Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice

J. Neurochem. (2012) 122, 1211-1218. ABSTRACT: In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4) ) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-) ) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4) . In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4) , increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.     

Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells

Tetrahydrobiopterin (BH4) is a member of the pterin family that has a core structure of pyrazino-2,3-d-pyrimidine rings. Because BH4 is an essential cofactor for the biosynthesis of nitric oxide (a major vasodilator), there is growing interest in BH4 biochemistry in endothelial cells (the cells that line blood vessels). BH4 is synthesized via de novo and salvage pathways from guanosine 5′-triphosphate (GTP) and 7,8-dihydrobiopterin, respectively, in animal cells. GTP cyclohydrolase-I (GTP-CH) is the first and rate-controlling enzyme in the de novo pathway. Available evidence shows that endothelial GTP-CH expression and BH4 synthesis are stimulated by a wide array of nutritional (phenylalanine and arginine), hormonal (insulin and estrogen), immunological (inflammatory cytokines including interleukin [IL]-1, interferon-γ, and tumor necrosis factor-α), therapeutic (statins and cyclosporin A), and endothelium-derived (basic fibroblast growth factor and H₂O₂) factors. In contrast, glucocorticoids and anti-inflammatory cytokines (IL-4, IL-10, and transforming growth factor [TGF]-β) inhibit endothelial BH4 synthesis. Because BH4 is oxidized to 7,8-dihydrobiopterin and 7,8-dihydropterin at physiological pH, endothelial BH4 homeostasis is regulated by both BH4 synthesis and its oxidation. Vitamin C, folate, and other antioxidants enhance endothelial BH4 bioavailability through chemical stabilization or scavenging of reactive oxygen species, thereby contributing to the maintenance of physiological homeostasis in the endothelium. New know ledge about the cellular and molecular mechanisms for the regulation of endothelial BH4 synthesis and bioavailability is beneficial for developing effective means to prevent and treat cardiovascular disorders, the leading cause, of death in developed nations.     

Mitochondrial glutathione protects against cell death induced by oxidative and nitrative stress in astrocytes

The major cellular antioxidant, glutathione, is mostly localized in the cytosol but a small portion is found in mitochondria. We have recently shown that highly selective depletion of mitochondrial glutathione in astrocytes in culture markedly increased cell death induced by the peroxynitrite donor, 3-morpholino-syndnonimine. The present study was aimed at characterizing the increase in susceptibility arising from mitochondrial glutathione loss and testing the possibility that elevating this metabolite pool above normal values could be protective. The increased vulnerability of astrocytes with depleted mitochondrial glutathione to Sin-1 was confirmed. Furthermore, these cells showed marked increases in sensitivity to hydrogen peroxide and also to high concentrations of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine. The increase in cell death was mostly due to necrosis as indicated by substantially increased release of lactate dehydrogenase and staining of nuclei with propidium iodide but little change in annexin V staining and caspase 3 activation. The enhanced cell loss was blocked by prior restoration of the mitochondrial glutathione content. It was also essentially fully inhibited by treatment with cyclosporin A, consistent with a role for the mitochondrial permeability transition in the development of cell death. Susceptibility to the classical apoptosis inducer, staurosporine, was only affected to a small extent in contrast to the response to the other substances tested. Incubation of normal astrocytes with glutathione monoethylester produced large and long-lasting increases in mitochondrial glutathione content with much smaller effects on the cytosolic glutathione pool. This treatment reduced cell death on exposure to 3-morpholino-syndnonimine or hydrogen peroxide but not S-nitroso-N-acetyl-pencillamine or staurosporine. These findings provide evidence for an important role for mitochondrial glutathione in preserving cell viability during periods of oxidative or nitrative stress and indicate that increases in this glutathione pool can confer protection against some of these stressors.     

Inducible nitric oxide synthase and cyclooxgenase-2 mediate protection of hydrogen peroxide preconditioning against apoptosis induced by oxidative stress in PC12 cells

The induction of inducible nitric oxide synthase (iNOS) in response to different stress is associated with simultaneous induction of cyclooxygenase-2 (COX-2) in various cell types. Both iNOS and COX-2 have been reported to mediate the late phase of cardioprotection induced by different preconditioning. However, whether both iNOS and COX-2 are mediators in the neuroprotection induced by preconditioning with hydrogen peroxide (H(2)O(2)) at low concentration is unknown. In this study, using the neurosecretory cell line-PC12 cells to set up the model of neuroprotection of preconditioning with H(2)O(2) against apoptosis, we first investigate what changes in expression of iNOS and COX-2 appear during H(2)O(2) preconditioning, then determine if both iNOS inhibitor and COX-2 inhibitor interfere with the neuroprotection elicited by preconditioning with H(2)O(2). We found that preconditioning with H(2)O(2) at 10 microM significantly protected PC12 cells against apoptosis induced by lethal H(2)O(2) (50 microM) and increased the expression of iNOS and COX-2 and that selective iNOS inhibitor, aminoguanidine (AG) and COX-2 inhibitor, NS-398 obviously blocked the protective effects induced by preconditioning with 10 microM H(2)O(2). The results of this study suggest that both iNOS and COX-2 are mediators of the neuroprotection induced by preconditioning with oxidative stress (H(2)O(2) at low concentration) in PC12 cells.     

Homocysteine induces oxidative stress by uncoupling of NO synthase activity through reduction of tetrahydrobiopterin

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.     

环加氧酶-2抑制剂尼美舒利通过改善内皮功能和增加NO含量对抗大鼠心肌氧化应激损伤

本文旨在研究冠状动脉内皮和NO在选择性环加氧酶2（cyclooxygenase2,COX-2）抑制剂尼美舒利（nimesulide）对抗心肌氧化损伤中的作用。离体大鼠心脏行Langendorff灌流,给予H2O2（140Bmol／L）观察心脏收缩功能。用U-46619灌流心脏,使冠状动脉预收缩后,观察冠状动脉对内皮依赖性舒张因子5-HT和内皮非依赖性舒张因子硝普钠（sodiumnitroprusside,SNP）的反应。结果显示：（1）与空白对照组（100％）相比,H202灌流20min后,左心室发展压[left ventriculardevelo pedpressure,LVDP,（54．8±4．0）％],和心室内压最大变化速率【±dp／dtmax（50．8±3．1）％和（46．2±2．9）％]明显降低。H2O2灌流前尼美舒利（5μmol/L）预处理10min,能够显著抑制H2O2引起的LVDP和μdp/dtmax下降[（79．9±2．8）％,（80．3±2．6）％和（81．4±2．6）％,P〈0．0l]。（2）与空白对照组相比,H2O2灌流后,5-HT和SNP引起内皮依赖性和内皮非依赖性血管舒张功能均明显下降;而尼美舒利预处理10min能明显对抗内皮依赖性血管舒张功能的下降[（-22．2±4．2）％vsH2O2组（-6．0±2．5）％,P〈0．0l],但对其内皮非依赖性血管舒张功能的下降没有明显作用[（-2．0±1．8）％vsH202组（-7．0±3．5）％,P〉0．05]。（3）一氧化氮合酶（nitric oxide synthase,NOS）抑制剂L-NAME能够部分取消尼美舒利预处理对H20,应激心脏心功能指标的改善作用ILVDP和±dp/dtmax分别为（60．2±2．1）％,（63．9±2．4）％和（63．1±2．9）％,P〈0．01]。同时尼美舒利预处理10min能使H202应激心肌NO含量增加[（2．63±0．40）vs（1．36±0．23）nmol／gprotein,P〈0．051,而L-NAME抑制此作用。（4）选择性COX-1抑制剂吡罗昔康（piroxicam）预处理不能抑制H202引起的LVDP和±dp/dtmax下降,但促进左心室舒张末压（1eftventricular end diastolicpressure,LVEDP）升高;吡罗昔康对H202引起的内皮依赖性和内皮非依赖性血管舒张功能下降无显著作用。以上结果提示,选择性COX-2抑制剂尼美舒利能够对抗大鼠离体心肌氧化应激损伤,其机制可能是通过改善内皮依赖性血管舒张功能和增加心肌NO含量起作用。     

Role of Superoxide Dismutase in Ischemic Brain Injury: A Study Using SOD-1 Transgenic Mice

1. Nitric oxide radicals (NO) play an important role in the pathophysiology of focal cerebral ischemia.2. Vascular NO can reduce ischemic brain injury by increasing CBF, whereas neuronal NO may mediate neurotoxicity following brain ischemia, mainly by its reaction with superoxide to generate peroxynitrite.3. These findings could contribute to a strategy for the treatment of cerebral ischemia.     

Pharmacological modulation of reactive oxygen species (ROS) improves the airway hyperresponsiveness by shifting the Th1 response in allergic inflammation induced by ovalbumin

Asthma is an allergic inflammation driven by the Th2 immune response with release of cytokines such as IL-4 and IL-13, which contribute to the airflow limitations and airway hyperresponsiveness (AHR). The involvement of oxidative stress in this process is well-established, but the specific role of the superoxide anion and nitric oxide in asthma are poorly understood. Thus, the aim of this study was to investigate the mechanisms underlying the superoxide anion/nitric oxide production and detoxification in a murine asthma model. BALB/c male mice were sensitised and challenged with ovalbumin (OVA). Pretreatments with either apocynin (14?mg/kg) or allopurinol (25?mg/kg) (superoxide anion synthesis inhibitors), aminoguanidine (50?mg/kg) (nitric oxide synthesis inhibitor) or diethyldithiocarbamate (100?mg/kg) (superoxide dismutase inhibitor) were performed 1?h before the challenge. Our data showed that apocynin and allopurinol ameliorated AHR and reduced eosinophil peroxidase, as well as IL-4 and IL-13 levels. Apocynin also abrogated leukocyte peribronchiolar infiltrate and increased IL-1β secretion. Aminoguanidine preserved lung function and shifted the Th2 to the Th1 response with a reduction of IL-4 and IL-13 and increase in IL-1β production. Diethyldithiocarbamate prevented neither allergen-induced AHR nor eosinophil peroxidase (EPO) generation. All treatments protected against oxidative damage observed by a reduction in TBARS levels. Taken together, these results suggest that AHR in an asthma model can be avoided by the down-regulation of superoxide anion and nitric oxide synthesis in a mechanism that is independent of a redox response. This down-regulation is also associated with a transition in the typical immunological Th2 response toward the Th1 profile.     

Vascular oxidative stress increases dendritic cell adhesion and transmigration induced by homocysteine

Atherosclerosis is a long-term chronic inflammatory and immunological disease. Endothelial dysfunction and the dendritic cell (DC) immune response are pivotal early events in atherogenesis. This study investigated the effects and possible mechanisms of action of homocysteine (Hcy) on DC adhesion to and transmigration between endothelial cells (ECs), and indicated a novel immunoregulatory mechanism by which Hcy induces atherogenesis. When ECs were stimulated with increasing concentrations of Hcy, immunofluorescence showed that endothelial reactive oxygen species (ROS) generation strikingly increased, while nitrite assay showed that nitric oxide (NO) release markedly decreased. Furthermore, DC adhesion and transmigration were significantly increased when ECs were activated by Hcy. However, pretreatment of ECs with antioxidant before Hcy markedly attenuated the induction of DC adhesion and transmigration, dependent on the intracellular ROS decrease and endothelial NO increase. In conclusion, DC adhesion and transmigration are significantly increased by vascular oxidative stress under conditions of elevated Hcy levels. These findings provide insight into the inflammatory processes and immune responses occurring in atherosclerosis induced by Hcy.     

Thymoquinone protects human retinal pigment epithelial cells against hydrogen peroxide induced oxidative stress and apoptosis

Oxidative stress in retinal pigment epithelium (RPE) cells may contribute to the progression of age-related macular degeneration. Thymoquinone (TQ), an active component derived from Nigella sativa, possesses antioxidative effect. However, the role of TQ in RPE cells under oxidative stress condition remains unclear. The present study aimed to examine the protective effect of TQ against hydrogen peroxide (H₂O₂)-induced oxidative stress in human RPE cells. Our results showed that TQ improved the cell viability and apoptosis in H₂O₂-induced ARPE cells. We also found that the levels of reactive oxygen species and malondialdehyde induced by H₂O₂ were reduced after the pretreatment of TQ. In addition, the inhibitory effect of H₂O₂ on the glutathione (GSH) level and superoxide dismutase activity was markedly attenuated by TQ pretreatment. Moreover, TQ enhanced the activation of Nrf2/heme oxygenase 1 (HO-1) signaling pathway in H₂O₂-induced ARPE cells. Knockdown of Nrf2 abolished the protective effect of TQ on H₂O₂-induced oxidative damage. These results suggested that TQ protected ARPE cells from H₂O₂-induced oxidative stress and apoptosis via the Nrf2/HO-1 signaling pathway.     

Endoplasmic reticulum stress induced by tunicamycin and thapsigargin protects against transient ischemic brain injury

Transient cerebral ischemia leads to endoplasmic reticulum (ER) stress. However, the contributions of ER stress to cerebral ischemia are not clear. To address this issue, the ER stress activators tunicamycin (TM) and thapsigargin (TG) were administered to transient middle cerebral artery occluded (tMCAO) mice and oxygen-glucose deprivation-reperfusion (OGD-Rep.)-treated neurons. Both TM and TG showed significant protection against ischemia-induced brain injury, as revealed by reduced brain infarct volume and increased glucose uptake rate in ischemic tissue. In OGD-Rep.-treated neurons, 4-PBA, the ER stress releasing mechanism, counteracted the neuronal protection of TM and TG, which also supports a protective role of ER stress in transient brain ischemia. Knocking down the ER stress sensor Eif2s1, which is further activated by TM and TG, reduced the OGD-Rep.-induced neuronal cell death. In addition, both TM and TG prevented PARK2 loss, promoted its recruitment to mitochondria, and activated mitophagy during reperfusion after ischemia. The neuroprotection of TM and TG was reversed by autophagy inhibition (3-methyladenine and Atg7 knockdown) as well as Park2 silencing. The neuroprotection was also diminished in Park2^+/? mice. Moreover, Eif2s1 and downstream Atf4 silencing reduced PARK2 expression, impaired mitophagy induction, and counteracted the neuroprotection. Taken together, the present investigation demonstrates that the ER stress induced by TM and TG protects against the transient ischemic brain injury. The PARK2-mediated mitophagy may be underlying the protection of ER stress. These findings may provide a new strategy to rescue ischemic brains by inducing mitophagy through ER stress activation.     

The interaction between dinitrosy iron complexes and intermediates of oxidative stress

The interaction between the glutathione-containing dinitrosyl iron complexes and the superoxide radical generated in mitochondria and in the xanthine-xanthine oxidase system was studied. Both superoxide and hydroxyl radicals proved to be involved in destruction of dinitrosyl iron complexes. However, the iron within dinitrosyl complexes is unlikely to catalyze decomposition of hydrogen peroxide yielding hydroxyl radical. It was found that iron dinitrosyl complexes with various anion ligands efficiently inhibited the formation of probucol phenoxyl radical in the hemin-H₂O₂ system, different components of these complexes being involved in the antioxidant action.     

Activation of stimulatory heterotrimeric G proteins increases glutathione and protects neuronal cells against oxidative stress

Oxidative glutamate toxicity in the neuronal cell line HT22 is a model for cell death by oxidative stress, where an excess of extracellular glutamate inhibits import of cystine, a building block of the antioxidant glutathione. The subsequent decrease in glutathione then leads to the accumulation of reactive oxygen species (ROS) and programmed cell death. We used pharmacological compounds known to interact with heterotrimeric G-protein signalling and studied their effects on cell survival, morphology, and intracellular events that ultimately lead to cell death. Cholera toxin and phorbol esters were most effective and prevented cell death through independent pathways. Treating HT22 cells with cholera toxin attenuated the glutamate-induced accumulation of ROS and calcium influx. This was, at least in part, caused by an increase in glutathione due to improved uptake of cystine mediated by the induction of the glutamate/cystine-antiporter subunit xCT or, additionally, by the up-regulation of the antiapoptotic protein Bcl-2. Gs activation also protected HT22 cells from hydrogen peroxide or inhibition of glutathione synthesis by buthionine sulfoximine, and immature cortical neurones from oxidative glutamate toxicity. Thus, this pathway might be more generally implicated in protection from neuronal death by oxidative stress.     

Progranulin protects against cerebral ischemia-reperfusion (I/R) injury by inhibiting necroptosis and oxidative stress

Ischemic stroke is a leading cause of mortality and disability worldwide. Nevertheless, its molecular mechanisms have not yet been adequately illustrated. Progranulin (PGRN) is a secreted glycoprotein with pleiotropic functions. In the present study, we found that PGRN expression was markedly reduced in mice after stroke onset through middle cerebral artery occlusion (MCAO). We also showed that necroptosis was a mechanism underlying cerebral I/R injury. Importantly, PGRN knockdown in vivo significantly promoted the infarction volume and neurological deficits scores in mice after MCAO surgery. Necroptosis induced by MCAO was further accelerated by PGRN knockdown, as evidenced by the promoted expression of phosphorylated receptor-interacting protein (RIP) 1 kinase (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), which was accompanied with increased expression of cleaved Caspase-8 and Caspase-3. However, PGRN over-expression was neuroprotective. Additionally, PGRN-regulated ischemic stroke was related to ROS accumulation that MCAO-mice with PGRN knockdown exhibited severe oxidative stress, as proved by the aggravated malondialdehyde (MDA) and lipid peroxidation (LPO) contents, and the decreased superoxide dismutase (SOD) activity. However, PGRN over-expression in mice with cerebral ischemia showed anti-oxidative effects. Finally, PGRN was found to attenuate oxidative damage partly via its regulatory effects on necroptosis. Therefore, promoting PGRN expression could reduced cerebral I/R-induced brain injury by suppressing neroptosis and associated reactive oxygen species (ROS) production. These data elucidated that PGRN might provide an effective therapeutic treatment for ischemic stroke.     

Nicotinic receptor activation by epibatidine induces heme oxygenase-1 and protects chromaffin cells against oxidative stress

Activation of neuronal nicotinic acetylcholine receptors (nAChR) provides neuroprotection against different toxic stimuli that often lead to overproduction of reactive oxygen species (ROS) and cell death. ROS production has been related with disease progression in several neurodegenerative pathologies such as Alzheimer's or Parkinson's diseases. In this context, we investigated here if the exposure of bovine chromaffin cells to the potent nAChR agonist epibatidine protected against rotenone (30 micromol/L) plus oligomycin (10 micromol/L) (rot/oligo) toxicity, an in vitro model of mitochondrial ROS production. Epibatidine induced a concentration- and time-dependent protection, which was maximal at 3 mumol/L after 24 h. Pre-incubation with dantrolene (100 micromol/L) (a blocker of the ryanodine receptor channel), chelerythrine (1 micromol/L) (a protein kinase C inhibitor), or PD98059 (50 micromol/L) (a MEK inhibitor), aborted epibatidine-elicited cytoprotection. Mitochondrial depolarization, ROS, and caspase 3 active produced by rot/oligo were also prevented by epibatidine. Epibatidine doubled the amount of heme oxygenase-1 (HO-1), a critical cell defence enzyme against oxidative stress. Furthermore, the HO-1 inhibitor Sn(IV) protoporphyrin IX dichloride reversed the epibatidine protecting effects and HO-1 inducer Co (III) protoporphyrin IX dichloride exhibited neuroprotective effects by itself. The results of this study point to HO-1 as the cytoprotective target of nAChR activation through the following pathway: endoplasmic reticulum Ca(2+)-induced Ca(2+)-release activates the protein kinase C/extracellular regulated kinase/HO-1 axis to mitigate mitochondrial depolarization and ROS production. This study provides a mechanistic insight on how nAChR activation translates into an antioxidant and antiapoptotic signal through up-regulation of HO-1.     

Inhibition of the AIF/CypA complex protects against intrinsic death pathways induced by oxidative stress

Delayed neuronal cell death largely contributes to the progressive infarct development and associated functional impairments after cerebral ischemia or brain trauma. Previous studies exposed a key role for the interaction of the mitochondrial protein apoptosis-inducing factor (AIF) and cytosolic cyclophilin A (CypA) in pathways of programmed cell death in neurons in vitro and in vivo. These studies suggested that pro-apoptotic activities of AIF, such as its translocation to the nucleus and subsequent DNA degradation, depend on the physical interaction of AIF with CypA. Hence, this protein complex may represent a new pharmacological target for inhibiting the lethal action of AIF on the brain tissue. In this study, we show that the AIF amino-acid residues 370–394 mediate the protein complex formation of AIF with CypA. The synthetic AIF(370–394) peptide inhibited AIF/CypA complex formation in vitro by binding CypA with a K_D of 12 μM. Further, the peptide exerted pronounced neuroprotective effects in a model of glutamate-induced oxidative stress in cultured HT-22 cells. In this model system of AIF-dependent cell death, the AIF(370–394) peptide preserved mitochondrial integrity, as detected by measurements of the mitochondrial membrane potential and quantification of mitochondrial fragmentation. Further, the AIF(370–394) peptide inhibited perinuclear accumulation of fragmented mitochondria, mitochondrial release of AIF to the nucleus and glutamate-induced cell death to a similar extent as CypA-siRNA. These data indicate that the targeting of the AIF-CypA axis is an effective strategy of neuroprotection.     

Ferulic acid ethyl ester protects neurons against amyloid beta- peptide(1-42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity

Alzheimer's disease (AD) is neuropathologically characterized by depositions of extracellular amyloid and intracellular neurofibrillary tangles, associated with loss of neurons in the brain. Amyloid beta-peptide (Abeta) is the major component of senile plaques and is considered to have a causal role in the development and progress of AD. Several lines of evidence suggest that enhanced oxidative stress and inflammation play important roles in the pathogenesis or progression of AD. The present study aimed to investigate the protective effects of ethyl-4-hydroxy-3-methoxycinnamic acid (FAEE), a phenolic compound which shows antioxidant and anti-inflammatory activity, on Abeta(1-42)-induced oxidative stress and neurotoxicity. We hypothesized that the structure of FAEE would facilitate radical scavenging and may induce protective proteins. Abeta(1-42) decreases cell viability, which was correlated with increased free radical formation, protein oxidation (protein carbonyl, 3-nitrotyrosine), lipid peroxidation (4-hydroxy-2-trans-nonenal) and inducible nitric oxide synthase. Pre-treatment of primary hippocampal cultures with FAEE significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular reactive oxygen species accumulation, protein oxidation, lipid peroxidation and induction of inducible nitric oxide synthase. Treatment of neurons with Abeta(1-42) increases levels of heme oxygenase-1 and heat shock protein 72. Consistent with a cellular stress response to the Abeta(1-42)-induced oxidative stress, FAEE treatment increases the levels of heme oxygenase-1 and heat shock protein 72, which may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells against Abeta(1-42)-induced toxicity. These results suggest that FAEE exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress directly and by inducing protective genes. These findings suggest that FAEE could potentially be of importance for the treatment of AD and other oxidative stress-related diseases.     

Propolis protects CYP 2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride

Induction of CYP 2E1 by carbon tetrachloride (CCl₄) is one of the central pathways by which CCl₄ generates oxidative stress in hepatocytes. Experimental liver injury was induced in rats by CCl₄ to determine toxicological actions on CYP 2E1 by microsomal drug metabolizing enzymes. In this report, ethanolic extract of propolis at a dose of 200 mg/kg (po) was used after 24 h of toxicant administration to validate its protective potential. Intraperitoneal injection of CCl₄ (1.5 ml/kg) induced hepatotoxicity after 24 h of its administration that was associated with elevated malonyldialdehyde (index of lipid peroxidation), lactate dehydrogenase and γ-glutamyl transpeptidase release (index of a cytotoxic effect). Hepatic microsomal drug metabolizing enzymes of CYP 2E1 showed sharp depletion as assessed by estimating aniline hydroxylase and amidopyrine N-demethylase activity after CCl₄ exposure. Toxic effect of CCl₄ was evident on CYP 2E1 activity by increased hexobarbitone induced sleep time and bromosulphalein retention. Propolis extract showed significant improvement in the activity of both enzymes and suppressed toxicant induced increase in sleep time and bromosulphalein retention. Choleretic activity of liver did not show any sign of toxicity after propolis treatment at a dose of 200 mg/kg (id). Histopathological evaluation of the liver revealed that propolis reduced the incidence of liver lesions including hepatocyte swelling and lymphocytic infiltrations induced by CCl₄. Electron microscopic observations also showed improvement in ultrastructure of liver and substantiated recovery in biochemical parameters. Protective activity of propolis at 200 mg/kg dose was statistically compared with positive control silymarin (50 mg/kg, po), a known hepatoprotective drug seems to be better in preventing hepatic CYP 2E1 activity deviated by CCl₄. These results lead us to speculate that propolis may play hepatoprotective role via improved CYP 2E1 activity and reduced oxidative stress in living system.     

PARK2-dependent mitophagy induced by acidic postconditioning protects against focal cerebral ischemia and extends the reperfusion window

Prompt reperfusion after cerebral ischemia is critical for neuronal survival. Any strategies that extend the limited reperfusion window will be of great importance. Acidic postconditioning (APC) is a mild acidosis treatment that involves inhaling CO₂ during reperfusion following ischemia. APC attenuates ischemic brain injury although the underlying mechanisms have not been elucidated. Here we report that APC reinforces ischemia-reperfusion-induced mitophagy in middle cortical artery occlusion (MCAO)-treated mice, and in oxygen-glucose deprivation (OGD)-treated brain slices and neurons. Inhibition of mitophagy compromises neuroprotection conferred by APC. Furthermore, mitophagy and neuroprotection are abolished in Park2 knockout mice, indicating that APC-induced mitophagy is facilitated by the recruitment of PARK2 to mitochondria. Importantly, in MCAO mice, APC treatment extended the effective reperfusion window from 2 to 4 h, and this window was further extended to 6 h by exogenously expressing PARK2. Taken together, we found that PARK2-dependent APC-induced mitophagy renders the brain resistant to ischemic injury. APC treatment could be a favorable strategy to extend the thrombolytic time window for stroke therapy.