supplementation restored the sensitivity to propofol.

Mitochondrial anti-oxidant protects IEX-1 deficient mice from organ damage during endotoxemia.

Sepsis, a leading cause of mortality in intensive care units worldwide, is often a result of overactive and systemic inflammation following serious infections. We found that mice lacking immediate early responsive gene X-1 (IEX-1) were prone to lipopolysaccharide (LPS) -induced endotoxemia. A nonlethal dose of LPS provoked numerous aberrations in IEX-1 knockout (KO) mice including pancytopenia, increased serum aspartate aminotransferase (AST), and lung neutrophilia, concurrent with liver and kidney damage, followed by death. Given these results, in conjunction with a proven role for IEX-1 in the regulation of reactive oxygen species (ROS) homeostasis during stress, we pre-treated IEX-1 KO mice with Mitoquinone (MitoQ), a mitochondrion-based antioxidant prior to LPS injection. The treatment significantly reduced ROS formation in circulatory cells and protected against pancytopenia and multiple organ failure, drastically increasing the survival rate of IEX-1 KO mice challenged by this low dose of LPS. This study confirms significant contribution of mitochondrial ROS to the etiology of sepsis.

Loss of Sirt3 limits bone marrow cell-mediated angiogenesis and cardiac repair in post-myocardial infarction.

Sirtuin-3 (Sirt3) has a critical role in the regulation of human aging and reactive oxygen species (ROS) formation. A recent study has identified Sirt3 as an essential regulator of stem cell aging. This study investigated whether Sirt3 is necessary for bone marrow cell (BMC)-mediated cardiac repair in post-myocardial infarction (MI). In vitro, BMC-derived endothelial progenitor cells (EPCs) from wild type (WT) and Sirt3KO mice were cultured. EPC angiogenesis, ROS formation and apoptosis were assessed. In vivo, WT and Sirt3 KO mice were subjected to MI and BMCs from WT and Sirt3 KO mice were injected into ischemic area immediately. The expression of VEGF and VEGFR2 was reduced in Sirt3KO-EPCs. Angiogenic capacities and colony formation were significantly impaired in Sirt3KO-EPCs compared to WT-EPCs. Loss of Sirt3 further enhanced ROS formation and apoptosis in EPCs. Overexpression of Sirt3 or treatment with NADPH oxidase inhibitor apocynin (Apo, 200 and 400 microM) rescued these abnormalities. In post-MI mice, BMC treatment increased number of Sca1+/c-kit+ cells; enhanced VEGF expression and angiogenesis whereas Sirt3KO-BMC treatment had little effects. BMC treatment also attenuated NADPH oxidase subunits p47phox and gp91phox expression, and significantly reduced ROS formation, apoptosis, fibrosis and hypertrophy in post-MI mice. Sirt3KO-BMC treatment did not display these beneficial effects. In contrast, Sirt3KO mice treated with BMCs from WT mice attenuated myocardial apoptosis, fibrosis and improved cardiac function. Our data demonstrate that Sirt3 is essential for BMC therapy; and loss of Sirt3 limits BMC-mediated angiogenesis and cardiac repair in post-MI.

Nicotine detoxification of rutin, quercitrin, and chlorogenic acid isolated from Houttuynia cordata by reducing reactive oxygen species and inducing conversion from nicotine to cotinine

The hypothesis of the present study is that methanol extract of Houttuynia cordata Thunb (MEH) and its targeted bioactive components including rutin, quercitrin, and chlorogenic acid can be effective in reducing reactive oxygen species (ROS) caused by nicotine and promoting nicotine to cotinine in HepG2 cell. Oxygen radical absorbance capacity (ORAC) of bioactive components and MEH was measured to assess free radical scavenging capacity. ROS inhibition ability of bioactive components and MEH was measured by 2′,7′-dichlorodihydrofluorescein diacetate assay. The conversion rate of nicotine to cotinine by bioactive components and MEH was determined by the direct barbiturate assay method. ORAC value confirmed that MEH and its bioactive components provided an antioxidant capacity ranging from 126 to 138 μMtrolox equivalents/100 g. Compared to nicotine only, pretreatment of MEH, rutin, and quercitrin was revealed to effectively inhibit ROS production in HepG2 cell by up to 9, 7.4, and 14%, respectively. Nicotine conversion to cotinine after 120 min incubation was 1.7 and 1.4 times higher in rutin and chlorogenic acid pretreatment than the control, respectively. H. cordata and its targeted bioactive components could be a valuable natural ingredient for inhibiting ROS formation by nicotine as well as enhancing the rate of nicotine to cotinine turnover.

SYNCRIP-dependent Nox2 mRNA destabilization impairs ROS formation in M2-polarized macrophages.

During sepsis, macrophages are alternatively activated toward an M2-like phenotype on contact with apoptotic cells (ACs) or their secretion products. Simultaneously, NADPH oxidase-dependent reactive oxygen species (ROS) formation is attenuated, thus contributing to immune paralysis. However, the exact mechanism remains elusive. Here, we provide mechanistic insights into diminished mRNA stability of the NADPH oxidase Nox2 on macrophage M2 polarization and therefore reduced ROS formation in sepsis. Murine J774A.1 macrophages were stimulated with conditioned medium (CM) of apoptotic T cells, which reduced Nox2 mRNA and protein expression, consequently decreasing ROS production. An mRNA pulldown approach coupled to mass spectrometry analysis identified the RNA-binding protein SYNCRIP attached to the Nox2 mRNA 3' untranslated region (3'UTR). The binding of SYNCRIP to the 3'UTR of Nox2 mRNA is attenuated after treatment with CM of apoptotic T cells, followed by Nox2 mRNA destabilization. In in vivo models of polymicrobial sepsis such as cecal ligation and puncture, SYNCRIP was strongly downregulated, which was associated with a decreased Nox2 expression in peritoneal macrophages. Downregulation of SYNCRIP in macrophages after contact to material of ACs destabilized Nox2 mRNA and impaired ROS formation, thereby contributing to an M2 phenotype shift of macrophages in sepsis. M2 polarization of macrophages in sepsis results in an attenuated SYNCRIP binding to the 3'UTR of Nox2 mRNA, destabilizing Nox2 mRNA abundance and expression. Consequently, ROS formation needed to fight against recurrent infections is impaired. In conclusion, SYNCRIP-regulated Nox2 mRNA degradation mediates the hypoinflammatory phase of sepsis.