Oxidative Stress-induced Increase Research Articles

The anti-aging effect of vitamin D and vitamin D receptor in Drosophila midgut.

Adult stem cells are pivotal for maintaining tissue homeostasis, and their functional decline is linked to aging and its associated diseases, influenced by the niche cells' environment. Age- and cancer-related reduction of vitamin D and its receptor levels are well documented in human clinical studies. However, the mechanisms through which the vitamin D/vitamin D receptor pathway contributes to anti-aging and extends life expectancy are not well understood. In this study, we aimed to determine the protective role of the vitamin D/vitamin D receptor pathway in differentiated enterocytes (ECs) during intestinal stem cell (ISC) aging. By utilizing a well- established Drosophila midgut model for stem cell aging biology, we revealed that vitamin D receptor knockdown in ECs induced ISC proliferation, EC death, ISC aging, and enteroendocrine cell differentiation. Additionally, age- and oxidative stress-induced increases in ISC proliferation and centrosome amplification were reduced by vitamin D treatment. Our findings suggest a direct evidence of the anti-aging role of the vitamin D/vitamin D receptor pathway and provides insights into the molecular mechanisms underlying healthy aging in Drosophila.

Circ-CARD6 inhibits oxidative stress-induced apoptosis and autophagy in ARPE-19 cells via the miR-29b-3p/PRDX6/PI3K/Akt axis

BackgroundOxidative stress-induced damage and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD) and hereditary retinopathy diseases (HRDs). This study aimed to elucidate the roles and mechanisms of circ-CARD6 and miR-29b-3p in oxidative stress-induced RPE and provide new ideas for the diagnosis and treatment of retinopathy disease (RD). MethodsA model of oxidative stress-induced RPE (ARPE-19) was established, and the level of malondialdehyde (MDA) and concentration of reactive oxygen species (ROS) were detected by a DCFH-DA fluorescent probe and MDA kit. The cell viability was measured by a CCK-8 assay. The expression of PRDX6/PI3K/Akt axis genes and proteins related to apoptosis and autophagy were determined by RT‒qPCR and Western blot analyses. The dual-luciferase reporter system confirmed the targeting relationship between miR-29b-3p and circ-CARD6 and between miR-29b-3p and PRDX6. ResultsIn H2O2-treated ARPE-19 cells, the expression of circ-CARD6 and PRDX6 was decreased, while the expression of miR-29b-3p was increased. The overexpression of circ-CARD6 inhibits oxidative stress-induced increases in ROS, apoptosis and autophagy in ARPE-19 cells. circ-CARD6 targets miR-29b-3p, miR-29b-3p targets PRDX6, and circ-CARD6 regulates PRDX6 via miR-29b-3p. Further studies showed that circ-CARD6 acts as a competitive endogenous RNA of miR-29b-3p to affect the expression of PRDX6, thereby inhibiting autophagy and apoptosis in ARPE-19 cells. Conclusioncirc-CARD6 can inhibit oxidative stress and apoptosis by regulating the miR-29b-3p/PRDX6/PI3K/Akt axis.

Cofilin Signaling in the CNS Physiology and Neurodegeneration.

All eukaryotic cells are composed of the cytoskeleton, which plays crucial roles in coordinating diverse cellular functions such as cell division, morphology, migration, macromolecular stabilization, and protein trafficking. The cytoskeleton consists of microtubules, intermediate filaments, and actin filaments. Cofilin, an actin-depolymerizing protein, is indispensable for regulating actin dynamics in the central nervous system (CNS) development and function. Cofilin activities are spatiotemporally orchestrated by numerous extra- and intra-cellular factors. Phosphorylation at Ser-3 by kinases attenuate cofilin’s actin-binding activity. In contrast, dephosphorylation at Ser-3 enhances cofilin-induced actin depolymerization. Cofilin functions are also modulated by various binding partners or reactive oxygen species. Although the mechanism of cofilin-mediated actin dynamics has been known for decades, recent research works are unveiling the profound impacts of cofilin dysregulation in neurodegenerative pathophysiology. For instance, oxidative stress-induced increase in cofilin dephosphorylation is linked to the accumulation of tau tangles and amyloid-beta plaques in Alzheimer’s disease. In Parkinson’s disease, cofilin activation by silencing its upstream kinases increases α-synuclein-fibril entry into the cell. This review describes the molecular mechanism of cofilin-mediated actin dynamics and provides an overview of cofilin’s importance in CNS physiology and pathophysiology.

Redox equilibrium of serum apolipoprotein E3: a buffering effect of disulfide-linked complexes against oxidative stress on apolipoprotein E3–containing lipoproteins

Reversible redox modification of cysteine thiols is crucial for protecting proteins from irreversible detrimental change. However, the physiological significance of the redox modification of apolipoprotein (apo) E is unclear. Here, we hypothesized that the disulfide-linked complexes of apoE3 corresponding to the representative reversible-modified apoE3 play a protective role against oxidative stress. The effects of disulfide bond formation on oxidative stress on apoE3 were evaluated with a band-shift assay. Maleimide-labeled apoE3 and unlabeled apoE3 were defined as the reduced (r)-apoE3 and non-reduced (nr)-apoE3 forms, respectively. Hydrogen peroxide-induced oxidation decreased for reduced-form apoE (r-apoE3) but increased for nr-apoE3. Induction of apoE3-AII complex formation with excess of apoAII markedly suppressed the oxidative stress-induced increase in nr-apoE3 (P<0.001) and enhanced homodimer formation. The apoE3-AII complex was more dominant in high-density lipoprotein (HDL) than in very low-density lipoprotein. Under oxidative stress, HDL showed a significant decrease, rather than an increase, in nr-apoE3 levels with a concomitant significant increase in apoE3-AII levels (P<0.005). This finding suggests that the majority of nr-apoE3 in HDL exists in a reversible oxidized form. The apoE3-AII complex, formed from the reversible oxidized apoE3, is beneficial for maintaining the redox equilibrium of apoE3 by preventing the modification of apoE3 to its irreversible oxidized form. The apoE3-AII complex may be possibly implicated in the pathophysiology of various apoE-related diseases.

The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation.

The reperfusion of ischemic brain tissue following a cerebral stroke causes oxidative stress, and leads to the generation of reactive oxygen species (ROS). Apart from inflicting oxidative damage, the latter may also trigger the upregulation of aquaporin 4 (AQP4), a water-permeable channel expressed by astroglial cells of the blood-brain barrier (BBB), and contribute to edema formation, the severity of which is known to be the primary determinant of mortality and morbidity. The mechanism through which this occurs remains unknown. In the present study, we have attempted to address this question using primary astrocyte cultures treated with hydrogen peroxide (H2O2) as a model system. First, we showed that H2O2 induces a significant increase in AQP4 protein levels and that this is inhibited by the antioxidant N-acetylcysteine (NAC). Second, we demonstrated using cell surface biotinylation that H2O2 increases AQP4 cell-surface expression independently of it’s increased synthesis. In parallel, we found that caveolin-1 (Cav1) is phosphorylated in response to H2O2 and that this is reversed by the Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). PP2 also abrogated the H2O2-induced increase in AQP4 surface levels, suggesting that the phosphorylation of tyrosine-14 of Cav1 regulates this process. We further showed that dominant-negative Y14F and phosphomimetic Y14D mutants caused a decrease and increase in AQP4 membrane expression respectively, and that the knockdown of Cav1 inhibits the increase in AQP4 cell-surface, expression following H2O2 treatment. Together, these findings suggest that oxidative stress-induced Cav1 phosphorylation modulates AQP4 subcellular distribution and therefore may indirectly regulate AQP4-mediated water transport.

Mitochondrial calcium uniporter in Drosophila transfers calcium between the endoplasmic reticulum and mitochondria in oxidative stress-induced cell death

Mitochondrial calcium plays critical roles in diverse cellular processes ranging from energy metabolism to cell death. Previous studies have demonstrated that mitochondrial calcium uptake is mainly mediated by the mitochondrial calcium uniporter (MCU) complex. However, the roles of the MCU complex in calcium transport, signaling, and dysregulation by oxidative stress still remain unclear. Here, we confirmed that Drosophila MCU contains evolutionarily conserved structures and requires essential MCU regulator (EMRE) for its calcium channel activities. We generated Drosophila MCU loss-of-function mutants, which lacked mitochondrial calcium uptake in response to caffeine stimulation. Basal metabolic activities were not significantly affected in these MCU mutants, as observed in examinations of body weight, food intake, body sugar level, and starvation-induced autophagy. However, oxidative stress-induced increases in mitochondrial calcium, mitochondrial membrane potential depolarization, and cell death were prevented in these mutants. We also found that inositol 1,4,5-trisphosphate receptor genetically interacts with Drosophila MCU and effectively modulates mitochondrial calcium uptake upon oxidative stress. Taken together, these results support the idea that Drosophila MCU is responsible for endoplasmic reticulum-to-mitochondrial calcium transfer and for cell death due to mitochondrial dysfunction under oxidative stress.

Vitamin D Reduces Oxidative Stress-Induced Procaspase-3/ROCK1 Activation and MP Release by Placental Trophoblasts.

Increased microparticle (MP) shedding by placental trophoblasts contributes to maternal vascular inflammatory response and endothelial dysfunction in preeclampsia. Vitamin D has beneficial effects in pregnancy; however, its effect on trophoblast MP release has not been investigated. To investigate if vitamin D could protect trophoblasts from oxidative stress-induced MP release. Placental trophoblasts were isolated from uncomplicated and preeclamptic placentas. Effects of vitamin D on MP release induced by oxidative stress inducer CoCl2 were studied. Annexin V+ MPs were assessed by flow cytometry. Expression of caveolin-1, endothelial nitric oxide synthase (eNOS), procaspase-3, cleaved caspase-3, and Rho-associated coiled-coil protein kinase 1 (ROCK1) in trophoblasts and trophoblast-derived MPs were determined by Western blot. Trophoblasts from preeclamptic pregnancies released significantly more MPs than cells from uncomplicated pregnancies (P < 0.01). CoCl2-induced increase in MP release was associated with upregulation of caveolin-1 and downregulation of eNOS expression in trophoblasts (P < 0.05), which could be attenuated by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 could also inhibit CoCl2-induced procaspase-3 cleavage and ROCK1 activation in trophoblasts. Consistently, CoCl2-induced upregulation of procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs were also reduced in cells treated with 1,25(OH)2D3. Placental trophoblasts from preeclamptic pregnancies released more MP than cells from uncomplicated pregnancies. Oxidative stress-induced increase in MP shedding is associated with upregulation of caveolin-1 and downregulation of eNOS expression in placental trophoblasts. Inhibition of caspase-3 cleavage and ROCK1 activation, together with upregulation of eNOS expression, could be the potential cellular/molecular mechanism(s) of vitamin D protective effects on placental trophoblasts.

Oxidative stress-induced increase of intracellular zinc in astrocytes decreases their functional expression of P2X7 receptors and engulfing activity.

Neuron-glia communication mediated by neuro- and glio-transmitters such as ATP and zinc is crucial for the maintenance of brain homeostasis, and its dysregulation is found under pathological conditions. It is reported that under oxidative stress-loaded conditions, astrocytes exhibit increased intra- and extra-cellular labile zinc, the latter triggering microglial M1 activation, while the pathophysiological role of the former remains unrevealed. In this study, we examined whether the oxidative stress-induced increase of intracellular labile zinc is involved in the P2X7 receptor (P2X7R)-mediated regulation of astrocytic engulfing activity. The exposure of cultured astrocytes to sub-lethal oxidative stress through their treatment with 400 μM H2O2 increased intracellular labile zinc, of which the concentration reached a peak level of approximately 2 μM at 2 h after the treatment. In astrocytes under sub-lethal oxidative stress, the uptake of YO-PRO-1 and latex beads as markers for P2X7R channel/pore activity and astrocytic engulfing activity, respectively, was decreased, and these decreased activities were accompanied by decreased expression of P2X7R at the plasma membrane via intracellular labile zinc-mediated translocation of it. With the oxidative stress, the expression level of full length P2X7R relative to that of its splice variants in astrocytes was decreased, leading to a decrease of the relative expression of the trimer consisting of full length P2X7R. Collectively, sub-lethal oxidative stress induces an astrocytic modal shift from the normal resting engulfing mode to the activated astrogliosis mode via an intracellular labile zinc-mediated decrease of the functional expression of P2X7R.

Role of NOX2 in mediating doxorubicin-induced senescence in human endothelial progenitor cells

Senescence exerts a great impact on both biological and functional properties of circulating endothelial progenitor cells (EPCs), especially in cardiovascular diseases where the physiological process of aging is accelerated upon clinical administration of certain drugs such as doxorubicin. EPC impairment contributes to doxorubicin-induced cardiotoxicity. Doxorubicin accelerates EPC aging, although mechanisms underlying this phenomenon remain to be fully clarified. Here we investigated if Nox2 activity is able to modulate the premature senescence induced in vitro by doxorubicin in human EPCs. Results showed that in conditioned media obtained from late EPC cultures, the levels of interleukin-6, isoprostanes and nitric oxide bioavailability were increased and reduced respectively after 3h of doxorubicin treatment. These derangements returned to physiological levels when cells were co-treated with apocynin or gp91ds-tat (antioxidant and specific Nox2 inhibitors, respectively). Accordingly, Nox2 activity resulted to be activated by doxorubicin. Importantly, we found that Nox2 inhibition reduced doxorubicin-induced EPC senescence, as indicated by a lower percentage of β-gal positive EPCs. In conclusion, Nox2 activity efficiently contributes to the mechanism of oxidative stress-induced increase in premature aging conferred by doxorubicin. The importance of modulation of Nox2 in human EPCs could reveal a useful tool to restore EPC physiological function and properties.

Carbocysteine restores steroid sensitivity by targeting histone deacetylase 2 in a thiol/GSH-dependent manner

Steroid insensitivity is commonly observed in patients with chronic obstructive pulmonary disease. Here, we report the effects and mechanisms of carbocysteine (S-CMC), a mucolytic agent, in cellular and animal models of oxidative stress-mediated steroid insensitivity. The following results were obtained: oxidative stress induced higher levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), which are insensitive to dexamethasone (DEX). The failure of DEX was improved by the addition of S-CMC by increasing histone deacetylase 2 (HDAC2) expression/activity. S-CMC also counteracted the oxidative stress-induced increase in reactive oxygen species (ROS) levels and decreases in glutathione (GSH) levels and superoxide dismutase (SOD) activity. Moreover, oxidative stress-induced events were decreased by the thiol-reducing agent dithiothreitol (DTT), enhanced by the thiol-oxidizing agent diamide, and the ability of DEX was strengthened by DTT. In addition, the oxidative stress-induced decrease in HDAC2 activity was counteracted by S-CMC by increasing thiol/GSH levels, which exhibited a direct interaction with HDAC2. S-CMC treatment increased HDAC2 recruitment and suppressed H4 acetylation of the IL-8 promoter, and this effect was further ablated by addition of buthionine sulfoximine, a specific inhibitor of GSH synthesis. Our results indicate that S-CMC restored steroid sensitivity by increasing HDAC2 expression/activity in a thiol/GSH-dependent manner and suggest that S-CMC may be useful in a combination therapy with glucocorticoids for treatment of steroid-insensitive pulmonary diseases.

Oxidative-stress induced increase in circulating fatty acids does not contribute to phospholipase A2-dependent appetitive long-term memory failure in the pond snail Lymnaea stagnalis.

BackgroundReactive oxygen species (ROS) are essential for normal physiological functioning of the brain. However, uncompensated increase in ROS levels may results in oxidative stress. Phospholipase A2 (PLA2) is one of the key players activated by elevated ROS levels resulting in the hydrolysis of various products from the plasmamembrane such as peroxidized fatty acids. Free fatty acids (FFAs) and fatty acid metabolites are often implicated to the genesis of cognitive impairment. Previously we have shown that age-, and experimentally induced oxidative stress causes PLA2-dependent long-term memory (LTM) failure in an aversive operant conditioning model in Lymnaea stagnalis. In the present study, we investigate the effects of experimentally induced oxidative stress and the role of elevated levels of circulating FFAs on LTM function using a non-aversive appetitive classical conditioning paradigm.ResultsWe show that intracoelomic injection of exogenous PLA2 or pro-oxidant induced PLA2 activation negatively affects LTM performance in our learning paradigm. In addition, we show that experimental induction of oxidative stress causes significant temporal changes in circulating FFA levels. Importantly, the time of training coincides with the peak of this change in lipid metabolism. However, intracoelomic injection with exogenous arachidonic acid, one of the main FFAs released by PLA2, does not affect LTM function. Moreover, sequestrating circulating FFAs with the aid of bovine serum albumin does not rescue pro-oxidant induced appetitive LTM failure.ConclusionsOur data substantiates previous evidence linking lipid peroxidation and PLA2 activation to age- and oxidative stress-related cognitive impairment, neuronal dysfunction and disease. In addition however, our data indicate that lipid peroxidation induced increased levels of circulating (per)oxidized FFAs are not a factor in oxidative stress induced LTM impairment.

Reactive Oxygen Species, NADPH Oxidases, and Hypertension

Reactive oxygen species (ROS) produced in the neuronal, renal, and vascular systems not only influence cardiovascular physiology but are also strongly implicated in pathological signaling leading to hypertension. Different sources of ROS have been identified, ranging from xanthine-xanthine oxidase and mitochondria to NADPH oxidase (Nox) enzymes. Of 7 Nox family members, Nox1, Nox2, and Nox4 (and Nox5 in humans) influence the cardiovascular system. Their activation processes and cell and tissue distribution vary widely, adding complexity to understanding their functional roles. Whether these systems act collectively or independently in disease conditions is unclear, but recently feed forward mechanisms have been established between ROS sources. Studies published in Hypertension over the last few years are the focus of this review, and they provide a framework with which to consider the roles of Nox enzymes in neuronal, renal, and vascular hypertensive mechanisms, as well as cardiac remodeling, and their relationships with other ROS-generating systems. ### Neuronal ROS in Hypertension Redox signaling in the central nervous system is well recognized in neuronal control of blood pressure (BP), as well as in response to angiotensin II (Ang II) and aldosterone, which are linked to ROS-dependent hypertension. Recently, new roles for ROS have been described in the hypothalamus and brain stem, nucleus tractus solitarius (NTS), subfornical organ (SFO), rostral ventrolateral medulla, and area postrema (Figure 1). Figure 1. Neuronal NADPH oxidase–dependent ROS involved in central regulation of hypertension. NADPH oxidase homologues, mainly Nox2 and Nox4, are found in different regions of the neuronal system and are reported to have a role in the neuropathogenesis of hypertension by enhancing the sympathetic nerve activity. Nox-induced ROS initiate a forward loop in cross-activation of different receptors and between Nox and mitochondrial ROS. OVLT indicates organum vasculosum of the lamina terminalis; PVN, paraventricular nucleus; PP, posterior pituitary; AP, area postrema; RVLM, rostral ventrolateral medulla. Several …

Melatonin blocks oxidative stress-induced increased asymmetric dimethylarginine

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of nitric oxide synthase, and its increase is associated with many systemic diseases. We recently found that increases in plasma and hepatic ADMA levels were associated with oxidative stress in young bile-duct-ligation (BDL) rats; these increases were prevented by melatonin therapy. Therefore, we used an in vivo BDL model and in vitro cultured hepatocytes to elucidate the protective mechanisms of melatonin against oxidative stress-induced increase in ADMA. We found that the presence of reactive oxygen species (ROS) in young rats with BDL leads to downregulation of dimethylarginine dimethyaminohydrolase (DDAH)-1 and -2 as well as DDAH activity. Melatonin prevented ADMA increases in the liver mainly by regulating DDAH-1 and -2. The expression and activity of DDAH were suppressed in vitro by superoxide and hydrogen peroxide (H 2O 2) in a time-dependent manner, whereas melatonin could block H 2O 2-induced downregulation of DDAH-2 as well as decreased DDAH activity, thereby preventing increases in hepatic ADMA. Our findings reveal a mechanistic basis of DDAH downregulation by ROS and suggest that melatonin might be a potential therapy for various diseases with elevated cellular ADMA.

Protective effect of 4,4′-diaminodiphenylsulphone against oxidative stress but not to apoptotic stress in human diploid fibroblasts

The antibiotic drug 4,4′-diaminodiphenylsulphone (DDS) is used to treat several dermatologic diseases, including Hansen's disease. This study confirmed the antioxidant nature of DDS in hydrogen peroxide (H2O2)-induced oxidative stress and assessed its role in other apoptotic stresses in human diploid fibroblasts (HDFs). Oxidative stress was effectively reduced by DDS in a dose-dependent manner. Moreover, the oxidative stress-induced increases in the levels of the p53 and p21 proteins were inhibited by pre-treatment with DDS. In addition, H2O2 and DDS increased the level of cytochrome P450 (CYP450) IIE1 in HDFs, implicating a role for DDS in H2O2 scavenging via the activation of CYP450. DDS treatment increased the activity of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the GSH/GSSG ratio, indicating activation of the glutathione system against oxidative stress. However, DDS showed no protective effects on HDFs against other apoptotic stimuli, such as thapsigargin and staurosporine, suggesting that DDS would act only against oxidative stress. Therefore, in addition to its antibiotic function, DDS is a potent antioxidant against H2O2-induced oxidative stress in HDFs.

Metallothionein‐3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions

Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

Ranolazine Inhibits An Oxidative Stress-induced Increase in Myocyte Sodium and Calcium Loading During Simulated-demand Ischemia

Ranolazine inhibits the late Na current and is proposed to reduce angina by decreasing [Na]i during ischemia, thereby reducing Ca influx via Na/Ca exchange (NCX). We sought to test this hypothesis and to determine whether oxidative stress during simulated-demand ischemia activates the late Na current. We measured [Ca]i and [Na]i in rabbit ventricular myocytes by flow cytometry during metabolic inhibition (MI) with 2 mM cyanide and 0 mM glucose at 37 degrees C plus pacing (P) at 0.5 Hz (P-MI), and in P-MI + 1, 10, or 50 microM ranolazine. In the clinically relevant concentration range (1-10 microM), ranolazine decreased Na and Ca loading and the development of myocyte contracture. P-MI caused an increase in fluorescence of the oxidative radical probe CM-H2DCFDA, which was inhibited by the radical scavenger Tiron 20 mM. The NCX inhibitor KB-R7943 (10 microM) and Tiron 20 mM reduced the rise in [Ca]i during P-MI and eliminated the effect of 10 microM ranolazine on [Ca]i. These results indicate that oxidative stress increases the late Na current during MI. Inhibition of the resulting increase in Na and Ca loading and contracture seems to account for the observed antiischemia effects of ranolazine.

Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3β signaling pathways and changes in protein expression

Previously we demonstrated that insulin protects against neuronal oxidative stress by restoring antioxidants and energy metabolism. In this study, we analysed how insulin influences insulin- (IR) and insulin growth factor-1 receptor (IGF-1R) intracellular signaling pathways after oxidative stress caused by ascorbate/Fe2+ in rat cortical neurons. Insulin prevented oxidative stress-induced decrease in tyrosine phosphorylation of IR and IGF-1R and Akt inactivation. Insulin also decreased the active form of glycogen synthase kinase-3β (GSK-3β) upon oxidation. Since phosphatidylinositol 3-kinase (PI-3K)/Akt-mediated inhibition of GSK-3β may stimulate protein synthesis and decrease apoptosis, we analysed mRNA and protein expression of “candidate” proteins involved in antioxidant defense, glucose metabolism and apoptosis. Insulin prevented oxidative stress-induced increase in glutathione peroxidase-1 and decrease in hexokinase-II expression, supporting previous findings of changes in glutathione redox cycle and glycolysis. Moreover, insulin precluded Bcl-2 decrease and caspase-3 increased expression. Concordantly, insulin abolished caspase-3 activity and DNA fragmentation caused by oxidative stress. Thus, insulin-mediated activation of IR/IGF-1R stimulates PI-3K/Akt and inhibits GSK-3β signaling pathways, modifying neuronal antioxidant defense-, glucose metabolism- and anti-apoptotic-associated protein synthesis. These and previous data implicate insulin as a promising neuroprotective agent against oxidative stress associated with neurodegenerative diseases.

Comparison of the Effects of Korean Ginseng and Heat-Processed Korean Ginseng on Diabetic Oxidative Stress

To investigate the effects of Korean ginseng (KG, Panax ginseng C.A. Meyer) and heat-processed Korean ginseng (H-KG) on diabetic renal damage, we used the streptozotocin-induced diabetic rat model in this study. The diabetes-induced physiological abnormalities at early-stage were attenuated by KG or H-KG administration through reducing the blood glucose level and improving renal function. The oxidative stress-induced increases in serum and renal thiobarbituric acid-reactive substance levels were significantly reduced by KG and H-KG administrations. Moreover, the protein expressions related to oxidative stress and advanced glycation endproducts were significantly reduced in diabetic rats and/or not significantly increased compared to normal rats by KG or H-KG administration. All of these beneficial effects of H-KG in diabetic rats were stronger than those of KG. Therefore, KG and H-KG may improve diabetic pathological conditions and prevent renal damage associated with diabetic nephropathy, and these protective effects of KG can be improved by heat-processing. This study provides scientific evidence that H-KG may be a potential therapeutic agent for pathological conditions associated with diabetic complications including diabetic nephropathy.

Mechanisms of Oxidative Stress-Induced Increase in Salt Sensitivity and Development of Hypertension in Sprague-Dawley Rats

High salt intake produces vascular changes that contribute to the development of hypertension in salt-sensitive individuals. Because reactive oxygen species play a role in the pathogenesis of cardiovascular diseases, we investigated whether oxidative stress contributes to salt-sensitive hypertension. Sprague-Dawley rats were divided in different groups and received tap water (vehicle), 30 mmol/L of l-buthionine sulfoximine ([BSO] an oxidant), high salt ([HS] 1% NaCl), and BSO plus HS without and with antioxidant tempol (1 mmol/L) in drinking water for 12 days. Compared with vehicle, BSO treatment caused oxidative stress and mild increase in blood pressure. Thoracic aortic rings from BSO-treated rats exhibited decreased response to endothelium-independent vasorelaxants. In HS-treated rats, the response to vasoactive agents, as well as blood pressure, was unaffected. Concomitant treatment of rats with BSO and HS produced a marked increase in blood pressure and a decreased response to both endothelium-dependent and endothelium-independent vasorelaxants with an increase in EC(50). Incubation of aortic tissue from BSO-treated rats with sodium nitroprusside showed decreased cGMP accumulation, whereas HS rats had decreased basal NO synthase activity. Tempol decreased oxidative stress, normalized blood pressure, and restored NO signaling and responses to vasoactive compounds in BSO and BSO plus HS rats. We conclude that BSO increases oxidative stress and reduces NO signaling, whereas HS reduces NO levels by decreasing the NO synthase activity. These phenomena collectively result in reduced responsiveness to both endothelium -dependent and endothelium- independent vasorelaxants and may contribute to salt-sensitive hypertension.

Induction of the Human Oxidized Base-specific DNA Glycosylase NEIL1 by Reactive Oxygen Species

NEIL1, a mammalian DNA glycosylase and ortholog of Escherichia coli Nei/Fpg, is involved in the repair of oxidatively damaged bases in mammalian cells. Exposure of HCT116 human colon carcinoma cells to reactive oxygen species, generated by glucose oxidase (GO), enhanced the levels of NEIL1 mRNA and polypeptide by 2-4-fold by 6 h after GO treatment. A similar oxidative stress-induced increase in human NEIL1 (hNEIL1) promoter-dependent luciferase expression in HCT116 cells indicates that reactive oxygen species activates NEIL1 transcription. The transcriptional start site of hNEIL1 was mapped, and the upstream promoter sequence was characterized via luciferase reporter assay. Two identical CRE/AP-1-binding sites were identified in the promoter that binds transcription factors c-Jun and CREB/ATF2. This binding was significantly enhanced in extracts of cells treated with GO. Furthermore, a simultaneous increase in the level of phosphorylated c-Jun suggests its involvement in up-regulating the NEIL1 promoter. Oxidative stress-induced activation of NEIL1 appears to be involved in the feedback regulation of cellular repair activity needed to handle an increase in the level of oxidative base damage.