Modulation of peroxynitrite-induced fibroblast injury by hesperetin: a role for intracellular scavenging and modulation of ERK signalling

Peroxynitrite is thought to contribute to the progression of many diseases including cardiovascular disease, cancer, and neurodegenerative disorders. We report that pre-treatment of fibroblasts with the citrus flavanone, hesperetin, prior to peroxynitrite exposure protects against peroxynitrite-mediated cytotoxicity. This protection was partially mediated by the intracellular scavenging of peroxynitrite by hesperctin as exposure of fibroblasts to peroxynitrite following hesperetin loading led to the formation of two intracellular nitrohesperetin derivatives. In addition, protection appeared to be mediated by hesperetin-induced changes in MAP kinase signalling. Exposure of fibroblasts to hesperetin led to concentration-dependent increases in the phosphorylation of ERK1/2 and was observed to restore peroxynitrite-mediated decreases in ERK1/2 phosphorylation. We propose that the protective potential of hesperetin in fibroblasts may be mediated both by intracellular scavenging of peroxynitrite and by modulation of fibroblast signalling. (c) 2006 Elsevier Inc. All rights reserved.

Involvement of ERK, Akt and JNK signalling in H2O2-induced cell injury and protection by hydroxytyrosol and its metabolite homovanillic alcohol

The olive oil polyphenol, hydroxytyrosol (HT), is believed to be capable of exerting protection against oxidative kidney injury. In this study we have investigated the ability of HT and its O-methylated metabolite, homovanillic alcohol (HVA) to protect renal cells against oxidative damage induced by hydrogen peroxide. We show that both compounds were capable of inhibiting hydrogen peroxide-induced kidney cell injury via an ability to interact with both MAP kinase and PI3 kinase signalling pathways, albeit at different concentrations. HT strongly inhibited death and prevented peroxide-induced increases in ERK1/2 and JNK1/2/3 phosphorylation at 0.3 microM, whilst HVA was effective at 10 microM. At similar concentrations, both compounds also prevented peroxide-induced reductions in Akt phosphorylation. We suggest that one potential protective effect exerted by olive oil polyphenols against oxidative kidney cell injury may be attributed to the interactions of HT and HVA with these important intracellular signalling pathways.

Substance P released by TRPV1-expressing neurons produces reactive oxygen species that mediate ethanol-induced gastric injury

Although neurokinin 1 receptor antagonists prevent ethanol (EtOH)-induced gastric lesions, the mechanisms by which EtOH releases substance P (SP) and SP damages the mucosa are unknown. We hypothesized that EtOH activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to release SP, which stimulates epithelial neurokinin 1 receptors to generate damaging reactive oxygen species (ROS). SP release was assayed in the mouse stomach, ROS were detected using dichlorofluorescein diacetate, and neurokinin 1 receptors were localized by immunofluorescence. EtOH-induced SP release was prevented by TRPV1 antagonism. High dose EtOH caused lesions, and TRPV1 or neurokinin 1 receptor antagonism and neurokinin 1 receptor deletion inhibited lesion formation. Coadministration of low, innocuous doses of EtOH and SP caused lesions by a TRPV1-independent but neurokinin 1 receptor-dependent process. EtOH, capsaicin, and SP stimulated generation of ROS by superficial gastric epithelial cells expressing neurokinin 1 receptors by a neurokinin 1 receptor-dependent mechanism. ROS scavengers prevented lesions induced by a high EtOH dose or a low EtOH dose plus SP. Gastric lesions are caused by an initial detrimental effect of EtOH, which is damaging only if associated with TRPV1 activation, SP release from sensory nerves, stimulation of neurokinin 1 receptors on epithelial cells, and ROS generation.

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis. (c) 2006 Elsevier Inc. All rights reserved.

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.

Platelet protein disulfide isomerase is required for thrombus formation but not essential for hemostasis in mice

Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and ATP secretion induced by thrombin, collagen, and ADP. Such defects were rescued by exogenously-added wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ERp57 and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca2+ mobilization, β3-talin interaction, and platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation, aggregation and ATP secretion of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under arteriolar shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time and blood loss in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.

Do mitochondriotropic antioxidants prevent chlorinative stress-induced mitochondrial and cellular injury?

Reactive chlorine species such as hypochlorous acid ( HOCl) are cytotoxic oxidants generated by activated neutrophils at the sites of chronic inflammation. Since mitochondria are key mediators of apoptosis and necrosis, we hypothesized that mitochondriotropic antioxidants could limit HOCl-mediated intracellular oxidative injury to human fetal liver cells, preserve mitochondrial function, and prevent cell death. In this current study, we show that recently developed mitochondria-targeted antioxidants ( MitoQ and SS31) significantly protected against HOCl-induced mitochondrial damage and cell death at concentrations >= 25 nM. Our study highlights the potential application of mitochondria-specific targeted antioxidants for the prevention of cellular dysfunction and cell death under conditions of chlorinative stress, as occurs during chronic inflammation.

Champagne wine polyphenols protect primary cortical neurons against peroxynitrite-induced injury

White wines are generally low in polyphenol content as compared to red wines. However, Champagne wines have been shown to contain relatively high amounts of phenolic acids that may exert protective cellular actions in vivo. In this study, we have investigated the potential neuroprotective effects of Champagne wine extracts, and individual phenolics present in these extracts, against peroxynitrite-induced injury. Organic and aqueous Champagne wine extracts exhibited potent neuroprotective activity against peroxynitrite-induced injury at low concentrations (0.1 mu g/mL). This protection appeared to be in part due to the cellular actions of individual components found in the organic extracts, notably tyrosol, caffeic acid, and gallic acid. These phenolics were observed to exert potent neuroprotection at concentrations between 0.1 and 10 mu M. Together, these data suggest that polyphenols present in Champagne wine may induce a neuroprotective effect against oxidative neuronal injury.

Diacylglycerol-induced protection against injury during ischemia and reperfusion can be achieved without activation of protein kinase C in the rat heart: Comparative studies with ischemic preconditioning

The role of protein kinase C (PKC) activation in ischemic preconditioning remains controversial. Since diacylglycerol is the endogenous activator of PKC and as such might be expected cardioprotective, we have investigated whether: (i) the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) can protect against injury during ischemia and reperfusion; (ii) any effect is mediated via PKC activation; and (iii) the outcome is influenced by the time of administration. Isolated rat hearts were perfused with buffer at 37°C and paced at 400 bpm. In Study 1, hearts (n=6/group) were subjected to one of the following: (1) 36 min aerobic perfusion (controls); (2) 20 min aerobic perfusion plus ischemic preconditioning (3 min ischemia/3 min reperfusion+5 min ischemia/5 min reperfusion); (3) aerobic perfusion with buffer containing DOG (10 μM) given as a substitute for ischemic preconditioning; (4) aerobic perfusion with DOG (10 μM) during the last 2 min of aerobic perfusion. All hearts then were subjected to 35 min of global ischemia and 40 min reperfusion. A further group (5) were perfused with DOG (10 μM) for the first 2 min of reperfusion. Ischemic preconditioning improved postischemic recovery of LVDP from 24±3% in controls to 71±2% (P<0.05). Recovery of LVDP also was enhanced by DOG when given just before ischemia (54±4%), however, DOG had no effect on the recovery of LVDP when used as a substitute for ischemic preconditioning (22±5%) or when given during reperfusion (29±6%). In Study 2, the first four groups of study were repeated (n=4–5/group) without imposing the periods of ischemia and reperfusion, instead hearts were taken for the measurement of PKC activity (pmol/min/mg protein±SEM). PKC activity after 36 min in groups (1), (2), (3) and (4) was: 332±102, 299±63, 521±144, and 340±113 and the membrane:cytosolic PKC activity ratio was: 5.6±1.5, 5.3±1.8, 6.6±2.7, and 3.9±2.1 (P=NS in each instance). In conclusion, DOG is cardioprotective but under the conditions of the present study is less cardioprotective than ischemic preconditioning, furthermore the protection does not appear to necessitate PKC activation prior to ischemia.

Motor imagery-induced EEG patterns in individuals with spinal cord injury and their impact on brain–computer interface accuracy

Objective. Assimilating the diagnosis complete spinal cord injury (SCI) takes time and is not easy, as patients know that there is no ‘cure’ at the present time. Brain–computer interfaces (BCIs) can facilitate daily living. However, inter-subject variability demands measurements with potential user groups and an understanding of how they differ to healthy users BCIs are more commonly tested with. Thus, a three-class motor imagery (MI) screening (left hand, right hand, feet) was performed with a group of 10 able-bodied and 16 complete spinal-cord-injured people (paraplegics, tetraplegics) with the objective of determining what differences were present between the user groups and how they would impact upon the ability of these user groups to interact with a BCI. Approach. Electrophysiological differences between patient groups and healthy users are measured in terms of sensorimotor rhythm deflections from baseline during MI, electroencephalogram microstate scalp maps and strengths of inter-channel phase synchronization. Additionally, using a common spatial pattern algorithm and a linear discriminant analysis classifier, the classification accuracy was calculated and compared between groups. Main results. It is seen that both patient groups (tetraplegic and paraplegic) have some significant differences in event-related desynchronization strengths, exhibit significant increases in synchronization and reach significantly lower accuracies (mean (M) = 66.1%) than the group of healthy subjects (M = 85.1%). Significance. The results demonstrate significant differences in electrophysiological correlates of motor control between healthy individuals and those individuals who stand to benefit most from BCI technology (individuals with SCI). They highlight the difficulty in directly translating results from healthy subjects to participants with SCI and the challenges that, therefore, arise in providing BCIs to such individuals

Motor imagery induced EEG patterns in spinal cord injury patients and their impact on Brain-Computer Interface accuracy

OBJECTIVE: Assimilating the diagnosis complete spinal cord injury (SCI) takes time and is not easy, as patients know that there is no 'cure' at the present time. Brain-computer interfaces (BCIs) can facilitate daily living. However, inter-subject variability demands measurements with potential user groups and an understanding of how they differ to healthy users BCIs are more commonly tested with. Thus, a three-class motor imagery (MI) screening (left hand, right hand, feet) was performed with a group of 10 able-bodied and 16 complete spinal-cord-injured people (paraplegics, tetraplegics) with the objective of determining what differences were present between the user groups and how they would impact upon the ability of these user groups to interact with a BCI. APPROACH: Electrophysiological differences between patient groups and healthy users are measured in terms of sensorimotor rhythm deflections from baseline during MI, electroencephalogram microstate scalp maps and strengths of inter-channel phase synchronization. Additionally, using a common spatial pattern algorithm and a linear discriminant analysis classifier, the classification accuracy was calculated and compared between groups. MAIN RESULTS: It is seen that both patient groups (tetraplegic and paraplegic) have some significant differences in event-related desynchronization strengths, exhibit significant increases in synchronization and reach significantly lower accuracies (mean (M) = 66.1%) than the group of healthy subjects (M = 85.1%). SIGNIFICANCE: The results demonstrate significant differences in electrophysiological correlates of motor control between healthy individuals and those individuals who stand to benefit most from BCI technology (individuals with SCI). They highlight the difficulty in directly translating results from healthy subjects to participants with SCI and the challenges that, therefore, arise in providing BCIs to such individuals.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Peroxynitrite induced formation of the neurotoxins 5-S-cysteinyl-dopamine and DHBT-1: implications for Parkinson's disease and protection by polyphenols

Mechanisms of nigral cell injury in Parkinson's disease remain unclear, although a combination of increased oxidative stress, the formation of catecholamine-quinones and the subsequent formation of neurotoxic cysteinyl-catecholamine conjugates may contribute. In the present study, peroxynitrite was observed to generate both 2-S- and 5-S-cysteinyl-dopamine and a dihydrobenzothiazine species, DHBT-1, following the reaction of dopamine with L-cysteine. The formation of 5-S-cysteinyl-dopamine and DHBT-1 in the presence of peroxynitrite induced significant neuronal injury. Pre-treatment of cortical neurons with pelargonidin, quercetin, hesperetin, caffeic acid, the 4'-O-Me derivatives of catechin and epicatechin (0.1-3.0 mu M) resulted in concentration dependant protection against 5-S-cysteinyl-dopamine-induced neurotoxicity. These data suggest that polyphenols may protect against neuronal injury induced by endogenous neurotoxins relevant to the aetiology of the Parkinson disease. (C) 2008 Elsevier Inc. All rights reserved.

The citrus flavanone naringenin inhibits inflammatory signalling in glial cells and protects against neuroinflammatory injury

Neuroinflammation plays an integral role in the progression of neurodegeneration. In this study we investigated the anti-inflammatory effects of different classes of flavonoids (flavanones, flavanols and anthocyanidins) in primary mixed glial cells. We found that the flavanones naringenin and hesperetin and the flavols (+)-catechin and (-)-epicatechin, but not the anthocyanidins cyanidin and pelargonidin, attenuated LPS/IFN-gamma-induced TNF-alpha production in glial cells. Naringenin also inhibited LPS/IFN-gamma-induced iNOS expression and nitric oxide production in glial cells, thus showing the strongest antiinflammatory activity among all flavonoids tested. Moreover, naringenin protected against inflammatory-induced neuronal death in a primary neuronal-glial co-culture system. Naringenin also inhibited LPS/IFN-gamma-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and downstream signal transducer and activator of transcription-1 (STAT-1) in LPS/IFN-gamma stimulated primary mixed glial cells. Taken together, our results suggest that naringenin may produce an anti-inflammatory effect in LPS/IFN-gamma stimulated glial cells that may be due to its interaction with p38 signalling cascades and the STAT-I trascription factor. (C) 2009 Elseiver Inc. All rights reserved.

Caffeic acid, tyrosol and p-coumaric acid are potent inhibitors of 5-S-cysteinyl-dopamine induced neurotoxicity

Parkinson's disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra. Recent investigations have shown that conjugates such as the 5-S-cysteinyl-dopamine, possess strong neurotoxicity and may contribute to the underlying progression of the disease pathology. Although the neuroprotective actions of flavonoids are well reported, that of hydroxycinnamates and other phenolic acids is less established. We show that the hydroxycinnamates caffeic acid and p-coumaric acid, the hydroxyphenethyl alcohol, tyrosol, and a Champagne wine extract rich in these components protect neurons against injury induced by 5-S-cysteinyl-dopamine in vitro. The protection induced by these polyphenols was equal to or greater than that observed for the flavonoids, (+)-catechin, (-)-epicatechin and quercetin. For example, p-coumaric acid evoked significantly more protection at 1muM (64.0+/-3.1%) than both (-)-epicatechin (46.0+/-4.1%, p<0.05) and (+)-catechin (13.1+/-3.0%, p<0.001) at the same concentration. These data indicate that hydroxycinnamates, phenolic acids and phenolic alcohol are also capable of inducing neuroprotective effects to a similar extent to that seen with flavonoids.