Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations

The dietary polyphenolic compound resveratrol, by activating the protein deacetylase enzyme silent information regulator 2/sirtuin 1 (SIRT1), prolongs life span in evolutionarily distant organisms and may mimic the cytoprotective effects of dietary restriction. The present study was designed to elucidate the effects of resveratrol on cigarette smoke-induced vascular oxidative stress and inflammation, which is a clinically highly relevant model of accelerated vascular aging. Cigarette smoke exposure of rats impaired the acetylcholine-induced relaxation of carotid arteries, which could be prevented by resveratrol treatment. Smoking and in vitro treatment with cigarette smoke extract (CSE) increased reactive oxygen species production in rat arteries and cultured coronary arterial endothelial cells (CAECs), respectively, which was attenuated by resveratrol treatment. The smoking-induced upregulation of inflammatory markers (ICAM-1, inducible nitric oxide synthase, IL-6, and TNF-alpha) in rat arteries was also abrogated by resveratrol treatment. Resveratrol also inhibited CSE-induced NF-kappaB activation and inflammatory gene expression in CAECs. In CAECs, the aforementioned protective effects of resveratrol were abolished by knockdown of SIRT1, whereas the overexpression of SIRT1 mimicked the effects of resveratrol. Resveratrol treatment of rats protected aortic endothelial cells against cigarette smoking-induced apoptotic cell death. Resveratrol also exerted antiapoptotic effects in CSE-treated CAECs, which could be abrogated by knockdown of SIRT1. Resveratrol treatment also attenuated CSE-induced DNA damage in CAECs (comet assay). Thus resveratrol and SIRT1 exert antioxidant, anti-inflammatory, and antiapoptotic effects, which protect the endothelial cells against the adverse effects of cigarette smoking-induced oxidative stress. The vasoprotective effects of resveratrol will likely contribute to its antiaging action in mammals and may be especially beneficial in pathophysiological conditions associated with accelerated vascular aging.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Fisetin induces Nrf2-mediated HO-1 expression through PKC-δ and p38 in human umbilical vein endothelial cells

Fisetin is a natural flavonoid from fruits and vegetables that exhibits antioxidant, neurotrophic, anti-inflammatory, and anti-cancer effects in various disease models. Up-regulation of heme oxygenase-1 (HO-1) expression protects against oxidative stress-induced cell death, and therefore, plays a crucial role in cytoprotection in a variety of pathological models. In the present study, we investigated the effect of fisetin on the up-regulation of HO-1 in human umbilical vein endothelial cells (HUVECs). Small interfering RNA and pharmacological inhibitors of PKC-δ and p38 MAPK attenuated HO-1 induction in fisetin-stimulated HUVECs. Fisetin treatment resulted in significantly increased NF-E2-related factor 2 (Nrf2) nuclear translocation, and antioxidant response element (ARE)-luciferase activity, leading to up-regulation of HO-1 expression. In addition, fisetin pretreatment reduced hydrogen peroxide (H(2)O(2))-induced cell death, and this effect was reversed by ZnPP, an inhibitor of HO-1. In summary, these findings suggest that induction of HO-1 expression via Nrf2 activation may contribute to the cytoprotection exerted by fisetin against H(2)O(2) -induced oxidative stress in HUVECs.     

<Emphasis Type="Italic">ENPP1</Emphasis> 121Q functional variant enhances susceptibility to coronary artery disease in South Indian patients with type 2 diabetes mellitus

Resveratrol is a dietary polyphenol that displays neuroprotective properties in several in vivo and in vitro experimental models, by modulating oxidative and inflammatory responses. Glutathione (GSH) is a key antioxidant in the central nervous system (CNS) that modulates several cellular processes, and its depletion is associated with oxidative stress and inflammation. Therefore, this study sought to investigate the protective effects of resveratrol against GSH depletion pharmacologically induced by buthionine sulfoximine (BSO) in C6 astroglial cells, as well as its underlying cellular mechanisms. BSO exposure resulted in several detrimental effects, decreasing glutamate-cysteine ligase (GCL) activity, cystine uptake, GSH intracellular content and the activities of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR). Moreover, BSO increased reactive oxygen/nitrogen species (ROS/RNS) levels and pro-inflammatory cytokine release. Resveratrol prevented these effects by protecting astroglial cells against BSO-induced cytotoxicity, by modulating oxidative and inflammatory responses. Additionally, we observed that pharmacological inhibition of heme oxygenase 1 (HO-1), an essential cellular defense against oxidative and inflammatory injuries, abolished all the protective effects of resveratrol. These observations suggest HO-1 pathway as a cellular effector in the mechanism by which resveratrol protects astroglial cells against GSH depletion, a condition that may be associated to neurodegenerative diseases.     

Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheochromocytoma (PC12) cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities. One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. Resveratrol (3,5,4'-trihydroxy-trans-stilbene), one of the major antioxidative constituents found in the skin of grapes, has been considered to be responsible in part for the protective effects of red wine consumption against coronary heart disease ('French Pardox'). In this study, we have investigated the effects of resveratrol on hydrogen peroxide-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. PC12 cells treated with hydrogen peroxide underwent apoptotic death as determined by characteristic morphological features, internucleosomal DNA fragmentation and positive in situ end-labeling by terminal transferase (TUNEL staining). Resveratrol pretreatment attenuated hydrogen peroxide-induced cytotoxicity, DNA fragmentation, and intracellular accumulation of ROS. Hydrogen peroxide transiently induced activation of NF-kappaB in PC12 cells, which was mitigated by resveratrol pretreatment. These results suggest that resveratrol has the potential to prevent oxidative stress-induced cell death.     

Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

Vascular superoxide and hydrogen peroxide production and oxidative stress resistance in two closely related rodent species with disparate longevity

Vascular aging is characterized by increased oxidative stress, impaired nitric oxide (NO) bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long-lived species exhibit lower production of reactive oxygen species (ROS) and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white-footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus), that show a more than twofold difference in maximum lifespan potential (MLSP = 8 and 3.5 years, respectively). We compared interspecies differences in endothelial superoxide (O2-) and hydrogen peroxide (H2O2) production, NAD(P)H oxidase activity, mitochondrial ROS generation, expression of pro- and antioxidant enzymes, NO production, and resistance to oxidative stress-induced apoptosis. In aortas of P. leucopus, NAD(P)H oxidase expression and activity, endothelial and H2O2 production, and ROS generation by mitochondria were less than in mouse vessels. In P. leucopus, there was a more abundant expression of catalase, glutathione peroxidase 1 and hemeoxygenase-1, whereas expression of Cu/Zn-SOD and Mn-SOD was similar in both species. NO production and endothelial nitric oxide synthase expression was greater in P. leucopus. In mouse aortas, treatment with oxidized low-density lipoprotein (oxLDL) elicited substantial oxidative stress, endothelial dysfunction and endothelial apoptosis (assessed by TUNEL assay, DNA fragmentation and caspase 3 activity assays). According to our prediction, vessels of P. leucopus were more resistant to the proapoptotic effects of oxidative stressors (oxLDL and H2O2). Primary fibroblasts from P. leucopus also exhibited less H2O2-induced DNA damage (comet assay) than mouse cells. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accords with the prediction of the oxidative stress hypothesis of aging.     

Paraquat-generated oxidative stress in rat liver induces heme oxygenase-1 and aminolevulinic acid synthase

The in vivo effect of the known herbicide, paraquat, on both hepatic oxidative stress and heme metabolism was studied. A marked increase in lipid peroxidation and a decrease in reduced glutathione (GSH) content were observed 1 h after paraquat administration. The activity of liver antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase was decreased 3 h after paraquat injection. Heme oxygenase-1 induction started 9 h after treatment, peaking at 15 h. delta-aminolevulinic acid synthase induction occurred once heme oxygenase had been enhanced, reaching its maximum (1.5-fold of control) at 16 h. delta-aminolevulinic acid dehydratase activity was 40% inhibited at 3 h showing a profile similar to that of GSH, while porphobilinogenase activity was not modified along the whole period of the assay. Administration of alpha-tocopherol (35 mmol/kg body weight) 2 h before paraquat treatment entirely prevented the increase in thiobarbituric acid reactive substances (TBARS) content, the decrease in GSH levels as well as heme oxygenase-1 and delta-aminolevulinic acid synthase induction. This study shows that oxidative stress produced by paraquat leads to an increase in delta-aminolevulinic acid synthase and heme oxygenase-1 activities, indicating that the herbicide affects both heme biosynthesis and degradation.     

Preconditioning with hyperbaric oxygen induces tolerance against oxidative injury via increased expression of heme oxygenase-1 in primary cultured spinal cord neurons

Hyperbaric oxygen (HBO) preconditioning can induce ischemic tolerance in the spinal cord. The effect can be attenuated by the administration of an oxygen free radical scavenger or by inhibition of antioxidant enzymes. However, the mechanism underlying HBO preconditioning of neurons against ischemic injury remains enigmatic. Therefore, in the present study primary cultured spinal cord neurons were treated with HBO and then subjected to a hydrogen peroxide (H(2)O(2)) insult. The results show that H(2)O(2) stimulation of the cultured spinal neurons caused severe DNA damage and decreased cell viability, and that these neurons were well protected against damage after a single exposure to HBO preconditioning (0.35 MPa, 98% O(2), 37 degrees C, 2 h). The protective effect started 4 h after pretreatment and lasted for at least 24 h. The cultured neurons after HBO treatment also exhibited increased heme oxygenase-1 (HO-1) expression at both the protein and mRNA levels, which paralleled the protective effect of HBO. Treatment with tin-mesoporphyrin IX (SnMP), a specific HO-1 inhibitor, before HBO pretreatment abolished the HBO-induced adaptive protection noted in the cultured spinal neurons. In conclusion, HBO preconditioning can protect primary cultured spinal cord neurons against oxidative stress, and the upregulation of HO-1 expression plays an essential role in HBO induced preconditioning effect.     

Resveratrol protects H9c2 embryonic rat heart derived cells from oxidative stress by inducing autophagy: role of p38 mitogen-activated protein kinase

Recently, many studies have attempted to illustrate the mechanism of autophagy in protection against oxidative stress to the heart induced by H(2)O(2). However, whether resveratrol-induced autophagy involves the p38 mitogen-activated protein kinase (MAPK) pathway is still unknown. This study aimed to investigate whether treating H9c2 cells with resveratrol increases autophagy and attenuates the cell death and apoptosis induced by oxidative stress via the p38 MAPK pathway. Resveratrol with or without SB202190, an inhibitor of the p38 MAPK pathway, was added 30 min before H(2)O(2). After H(2)O(2) treatment, the cells were incubated under 5% CO(2) at 37 °C for 24 h to assess cell survival and death or incubated for 20 min for Western blot and transmission electron microscopy. Flow cytometry was used to detect apoptosis after 6 h of H(2)O(2) treatment. Resveratrol at 20 μmol/L protected H9c2 cells treated with 100 μmol/L H(2)O(2) from oxidative damage. It increased cell survival and markedly decrease lactate dehydrogenase release. In addition, resveratrol increased autophagy and decreased H(2)O(2)-induced apoptosis. Furthermore, the protective effects of resveratrol were inhibited by 10 μmol/L SB202190. Thus, resveratrol protected H(2)O(2)-treated H9c2 cells by upregulating autophagy via the p38 MAPK pathway.     

Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG

Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are unknown. We investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione, and upregulated Cu/Zn superoxide dismutase and heme oxygenase-1 (2 antioxidant enzymes), but it failed to alter the expression of catalase and glutathione peroxidase. In addition, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a proapoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (2 apoptogenic proteins) but was independent of caspase cascade activation. Whereas N-acetylcysteine (an antioxidant) effectively reversed the SE-induced increase in ROS and depletion of glutathione, it also suppressed SE-induced nuclear translocation of either AIF or EndoG and prevented the enhanced DNA fragmentation that would have resulted from this. We conclude that 1) although SE upregulates Cu/Zn superoxide dismutase and heme oxygenase-1, it nevertheless increases intracellular oxidative stress in HPAECs, and 2) SE promotes oxidative stress-mediated caspase-independent HPAEC apoptosis that involves mitochondrial-to-nuclear translocation of AIF and EndoG. Thus modulations of the expression of antioxidant enzymes and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.     

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial O2(-) and H2O2 production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial O2(-) and H2O2 production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular O2(-) and H2O2 production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress.     

Opposing roles of prion protein in oxidative stress- and ER stress-induced apoptotic signaling

Although the prion protein is abundantly expressed in the CNS, its biological functions remain unclear. To determine the endogenous function of the cellular prion protein (PrP(c)), we compared the effects of oxidative stress and endoplasmic reticulum (ER) stress inducers on apoptotic signaling in PrP(c)-expressing and PrP(ko) (knockout) neural cells. H(2)O(2), brefeldin A (BFA), and tunicamycin (TUN) induced increases in caspase-9 and caspase-3, PKCdelta proteolytic activation, and DNA fragmentation in PrP(c) and PrP(ko) cells. Interestingly, ER stress-induced activation of caspases, PKCdelta, and apoptosis was significantly exacerbated in PrP(c) cells, whereas H(2)O(2)-induced proapoptotic changes were suppressed in PrP(c) compared to PrP(ko) cells. Additionally, caspase-12 and caspase-8 were activated only in the BFA and TUN treatments. Inhibitors of caspase-9, caspase-3, and PKCdelta significantly blocked H(2)O(2)-, BFA-, and TUN-induced apoptosis, whereas the caspase-8 inhibitor attenuated only BFA- and TUN-induced cell death, and the antioxidant MnTBAP blocked only H(2)O(2)-induced apoptosis. Overexpression of the kinase-inactive PKCdelta(K376R) or the cleavage site-resistant PKCdelta(D327A) mutant suppressed both ER and oxidative stress-induced apoptosis. Thus, PrP(c) plays a proapoptotic role during ER stress and an antiapoptotic role during oxidative stress-induced cell death. Together, these results suggest that cellular PrP enhances the susceptibility of neural cells to impairment of protein processing and trafficking, but decreases the vulnerability to oxidative insults, and that PKCdelta is a key downstream mediator of cellular stress-induced neuronal apoptosis.     

Vitamin C, resveratrol and lipoic acid actions on isolated rat liver mitochondria: all antioxidants but different

Modulating mitochondrial antioxidant status is a nutritional issue of great interest in the treatment or prevention of several oxidative stress related diseases such as obesity. Thus, the aim of the present study was to analyze the effects of three antioxidants on hepatic mitochondrial function and antioxidant status. Isolated rat liver mitochondria were incubated with vitamin C, resveratrol and lipoic acid. The activity of antioxidant enzymes (manganese superoxide dismutase and glutathione peroxidase), ROS generation and respiratory parameters (RCR, P/O ratio and respiratory states) were measured. Vitamin C influenced mitochondrial function by decreasing of ROS generation (P < 0.0001), by stimulating the activity of manganese superoxide dismutase (197.60 ± 35.99%; P < 0.001) as well as glutathione peroxidase (15.70 ± 5.76%; P < 0.05) and by altering the activity of the electron transport chain, mainly by decreasing the P/O ratio (P < 0.05). Resveratrol induced a significant increase in manganese superoxide dismutase activity (160 ± 11.78%; P < 0.0001) and a decrease in ROS generation (P < 0.05 to P < 0.0001). By contrast, lipoic acid inhibited glutathione peroxidase activity (16.48 ± 3.27%; P < 0.05) and induced the uncoupling of the electron transport chain (P < 0.01). Moreover, this antioxidant induced a strong decrease in the P/O ratio (P < 0.05 to P < 0.0001). In conclusion, our results suggest that the three tested antioxidants produced direct effects on mitochondrial function, although the magnitude and intensity of these actions were significantly different, which may have implications when administrated as antioxidants.     

Involvement of heme oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE (antioxidant response element) signaling has been considered as a promising strategy to combat with oxidative stress-related diseases. In the present study, we tested for potential effects of sesamin, a major lignan contained in sesame seeds, its stereoisomer episesamin, and their metabolites on Nrf2/ARE activation in rat pheochromocytoma PC12 cells. Luciferase reporter assays showed that primary metabolites of sesamin and episesamin, SC-1 and EC-1 were the most potent ARE activators among all tested compounds. SC-1 {(1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane} enhanced nuclear translocation of Nrf2 and up-regulated expression of phase II antioxidant enzymes including heme oxygenase-1 (HO-1). Treatment with SC-1 resulted in increased phosphorylation of p38 MAP kinase and transient increase in intracellular ROS levels. N-acetylcysteine (NAC) treatment abolished p38 phosphorylation as well as HO-1 induction caused by SC-1, indicating that ROS are upstream signals of p38 in Nrf2/ARE activation by SC-1. Furthermore, preconditioning with SC-1 attenuated H(2)O(2)-induced cell death in a dose-dependent manner. Finally, treatment with a HO-1 inhibitor, Zn-protoporphyrin (ZnPP), and overexpression of a dominant-negative mutant of Nrf2 diminished SC-1-mediated neuroprotection. Our results demonstrate that SC-1 is capable of protecting against oxidative stress-induced neuronal cell death in part through induction of HO-1 via Nrf2/ARE activation, suggesting its potential to reduce oxidative stress and ameliorate oxidative stress-related neurodegenerative diseases.     

Total antioxidant capacity and nuclear DNA damage in keratinocytes after exposure to H2O2

Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glutathione peroxidase) were studied. Exposure to H2O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.