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Feeding rats a hyperlipidic diet in which animals were offered daily a variety of high-energy food resulted in a significant increase of serum free fatty acids and a decrease of phospholipids with respect to controls. On the contrary, there were no significant differences in erythrocyte membrane total lipid composition between the two groups. Erythrocyte membranes showed a significant decrease in saturated fatty acid content and a significant increase in (n-6) polyunsaturated fatty acid content; (n-3) polyunsaturated fatty acids significantly decreased. Membrane fluidity, investigated by fluorescence polarization of diphenylhexatriene, significantly increased in the erythrocyte membranes of the experimental group. These results seem compatible with the decreased saturated/unsaturated fatty acid ratio. A significant decrease of (Na+-K+)ATPase activity occurred in erythrocyte membranes of the experimental group rats with respect to the controls.

The benzodiazepine receptor from rat brain was solubilised and purified 5200-fold by affinity chromatography. The affinity column contained an immobilized benzodiazepine (delorazepam) and biospecific elution with 6 mm-chlorazepate was achieved. The purified receptor is apparently homogeneous in SDS-polyacrylamide gel electrophoresis. The native protein had a molecular weight of 240,000, and the subunit one of 60,000. The dissociation constant (KD) is 8 nm for [3H]diazepam. A correlation exists between the value of affinity obtained for benzodiazepine derivatives and their known pharmacological effectiveness.

Acute, exhaustive exercise leads to a burst of reactive oxygen species (ROS) generationthat increases the GSSG/GSH ratio in plasma, and causes structural damage to muscle cells as evidenced by an increase in plasma activity of cytosolic enzymes such as lactate dehydrogenase (LDH) and creatine kinase (CK). Acute exhaustive exercise can be considered an interesting model of oxidative stress and muscle damage. To prevent and counteract ROS generation and oxidative stress during exercise, many studies have been focused on the use of natural compounds with ROS scavenging properties acting as direct antioxidants, but the overall results are still inconclusive. In this study we have investigated the possibility to counteract exhaustive exercise induced oxidative stress and muscle damage in rats by treating animals with sulforaphane (SF). SF is a naturally occurring isothiocyanate present in the human diet and originating from the ingestion of Cruciferous vegetables. SF is known to act as a chemical inducer in many tissues, but no data on its ability to protect skeletal muscle tissues from oxidative stress induced by acute exhaustive exercise are available. MATERIALS AND METHODS. Male Wistar rats (age 4 months, weight 230±20g) were treated every 24 hours with SF (25 mg/kg bw i.p.) for three days before undergoing an acute exhaustive exercise protocol. The exercise protocol consisted in running on a treadmill at 24 m/min and 7% gradient, immediately at the end of the exercise animals were sacrified, and LDH and CK activities were determined on plasma samples. The activities and expressions of phase 2 enzymes such as NAD(P)H:quinone oxidoreductase (NQO1), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), thioredoxin reductase (TR) and the Total antioxidant activity (TAA) were evaluated in homogenates from freshly excised vastus lateralis skeletal muscle by spectrophotometric and immunoblotting techniques. RESULTS. Acute exhaustive exercise increased plasma LDH and CK activities, two well known biomarkers of tissue damage, and significantly reduced muscle tissue TAA, that provides an indication of the tissue antioxidant capacity. SF treatment significantly reduced LDH and CK release and was also able to counteract the decrease in TAA due to exhaustive exercise, evidencing a protection of muscle tissue against exhaustive induced damage. Acute exhaustive exercise induced a reduction in NQO1, an antioxidant enzyme protecting cells against the toxicity of quinones, while had no effect on GST, GR, GPx and TR expression and activity. On the other hand, SF treatment induced NQO1, GST and GR expression and activities in muscle homogenates, with no effect on GPx and TR. Our data demonstrate that SF could play a critical role in the modulation of muscle redox environment leading to the prevention of exhaustive exercise induced muscle damage. These results suggest that SF could become an interesting natural compound in the development of a dietary intervention that promotes oxidant scavenging through phase 2 protein induction, and in the development of new dietary supplements for physical active people. Supported by Fondazione del Monte di Bologna e Ravenna (Italy)

The effect of broccoli extract (BE)-enriched diet was studied in order to evaluate its ability to counteract liver cholesterol oxidation products (COPs) induced by acute strenuous exercise in rats. Thirty-two female Wistar rats were randomly divided into four groups: control diet without exercise (C), BE-enriched diet without exercise (B), control diet with acute exhaustive exercise (S) and BE-enriched diet with acute exhaustive exercise (BS). The study lasted 45 days and on the last day, rats of S and BS groups were forced to run until exhaustion on a treadmill. Glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and cholesterol oxidation products (COPs) were determined in liver. Exhaustive exercise was clearly responsible for tissue damage, as evidenced by the increase of lactate dehydrogenase (LDH) plasma activity increase in the S group rats. Moreover, the exercise protocol reduced CAT activity in liver, while it did not affect GST, GR and GPx. The BE-enriched diet increased raised GST, GR and CAT activities in rats of BS group. The main COPs found were 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholestanetriol, 24-hydroxycholesterol and 27-hydroxycholesterol. The BE-enriched diet led to reduced cholesterol oxidation following exhaustive exercise; the highest level of COPs was found in the S group, whereas the BS rats showed the lowest valueamount. This study indicates that the BE-enriched diet increases antioxidant enzyme activities and exerts an antioxidant effect towards cholesterol oxidation in rat liver, suggesting the use of phytochemicals in the prevention of oxidative damage and in the modulation of the redox environment.

Oxidative stress plays a major role in the pathophysiology of cardiac disorders, but the experimental data on the protective effects of exogenous antioxidants are controversial. A promising cardioprotective strategy may be through the induction of the endogenous antioxidants and phase II enzymes by chemical inducers. Sulforaphane is an isothiocyanate derived from cruciferous vegetables, and it has gained attention mainly as a potential chemopreventive agent in part through the induction of detoxifying enzymes. Accordingly, this study was undertaken to investigate the time-dependent induction of gene transcription, protein expression, and enzyme activity of antioxidant and phase II enzymes [glutathione reductase, glutathione-S-transferase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase-1, thioredoxin reductase] by sulforaphane in cultured rat neonatal cardiomyocytes. The potential cardioprotective action of sulforaphane was confirmed by the decrease in intracellular reactive oxygen species production, the increase in cell viability, and the decrease in DNA fragmentation after long-term treatment accompanied by the induction of antioxidants and phase II enzymes in cardiomyocytes.

Apha-1-adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha-1-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of beta and delta isoforms, but not alpha and epsilon, supporting the notion that GTE directly affects PKC activation and is a beta and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/counteraction of these diseases.

Recent in vivo studies have highlighted an important role for the neuroprotective actions of dietary anthocyanins. However, one consistent result of these studies is that the systemic bioavailability of anthocyanins, including cyanidin 3-O-glucopyranoside (Cy-3G), is very poor. Cy-3G has been demonstrated to be highly instable at physiological pH, so its in vivo metabolites, such as the aglycon cyanidin (Cy) and protocatechuic acid (PA), may be responsible for both the antioxidant activitiy and the neuroprotective effects observed in vivo. Therefore, we investigated the protective effects of Cy-3G, Cy and PA against H(2)O(2)-induced oxidative stress in a human neuronal cell line (SH-SY5Y). We determined their ability to counteract reactive oxygen species (ROS) formation and to inhibit apoptosis in terms of mitochondrial functioning loss and DNA fragmentation induced by H(2)O(2). We demonstrated that pretreatment of SH-SY5Y cells with Cy-3G, Cy and PA inhibits H(2)O(2)-induced ROS formation at different cellular levels: Cy-3G at membrane level, PA at cytosolic level and Cy at both membrane and cytosolic levels. In addition, Cy showed a higher antioxidant activity at membrane and cytosolic level than Cy-3G and PA, respectively. Interestingly, both Cy and PA, but not Cy-3G, could inhibit H(2)O(2)-induced apoptotic events, such as mitochondrial functioning loss and DNA fragmentation. These results suggest that Cy and PA may be considered as neuroprotective molecules and may play an important role in brain health promotion. These in vitro findings should encourage further research in animal models of neurological diseases to explore the potential neuroprotective effects of compounds generated during in vivo metabolism of anthocyanins.

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H2O2-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H2O2 cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H2O2 cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways. (c) 2006 Elsevier Masson SAS. All rights reserved.

Introduction: Sulforaphane (SF), an isothiocyanate from cruciferous vegetables, has proven to counteract oxidative stress induced apoptosis and to act as an indirect antioxidant in many organs and tissues. Since acute exhaustive exercise promotes ROS generation, the aim of this study was to evaluate the effect of a broccoli extract enriched diet on cholesterol oxidation in liver of rats subjected to exhaustive exercise. Materials and Methods: Thirty two male Wistar rats (age: 4 months, average weight: 230 g) were randomly divided into four groups: control diet without exercise (CC), enriched diet (average daily consumption: 0.15 mg of SF per rat) without exercise (CSF), control diet with acute exhaustive exercise (CW) and enriched diet with acute exhaustive exercise (WS). The study lasted 45 days and on the ultimate day, rats of groups CW and WS were forced to run until exhaustion on a treadmill. Immediately after the exercise protocol, all rats were slaughtered, and their livers were isolated and grouped into four pools of 2 livers each per single treatment. Livers were subjected to direct cold saponification, SPE-NH2 purification and the cholesterol oxidation products (COPs) fraction was injected into FAST GC/MS. Results: The main COPs found were 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholestanetriol, 24-hydroxycholesterol and 27-hydroxycholesterol. In general, the SF-enriched diet led to reduced cholesterol oxidation. The highest level of COPs (0.985±0.155 microg/g liver) was found in the CW group, whereas the WS rats showed the lowest value (0.237±0.080 microg/g liver). Moreover, the CSF group had a higher content of COPs (0.397±0.021 microg/g liver) than CC (0.503±0.064 microg/g liver). Conclusions: This study suggests that SF had an antioxidant effect towards cholesterol oxidation in liver of rats, particularly evident under exhaustive exercise conditions, and thus could play a critical role in the modulation of the redox environment and on the prevention of oxidative damage.

This study investigated potential cardiotoxicity as exerted by Erbicin-derived-immunoagents (EDIAs), novel human anti-ErbB2 immunoagents engineered by fusion of a human anti-ErbB2 scFv, Erbicin, with either a human RNase or the Fc region of a human IgG1. EDIAs are strongly cytotoxic on ErbB2-positive cells in vitro and in vivo and bind to an epitope different from that of Herceptin, a humanized anti-ErbB2 mAb effective in the therapy of breast carcinoma, but cardiotoxic in a high percentage of cases. Toxicity and apoptosis were tested in vitro by 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT), DNA fragmentation, and immunoblotting analyses. Echocardiography was measured in mice after treatment with each immunoagent. Cardiac fibrosis and detection of apoptosis were examined by Sirius red staining of collagen and TUNEL assay, respectively. EDIAs were found in vitro to have no adverse effects on cardiac cells for which Herceptin is severely toxic. In vivo studies on a mouse model showed that the EDIAs did not alter cardiac function, whereas Herceptin and doxorubicin, used as positive controls, significantly reduced the fractional shortening parameter. Cardiac fibrosis and apoptosis were not significantly affected in mice treated with EDIAs. Thus, EDIAs could fulfill the therapeutic need of patients ineligible for Herceptin treatment due to cardiac dysfunction.

Consumers consider plant food products from organic origin healthier than the corresponding conventional plant foods. Clear experimental evidence supporting this assumption is still lacking. To determine if the organic red oranges have a higher phyto–chemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and in vitro bioactivity, in terms of protective effect against oxidative damage at cellular level, than nonorganic red oranges. Total phenolics were measured using the Folin Ciocalteau assay, while total anthocyanins and ascorbic acid levels were determined by spectrophotometric and HPLC analysis, respectively. In addition, the total antioxidant activity of red orange extracts was measured by the ABTS•+ test. The ability of red orange extracts to counteract conjugated diene containing lipids and free radical production in cultured rat cardiomyocytes and differentiated Caco–2 cells, respectively, was assessed. Organic oranges had significantly higher total phenolics, total anthocyanins and ascorbic acid levels than the corresponding non–organic oranges (all p < 0.05). Moreover, the organic orange extracts had a higher total antioxidant activity than non–organic orange extracts (p < 0.05). In addition, our results indicate that red oranges have a strong capacity of inhibiting the production of conjugated diene containing lipids and free radicals in rat cardiomyocytes and differentiated Caco–2 cells, respectively. Statistically higher levels of antioxidant activity in both cell models were found in organically grown oranges as compared to those produced by integrated agriculture practice. Our results clearly show that organic red oranges have a higher phytochemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and bioactivity than integrated red oranges. Further studies are needed to confirm whether the organic agriculture practice is likely to increase the antioxidant activity of other varieties of fruits and vegetables.

Since in the aged heart an increased basal production of reactive oxygen species (ROS) has been demonstrated, and the resistance to ROS attack could be ameliorated by antioxidant supplementation, we verified the protective effect of selenium, as sodium selenite (SS) or seleno methionine (SM), in cultured rat cardiomyocytes aged in vitro. In normoxia, glutathione peroxidase (GPx) activity and total antioxidant activity were higher in old than in young cardiomyocytes, suggesting the existence of a compensatory increase of antioxidant defenses. When aged cells were submitted to hypoxia/reoxygenation, GPx activity was not modified; while total antioxidant activity decreased, conjugated diene level increased. Selenium supplementation, particularly as SM, was able to increase GPx, and consequently total antioxidant activity, and to decrease conjugated diene production. The observed ability of selenium supplementation to protect aged cardiomyocytes from hypoxia/reoxygenation damage underlines the importance of an optimal selenium dietary intake, particularly in the elderly.

Olive tree leaves are a source of bioactive compounds, as oleuropein, flavonoids, and triterpenes. Hibiscus, used for centuries in the treatment of hypertension and hyperlipidemia, is known to be a rich source of anthocyanins. We evaluated the cardiovascular protective effects of Olea Europea leaves (OE) and Hibiscus flower (HE) extracts and a 26:4 w/w combination of the two (MIX). The extracts were characterized in their bioactive composition by HPLC-DAD-MS analysis. OE, HE and MIX reduced intracellular reactive oxygen species formation and improved cell viability following oxidative stress in dose dependent manner in HUVEC cell cultures. In isolated guinea pig left and right atria and aorta the effect on cardiovascular parameters was evaluated. OE, HE and MIX showed a similar negative inotropic effect. MIX, in contrast to OE and HE, showed a significant negative chronotropic activity. On vascular smooth muscle, OE and HE reduced the contraction induced by 80 mM K+ in a dose-dependent manner and MIX evidenced a miorelaxant activity higher than OE and HE. So, the combination of OE and HE could represent a nutraceutical approach in the protection of cardio and vascular health.

The endogenous neurotoxin 5-S-cysteinyl-dopamine (CysDA) has been linked to the degeneration of dopaminergic neurons in the Substantia nigra. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables that has been proposed as a potential chemopreventive agent, although its effects on other cells are less clear. This study was undertaken to investigate the protective effects of SFN against CysDA-induced injury in primary cortical neurons. The results indicate that SFN protects primary cortical neurons against CysDA-induced injury by a mechanism involving the increased nuclear translocation of Nrf2 and the increased activity of phase II enzymes such as glutathione-S-transferase, glutathione reductase, and thioredoxine reductase. Moreover, the protection exerted by SFN appears to be mediated by the activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and Akt/protein kinase B (PKB) pathways.

Vascular smooth muscle cell (SMC) dysfunction due to enhanced oxidative stress contributes to age-related cardiovascular diseases such as atherosclerosis and calcification. Klotho, a protein with multiple pleiotropic effects associated with anti-ageing, can extend lifespan when overexpressed while its deficiencies result in rapid aging. It exists in membrane and secreted forms with distinct pleiotropic functions, with the secreted form regulating ion channels, suppressing growth factor signaling and oxidative stress while the transmembrane protein forms a co-receptor for FGF23, although the mechanisms involved in its actions remain to be fully elucidated. Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables that can induce antioxidant defence enzymes such as heme oxygenase-1 (HO-1) and peroxiredoxin-1 (Prx-1). The present study investigates whether SFN or klotho increases antioxidant defences in cultured human aortic SMC. Cells were treated (0-24 h) with either SFN (0-5 µM) or klotho (0-1 nM) and HO-1, Prx-1 expression determined by western blot analyses and glutathione (GSH) levels measured using the o-phthalaldehyde fluorescence assay. SFN significantly (p

The DNA-damage response (DDR) initiates cellular processes that can lead to a difficult choice: DNA repair, senescence or apoptosis. Differences in DDR efficiency may contribute to species-specific differences in lifespan and partially explain the exceptional longevity of some mammal species. To better understand the differences in genomic stability between species we have examined, in fibroblast cultures from several mammals, the appearance of micronuclei and the recruitment of 53BP1 in nuclear structures termed foci, after a genotoxic insult represented by the treatment with Etoposide and Neocarcinostatin. Quantification of 53BP1 foci formation together with micronuclei appearance up to three days after damage showed that cells from long lived species appear to be better equipped in the control of DNA damage repair system and the control of progression into the cell cycle. This capacity may be the consequence of a better capacity to detect DNA damage. We propose a newer interpretation of nuclear foci: they do not simply represent the presence of DNA damage but rather the cell awarness of it. We suggest that a key element for a long lived species is the capacity to detect damage and to take the necessary time to make an accurate choice between repair, senescence or apoptosis.

It is known that an increase in the intracellular level of reactive oxygen species (ROS), in particolar hydrogen peroxide (H2O2) is able to affect signalling pathways regulating proliferation and cancer development. H2O2 has been long thought to freely permeate across biological membranes, but recently, new evidence demonstrated that membrane H2O2-permeability is limited and that specific acquaporin (AQP) isoforms are able to funnel H2O2 across membranes. Data here reported show that AQP inhibition cause a decrease in intracellular H2O2 accumulation in leukaemia cells. Furthermore, AQP8 overexpression or silencing by means of siRNA resulted in the modulation of VRGF ability of triggering H2O2 intracellular level increase or decrease, respectively. Finally, we show that AQP8-facilitated H2O2 transport is able to increase cell proliferation through a mechanism dependent on PI3K and p38MAPK. In summary, our findings indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation. Therefore, the development of new drugs targeting specific AQP isoforms might be and interesting novel anti-cancer strategy.

Food bioactive peptides are currently investigated as they display functional activities for health promotion. Although high in carbohydrates, cereals contain substantial amount of proteins thus representing a possible dietary source of bioactive peptides. In the search of bioactive peptides in different wheat varieties a 7 kDa peptide was observed by means of HPLC-UV and nanoLC-nanoESI-QTof analyses in wheat flour extracts. Through data base searches the 7 kDa signal was ascribed to a Non-specific lipid-transfer protein (NsLTP) type 2 (sequence coverage of 92%). As no data regarding NsLTP type 2 biological activity in mammalian cells are present in the literature, experiments were aimed at the analysis of potential mechanisms by which this peptide can contribute to cardiovascular health using HUVEC cell cultures. In particular antioxidant activity and cytoprotective effects following oxidative or inflammatory stimulation were evaluated. Moreover, NsLTP type2 was also analysed for its ability to inhibit Angiotensin converting enzyme, a therapeutic target for the treatment of hypertension. This work was supported by BACCHUS project (FP7 European Commission Grant Agreement 312090).

Aquaporins (AQP) are channels that transport water and other small solutes across plasma membranes. In human, 13 different AQP isoforms have been discovered and three of them (AQP1, AQP3 and AQP8) have recently been reported to channel hydrogen peroxide (H2O2). Interestingly, the three AQP isoforms that possess the capacity of channel H2O2 are also widely expressed in blood cells. It is known that H2O2 can act as a messenger in cell signalling pathways and that an increase in intracellular ROS level supports pro-survival pathways contributing to cancer development. We previously demonstrated that H2O2 produced by NAD(P)H oxidases (Nox) promotes cellular proliferation in leukaemia cells. The aim of this work was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In the present study we report that specific AQP isoforms (AQP8 and AQP3, in a lesser extent) constitute a possible way through which Nox-derived H2O2 can enter leukaemia cells. Moreover, we demonstrate that AQP8 overexpression is able to enhance intracellular H2O2 accumulation induced by VEGF. Furthermore, we show that AQP8 is capable of amplifying phosphorylation triggered by H2O2 of both PI3K and p38 MAPK. In summary, results here reported indicate that AQP8 is able to modulate H2O2 transport through plasma membrane contributing to the aberrant proliferation of leukaemia cells.

It is widely accepted that oxidative injury and inflammation are intimately involved in the aging process and the development of age-related diseases. Among the age-related diseases, cardiovascular diseases (CHD) represents the leading cause of death in industrialized and developing countries. Although we lack the power to change some risk factors such as family history, sex or age, there are some key prevention steps we can take. CHD should be targeted as a major set of preventable causes of illness, with effects not only on premature mortality but also on well-being in older ages. Many intervention and epidemiological studies demonstrated that following a heart-healthy diet may prevent or delay the onset of CHD. There is convincing evidence that increasing consumption of vegetables and fruit reduces the risk of disease. These are the main constituents of the Mediterranean diet (UNESCO Intangible Cultural Heritage of Humanity), characterized by high levels of nutraceutical bioactive compounds. Nutraceutical, a term combining the words “nutrition” and “pharmaceutical”, is a food constituent, mainly a phytochemical component, that provides health benefits, including the prevention and treatment of disease. So, a healthy dietetic approach specifically formulated for elderly people, with a defined pattern of nutraceutical bioactive compounds, may represent a key strategy to improve the aging process. To date, most dietary anti-aging and cardioprotective strategies have only focused on the delivery of exogenous antioxidants to boost antioxidant status in an effort to protect against toxicant-induced oxidative and inflammatory stress as a means to prevent or combat the negative effects of age-related diseases. A promising new strategy intends to identify nutraceutical bioactive compounds with the ability to directly target and enhance intrinsic cytoprotective mechanisms, including modulation of the expression of genes involved in the detoxification of xenobiotics and their metabolites, genes involved in the synthesis and regulation of intrinsic antioxidants and antioxidant enzymes, genes involved in the regulation of inflammation. In particular, nutraceutical compounds as quercetin, the main polyphenol of the western diet, abundant in apples and onions, may prevent heart damage by inducing multiple cytoprotective pathways, upregulating antioxidant and phase II enzymes, intercepting and detoxifying damaging compounds, and efficiently removing said toxicants before they can initiate further damage (1-3). One more interesting compound is sulforaphane, derived from the hydrolysis of the Cruciferous vegetables glucosinolates, that is able to modulate the expression of genes and proteins related to the Nrf2/phase II detoxification and inflammatory pathways in cardiomyocytes, acting as a “second level” antioxidant (4,5). But individual nutraceuticals can have greater or lesser effects on specific Nrf2- and inflammation-related genes in various tissues and experimental models. Therefore, only using a combination of nutraceuticals, as those naturally present in fruits and vegetables, it would be possible to modulate the greatest diversity of Nrf2- and inflammation related genes in the greatest number of tissues to achieve the most dramatic protective effects against oxidative damage, toxicants, and inflammation, and to provide the most robust preventive/protective and anti-aging benefits.

This work was aimed at evaluating the cardioprotective effects ofCastanea sativaMill. (CSM) bark extract characterized in its phenolic composition by HPLC-DAD-MS analysis. The study was performed using primary cultures of neonatal rat cardiomyocytes to investigate the antioxidant and cytoprotective effects of CSM bark extract and isolated guinea pig left and right atria, left papillary muscle, and aorta to evaluate its direct effect on cholinergic and adrenergic response. In cultured cardiomyocytes the CSM bark extract reduced intracellular reactive oxygen species formation and improved cell viability following oxidative stress in dose-dependent manner. Moreover, the extract decreased the contraction induced by noradrenaline (1 μM) in guinea pig aortic strips and induced transient negative chronotropic and positive inotropic effects without involvement of cholinergic or adrenergic receptors in the guinea pig atria. Our results indicate that CSM bark extract exhibits antioxidant activity and might induce cardioprotective effect.

Steviol glycosides, primarily stevioside and rebaudioside A, are natural constituents of the plant Stevia rebaudiana Bertoni. Steviosides are already in use as non-caloric sweeteners (1). Several studies have reported hypoglycemic effects of Stevia extracts, particularly among individuals affected by type 2 diabetes, a disease associated to impaired insulin secretion and insulin resistance in peripheral tissues, leading to hypoinsulinemia and hyperglycemia (2). The aim of this study was to investigate the effect of rebaudioside A and three different mixtures of steviol glycosides on the regulation of glucose uptake in different cell types and their ability in activating a signaling cascade similar to insulin pathway. The effect of four Stevia extracts on glucose transport activity was evaluated in HL-60 human leukaemia cells, expressing principally the basal glucose transporter (GLUT)1, and in SHSY-5Y human neuroblastoma cells, expressing also GLUT4, the insulin sensitive one (3). The influence of Stevia extracts on glucose transport activity was compared to the effect of insulin and of methylglyoxal, inhibitor of the insulin receptor/PI3K/Akt pathway. The treatment with insulin or steviol glicosides causes similar effects on the glucose uptake. The concomitant treatment with insulin and Stevia extracts causes a rise of glucose transport higher than the increase due to insulin and steviol glicosides alone. Methylglyoxal incubation induces a significant decrease in glucose transport and the subsequent treatment with Stevia extracts (and insulin) is able to raise the glucose uptake. Taken together these results corroborate the hypothesis that Stevia extracts could mimic insulin effects on glucose uptake modulating PI3K/Akt pathway.

Advanced glycation end products (AGEs) are found in various intraneuronal protein deposits such as neurofibrillary tangles in Alzheimer’s disease and Lewy bodies in Parkinson’s disease. Among AGE precursors, methylglyoxal (MG) is most likely to contribute to intracellular AGE formation, since it is extremely reactive and constantly produced by degradation of triosephosphates. MG accumulation often occurs under hyperglycemic conditions, impaired glucose metabolism and oxidative stress. MG is efficiently metabolized by the cytosolic glyoxalase system that is composed of two enzymes, glyoxalase (GLO) 1 and GLO2, and a catalytic amount of GSH. Isothiocyanates are metabolites derived from the hydrolysis of glucosinolates, a class of compounds present in Brassica vegetables. In particular, sulforaphane (SF), an isothiocyanate produced from glucoraphanin by the enzyme myrosinase, is known for its chemopreventive, anti-inflammatory, cardioprotective and neuroprotective actions. This study was designed to investigate the potential protective effects of SF on MG induced damage in SH-SY5Y neuroblastoma cells. MG caused neuronal cell death as measured by MTT cell viability assay and LDH release and induced apoptosis via activation of caspase 3 and modulating signaling kinases involved in survival and cell death. MG significantly increased intracellular ROS production and decreased GSH levels and glucose uptake. SF treatment significantly counteracted cell death and apoptosis induced by MG and partially inhibited MG negative modulation of signaling kinases. SF treatment reduced MG induced oxidative stress decreasing intracellular ROS production and increasing GSH levels. Moreover, SF was able to increase glucose uptake in MG treated cells and significantly increased GLO1 protein expression and activity, eliciting a fundamental role in MG detoxification.

INTRODUCTION: Aging is associated with a phenomenon termed ‘inflammaging’, which is characterised by chronic, low-grade pro-inflammatory conditions leading to long-term tissue damage. Reducing chronic low-grade inflammation may be a way to prevent or reduce the severity of age-related diseases. It has been proposed that the age-related changes in gut microbiota, associated with an increase in systemic inflammation may also contribute to the progression of disease and frailty in the elderly. Evidence exists that inflammation, oxidative stress and gut microbiota are influenced by diet or nutraceutical dietary supplements. AIMS&METHODS: To assess the impact of a personalized diet, with or without the addition of VSL#3 probiotic blend, on markers of inflammation, oxidative stress and gut microbiota in elderly individuals. Open label, randomized multicentre study. Setting: Community. Primary endpoint: baseline change in hsCRP (high-sensitivity C-reactive protein). As a part of a larger study, 62 participants were randomized to follow a personalized diet created through a web-based platform (RISTOMED) and optimized to reduce inflammation and oxidative stress, either with or without supplementation with VSL#3, for 8 weeks. RESULTS: There was no significant change in hsCRP amount in either study arm. However, a subgroup of 17 participants were classified as having high inflammation at baseline, and hsCRP fell significantly in this group (p=0.022). VSL#3 plus diet, but not diet alone, was associated with a significant reduction in homocysteine levels and increases in folate (p

Activation of DNA-damage response (DDR) is a crucial process for the maintenance of genomic stability. DDR activation initiates cellular processes that can lead to apoptosis, cell-cycle arrest, DNA repair and cellular senescence. Differences in the DDR efficiency and in the DNA damage sensing machinery may contribute to species-specific differences in lifespan and partially explain the exceptional longevity of some species, like human. To better understand the differences in genomic stability and in damage response pathways between species, in fibroblast cultures from several mammals, we have examined the appearance of micronuclei and the recruitment of 53BP1 in nuclear structures termed foci, after a genotoxic insult. We used Etoposide, a hemotherapeutic agents that forms a ternary complex with DNA and the topoisomerase II enzyme and prevents re-ligation of the DNA strands causing DNA breaks. Quantification of 53BP1 foci formation together with micronuclei appearance up to three days after damage showed that cells from long lived species appear better equipped to control progression into the cell cycle. This capacity may be the consequence of a better capacity to detect DNA damage. We propose that a key element for a long lived species is the capacity of cells to take their time to repair the endured damage and to make accurate choices regarding their destiny: proliferation, senescence or apoptosis.

NAD(P)H oxidases (Nox) are a family of membrane-associated enzymatic complexes that, transferring electrons from NAD(P)H inside the cell to molecular oxygen across the membrane, generate superoxide anion (O2-) and/or hydrogen peroxide (H2O2) outside the cell [1]. Nox-produced ROS can act as messengers in redox signalling and hydrogen peroxide is considered the main effector due to its relative stability. Although it is now confirmed the involvement of ROS in biosignalling, it still remains unclear whether the ROS generated extracellularly by Nox can permeate through the plasma membrane by simple diffusion or mediated by transport proteins. Recent experimental evidence suggest that two isoforms of aquaporins (AQP3 and AQP8) have the ability to channel H2O2 across the membranes and that they may have an important role in redox signalling [2]. Our attention has been focused on ROS involved in signalling transduction pathways contributing to cancer development. In particular, we have previously demonstrated that Nox-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cell lines [3]. In this study, we assess whether specific aquaporin isoforms play a role in facilitating Nox-produced H2O2 transport through the plasma membrane affecting downstream pathways linked to cell proliferation in leukaemia cells. We demonstrated that the treatment with AgNO3, a potent aquaporin inhibitor, causes a decrease in intracellular ROS level both when H2O2 is produced by Nox enzymes and when it is added exogenously to the medium; on the contrary, overexpression of AQP8 and AQP3 causes an increase in the intracellular ROS level when hydrogen peroxide is added. Furthermore, aquaporin inhibition causes a significant decrease in glucose transport in HL60 and B1647 cells and VEGF-induced intracellular ROS accumulation is augmented when AQP3 or AQP8 are overexpressed. In conclusion, our data suggest that AQP3 and AQP8, but not AQP1, are able to transport Nox-generated H2O2 across the plasma membrane supporting leukaemia cells proliferation.

Traumatic brain injury (TBI) is one of the leading cause of disability due to neurologic impairment in children and young adults. Acute and chronic cerebral lesions in response to an insult may lead to acute or delayed neuronal death, apoptotic cell death, oxidative stress, B-amyloid deposition, neuronal degeneration and gliosis. Chronic neuroinflammation has been postulated as a relevant feature of the response to TBI. This raises the possibility of ongoing brain damage in long-term survivors, such that their cognitive function and motor function continues to deteriorate for months and years after the injury. Much of the disabilities results from a spectrum of cognitive impairments that in several cases get worse in time, sometimes progressing to a severe neurocognitive disorder and even to Alzheimer’s Disease (AD). TBI and Alzheimer’s disease have similarities as to protein and cellular responses and genetic influences. Severe TBI may hasten or even trigger the onset of AD even after many years, irrespective of age when the TBI occurred. In many cases the location and the extent of focal brain injuries does not fully explain the neurocognitive impairments. Altered neurochemistry and diffuse cellular injury (DAI) are important contributors to brain dysfunction, associated with poor outcome after TBI. In order to explore a putative set of criteria predicting to some extent the cognitive outcome, series of clinical and neuropsychological assessments in a sample of TBI patients were correlated with neuroimaging studies and bio-umoral profiles (every 3 months for 1 year). In particular, the plasma levels of the main antioxidant/detoxifying enzymes have been evaluated together with the levels of inflammatory markers. The neuroimaging studies were performed at 2 time points (< 2 months and > 6 months) and include: (i) MR Spectroscopy (MRS) to explore brain metabolites, particularly N-Acetyl-Aspartate (NAA), Choline (Cho), Creatine (Cr); (ii) Diffusion Tensor Imaging (DTI) by Fractional Anisotropy (FA); (iii) Functional MRI (fMRI) by studying both resting state and activation in Vegetative (VS) or Minimally Conscious (MCS) states; (iv) Arterial Spin Labelling (ASL) that allows the absolute quantitative evaluation of the brain flow values. The patients were enrolled among the subjects admitted to an acute neurorehabilitation facility in Bologna and were aging from 18 to 65. The first assessment was conducted 30 days after the trauma and the follow up lasted at least 12 months. A cohort of 15 patients is already included in the study and 12 of them had also functional neuroimaging studies. The cognitive recovery had different patterns in time: for about ¼ of the patients an early recovery was followed by progressive worsening, while about 75% of the cases show a continuous but more or less rapid recovery of consciousness and cognitive functioning. The relationship between these 2 different trends and the biochemical and neuroimaging markers will be discussed.

Heart failure (HF) is characterized by inflammatory conditions which cause endothelium dysfunctions and promote a worsening of the disease. We have previously shown that the serum of HF patients has a pro-apoptotic effect on human umbilical vein endothelial cells (HUVECs) which correlates with circulating levels of TNF-α. TNF-α dysregulates Notch signalling in HUVECs causing apoptosis. To establish whether the pro-apoptotic effect of HF serum in HUVECs could be due to dysregulation of Notch signalling, HUVECs were treated with serum of patients with mild and moderate/severe HF (New York Heart Association (NYHA) class I/II and III/IV respectively) or healthy controls and the effects on Notch signalling were evaluated. Western blot analyses showed reduced levels of Notch4 receptor in cells treated with serum from NYHA class I/II patients (3/12) and NYHA class III/IV (7/12) compared to control. Similarly, in serum-treated cells the mature form of Notch ligand Delta Like-4 (Dll4) was absent (2/12 class NYHA I/II and 7/12 class III/IV). Only with serum from class III/IV patients we found both reduced levels of Notch 4 and absence of Dll4 (4/12). Notch 1, 2 as well as Jagged1 ligand protein levels were unchanged by treatment. qRT-PCR showed increased Hey2 and reduced Hes1 and Hey1 mRNAs in treated cells compared to controls. These differences in mRNAs didn’t correlate with the severity of disease. In conclusion Notch signalling in HUVECs is modulated by inflammatory cytokines in serum of HF patients indicating that dysregulation of this pathway could be part of a mechanism by which these cytokines induce endothelial dysfunctions in HF patients.

Recently, the Nox4 inhibitory activity shown by some oxindole derivatives has been described. in This study, a panel of new synthesized derivatives against different leukemia cell lines in the NCI assays were tested for the ability to inhibit Nox4 in B1647 cells, a human acute myeloid leukemia cell line. Nox4 is known to be costitutively active and to play the major role in the generation of ROS requird for sustaining cellular growth and proliferation of B1647 cells. MTT assay confirmes the anti-proliferative activity of alla the tested molecules in a dose dependent manner at a concentration of 5 and 10 microM. ROS production was measured in B1647 cells after an acute treatment (30 min) with the selected compounds. Results indicate that two compounds (C5 and C7) are able to inhibit ROS generation in a dose-dependent manner. As B1647 cells express Nox2 and Nox4, in order to rule out the role of Nox2-derived ROS, the acute inhibition of ROS production was measured following RNA silencing of Nox2. Data show that the two selected compounds were able to inhibit ROS production at the same extent either in Nox2 silenced cells or in the control. Finally, Wester blotting analysis was performed to investigate Nox4 expression inB1647 cells after 24 h-incubation with the compounds. Data show that treatments with C5 and C7 were able to downregulate Nox4. In conclusion, results here reported indicate that Nox4 could be a target for two of the new synthesized oxindole derivatives and the Nox4 inhibition seems to contribute to the anti-proliferative effect of the compounds in acute leukemia B1647 cells and to be a promising strategy for a novel antileukemic therapy.

Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention as they have strong antioxidant properties and can protect against many degenerative diseases. Aim of this study was to profile grain phenolic extracts of one modern and five old common wheat (Triticum aestivum L.) varieties and to evaluate their potential antiproliferative or cytoprotective effect in different cell culture systems. Wheat extracts were characterized in terms of antioxidant activity and phenolic composition (HPLC/ESI-TOF-MS profile, polyphenol and flavonoid contents). Results showed that antioxidant activity (FRAP and DPPH) is mostly influenced by flavonoid (both bound and free) content and by the ratio flavonoids/polyphenols. Using a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes, the potential antiproliferative or cytoprotective effects of different wheat genotypes were evaluated in terms of intracellular reactive oxygen species levels and cell viability. All tested wheat phenolic extracts exerted dose-dependent cytoprotective and antiproliferative effects on cardiomyocytes and HL60 cells, respectively. Due to the peculiar phenolic pattern of each wheat variety, a significant genotype effect was highlighted. On the whole, the most relevant scavenging effect was found for the old variety Verna. No significant differences in terms of anti-proliferative activities among wheat genotypes was observed. Results reported in this study evidenced a correspondence between the in vitro antioxidant activity and potential healthy properties of different extracts. This suggests that an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases., This work was supported by grants from the Italian Ministry of Economic Development (Project “Made in Italy-over 50”), Emilia Romagna region, Italy (Project “BioPane”, CUP J31J09000430002) and the Italian Ministry of Agriculture (Project “Pane della salute” MiPAF-OIGA, CUP 31J09000370001) and by Fondazione del Monte di Bologna e Ravenna (Italy).

Negli ultimi anni è notevolmente aumentata l’incidenza di patologie cronico-degenerative quali Parkinson, Alzheimer, patologie cardiovascolari e cancro Per queste patologie, così come per l’invecchiamento, è stato evidenziato il ruolo di primaria importanza svolto dallo stress ossidativo, una condizione di squilibrio tra specie reattive dell’ossigeno (ROS) e difese antiossidanti endogene. Più recentemente, alcuni studi hanno evidenziato anche il ruolo svolto dallo stress carbonilico, definito alla stregua di quello ossidativo come una condizione di squilibrio tra specie pro-glicanti e difese enzimatiche anti-glicanti, a favore degli agenti pro-glicanti. Le principali specie reattive dicarboniliche presenti nei sistemi fisiologici e responsabili della glicazione sono metilgliossale (MG), gliossale e 3-desossiglucosone. Questi composti causano modificazioni irreversibili alle macromolecole biologiche che conducono alla formazione e all’accumulo di advanced glycation end-products (AGE) quali prodotti terminali della glicazione non-enzimatica nei sistemi fisiologici. Una strategia chiave per difendere l’organismo dai danni indotti da MG consiste nel potenziare le difese endogene nei confronti della glicazione. Il sistema delle gliossalasi formato dagli enzimi gliossalasi (GLO) 1 e 2 rappresenta la difesa enzimatica primaria nei confronti della glicazione indotta da MG. Recentemente è stato dimostrato che l’espressione di GLO1 è regolata dal fattore di trascrizione “nuclear factor E2-related factor 2” (Nrf2). Il sulforafane (SF) è un composto naturale che si forma per idrolisi dai glucosinolati, un gruppo di fitocomponenti presenti nelle piante appartenenti alla famiglia delle Brassicaceae. SF, negli ultimi anni, è stato principalmente studiato per le sue proprietà chemiopreventive e per la sua capacità di indurre gli enzimi di fase II attraverso la modulazione del fattore di trascrizione Nrf2. Scopo di questo studio è stato valutare il potenziale effetto cardioprotettivo di SF nei confronti della glicazione non enzimatica indotta da MG in colture primarie di cardiomiociti di ratti neonati. L’effetto protettivo di SF nei confronti della glicazione è stato valutato tramite il test di vitalità del MTT e il dosaggio dell’attività e dell’espressione proteica dell’enzima pro-apototico caspasi-3 attraverso un saggio spettrofluorimetrico e un’analisi immunoblotting, rispettivamente. La capacità di ridurre la formazione endogena delle ROS è stata valutata con il test della diclorofluoresceina diacetato. L’attività dell’enzima GLO1 è stata valutata con un saggio spettrofotometrico mentre la sua espressione tramite analisi immunoblotting. Dai dati è emerso che l’esposizione dei cardiomiociti a MG 1 mM riduce la vitalità cellulare, induce morte apoptotica e incrementa la produzione delle ROS. Il pre-trattamento con sulforafane 5 µM incrementa in maniera significativa la vitalità cellulare, riduce la morte apoptotica e la produzione delle ROS di cardiomiociti esposti da MG. Inoltre, SF è in grado di incrementare l’espressione proteica e l’attività dell’enzima gliossalasi 1. Quest’ultimo dato rappresenta un risultato estremamente innovativo in quanto, per la prima volta, viene dimostrata la possibilità di indurre la GLO1 attraverso la supplementazione di SF, composto naturale a valenza nutraceutica, rappresentando un approccio integrato ed efficace per la protezione dallo stress glicativo a livello cardiaco. Tali risultati aprono la strada a nuovi ed importantissimi studi in quanto la glicazione è coinvolta, non solo nella patologia cardiovascolare indotta dal diabete, ma anche in numerose patologie cronico-degenerative e nell’invecchiamento per cui la supplementazione di SF potrebbe rappresentare, in tal senso, un’efficace strategia terapeutica.

The purpose of this work was the formulation of liposomes for nasal administration of tacrine-HCl. This route has many advantages; in particular is possible to convey the drug directly to the Central Nervous System, through the olfactory bulb. Part of Tacrine administered can also be absorbed into the systemic circulation avoiding hepatic first pass effect. Liposomes were formulated with ingredients that have a beneficial effect against the disease. In particular, the classical excipients cholesterol and phosphatidylcholine were enriched with α-tocopherol and polyunsaturated fatty acid EPA and DHA. This formulative approach allowed us to obtain 100% active liposomes against symptoms of Alzheimer's disease.

Phenolic compounds present in whole grains have gained attention as they have strong antioxidant properties, potentially protective against many degenerative diseases. Aim of this study was to characterize the phytochemical profile of modern and old common wheat (Triticum aestivum L.) varieties in terms of antioxidant activity and phenolic composition and to evaluate their antiproliferative or cytoprotective effects in different cell culture systems: a leukemic cell line, HL60, and primary cultures of neonatal rat cardiomyocytes. The evaluation of the effect on cell viability and ROS intracellular level in in vitro models allowed to highlight differences in cyotoprotective/antiproliferative properties between cultivars and to correlate them to peculiar phytochemical/nutraceutical profile of the extracts. Data reported in this paper support the healthy role of cereals in human nutrition due to the presence of nutraceutical components and their possible synergistic effect and, above all, underline the importance of a re-discovery of ancient varieties in the development of functional food. Thus, an increased intake of wheat grain derived products could represent an effective strategy to achieve both chemoprevention and protection against oxidative stress related diseases, thanks to their antioxidant phytochemicals content.

Numerose ricerche confermano l'appartenenza dei cereali alla categoria di alimenti che, nel contesto di una dieta equilibrata, possono avere un'azione protettiva sulla salute dell'uomo e fornire quindi un importante contributo nel determinare una dieta salutare. Le linee guida nazionali pongono infatti i cereali integrali ed i prodotti derivati alla base della piramide alimentare. In accordo a dati epidemiologici che dimostrano che la dieta svolge un ruolo cruciale nella prevenzione di malattie croniche con riconosciuta componente ossidativa, quali malattie cardiovascolari, cancro, diabete e neurodegenerazione, vi è un crescente interesse nell’individuazione di nuove strategie dietetiche finalizzate alla prevenzione / neutralizzazione del danno ossidativo e la ricerca è sempre più focalizzata sul potenziale ruolo citoprotettivo di antiossidanti esogeni. La maggior parte della letteratura sulle sostanze fenoliche vegetali si concentra principalmente su quelle presenti in frutta e verdura, vino e tè. Tuttavia, molti dei composti fenolici di frutta e verdura (ad esempio acidi fenolici e flavonoidi) sono presenti anche nei cereali. L’obiettivo principale di questo lavoro è stato quello di dimostrare come alcune varietà di frumento, possiedano, in ragione di un peculiare profilo fitochimico, un migliore effetto antiossidante e citoprotettivo su cellule normali e un maggior effetto antiossidante e antiproliferativo su cellule tumorali. A questo scopo sono stati utilizzati due differenti modelli cellulari: culture primarie di cardiomiociti neonatali di ratto ed una linea cellulare leucemica HL60. Diversi approcci sperimentali sono comunemente utilizzati per la valutazione del valore nutraceutico di alimenti e ingredienti. L'uso di sistemi cellulari in vitro modello rappresenta un potente strumento informativo per la definizione della capacità antiossidante e relativi effetti citoprotettivi. Sono state valutate le proprietà nutraceutiche di 6 differenti estratti polifenolici di sfarinati di diverse varietà di frumento tenero: 5 varietà antiche (Andriolo, Frassineto, Gentil Rosso, Inallettabile, Verna) e una varietà moderna (Palesio). La caratterizzazione della composizione quantitativa in polifenoli totali e flavonoidi totali degli estratti ha rivelato una maggiore concentrazione di fitocomponenti antiossidanti nelle varietà antiche rispetto alla varietà moderna che, in accordo con questo dato, ha mostrato una capacità antiossidante significativamente inferiore in un sistema privo di cellule. La valutazione dell’effetto antiossidante degli estratti, analizzato nei due sistemi modello come diminuzione dei livelli intracellulari di specie reattive dell’ossigeno ed influenza sulla vitalità cellulare, hanno permesso di evidenziare le differenze nelle proprietà citoprotettive/antiproliferative esistenti tra le diverse cultivar. I risultati riportati in questo lavoro evidenziano quindi potenziali effetti nutraceutici del frumento che possono essere trasferiti dall’ingrediente ad un prodotto finito. L’utilizzo di varietà antiche di frumento consentirebbe di ottenere nuovi prodotti commerciali capaci di differenziarsi per il particolare valore aggiunto legato alle caratteristiche nutrizionali intrinseche in grado di conferire al prodotto proprietà salutistiche.

Methyl glyoxal is a reactive dicarbonyl precursor of advanced glycation end products (AGEs). AGEs play an important role in the development and progression of cardiovascular diseases in diabetes. Serum levels of AGEs in patients with type 2 diabetes with coronary heart disease 8CHD) are increased compared to patients without CHD, and correlate with CHD severity. Methylglyoxal is effectively detoxifies by the glyoxalase system, that comprises glyoxalase 1 and 2. overexpression of glyoxalase 1 prevents the accumulation of intracellular methylglyoxal and AGEs in endothelial cells exposed to high glucose concentration. Recently it has been demonstrated that glyoxalase 1 expression is regulated by the nuclear factor E2-related factor 2 (Nrf2). Sulforaphane is an isothacianate formed form glucoraphanin present in Cruciferous vegetables. As SF is a potent inducer of Nrf2-modulated phase II enzymes, we investigated its protective effect against methylglyoxal-induced damage in primary cultures of rat cardiomyocytes. Cell viability was evaluated by MTT assay, caspase 3 activity by a fluorimetric method, caspase 3 and glyoxalase 1 expression by immunoblotting, glyoxalase 1 activity spectrophotometrically and ROS production by the 2’,7’ dichlorofluorescein-diacetate assay. We observed that methylglyoxal reduced cardiomyocyte viability, induced apoptotic cell death and increased ROS production. Sulforaphane treatment significantly increased cell viability, and reduced apoptotic cell death and ROS production of methylglyoxal-damaged cardiomyocytes. Moreover, for the first time, we demonstrated the sulforaphane is able to significantly increase glyoxalase1 protein expression and activity. These findings suggest that targeting glyoxalase system might be a promising therapeutic strategy to counteract cardiovascular diseases of diabetic patients.

Advanced glycation end products (AGEs) are a group of molecules that are generated through non-enzymatic glycation and accumulate under diabetic condition. The most investigated AGEs precursor is methylglyoxal, a metabolic compound formed mainly from the glycolytic intermediate glyceraldehyde-3-phosphate. Methylglyoxal is more reactive than glucose and shows a stronger ability to cross-link with protein amino groups to form AGEs. Methylglyoxal -induced cytotoxicity may be at least partially responsible for diabetes-related impairments of cardiovascular function. methylglyoxal omeostasis is controlled by the glyoxalase system that consists of two enzymes, glyoxalase 1 (GLO1) and glyoxalase 2. In a recent study, Xue et al. demonstrated that the transcriptional levels of GLO1 are controlled by NF-E2-related factor 2 (Nrf2). The isothiocyanate sulforaphane, derived from the hydrolysis of glucoraphanin abundantly present in broccoli, represents one of the most potent inducers of phase II enzymes through the Keap1–Nrf2 pathway. Aim of this study was to investigate, in primary cultures of rat cardiomyocytes, the biological effects of SF on methylglyoxal-induced cardiotoxicity focusing on the signaling pathways of apoptosis and the modulation of the glyoxalase system. Cell viability was evaluated by MTT assay, caspase 3 activity by a fluorimetric method, ERK1/2, p38 MAPK, JNK and Akt phosphorylation and GLO1 expression by immunoblotting, GLO1 activity spectrophotometrically. We observed that sulforaphane treatment significantly counteracted cell death and apoptosis induced by methylglyoxal, inhibited methylglyoxal induced activation of the pro-apoptotic kinases JNK and p38. Moreover, for the first time, we demonstrated that sulforaphane is able to significantly increase GLO1 protein expression and activity. These findings suggest that targeting the glyoxalase system might be a promising therapeutic strategy to counteract cardiovascular diseases of diabetic patients.

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules and occurs in a biphasic pattern: an early phase after 1–2 h and a late phase after 12–24 h. While it is widely accepted that reactive oxygen species are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H2O2-induced cardioprotection in rat neonatal cardiomyocytes. We focused on antioxidant and phase II enzymes and their modulation by protein kinase signaling pathways and nuclear-factor-E2-related factor-1 (Nrf1) and Nrf2. H2O2preconditioning was able to counteract oxidative stress more effectively in the late than in the early phase of adaptation. In particular, H2O2preconditioning counteracted oxidative stress-induced apoptosis by decreasing caspase-3 activity, increasing Bcl2 expression and selectively increasing the expression and activity of antioxidant and phase II enzymes through Nrf1 and Nrf2 translocation to the nucleus. The downregulation of Nrf1 and Nrf2 by small interfering RNA reduced the expression level of phase II enzymes. Specific inhibitors of phosphatidylinositol 3-kinase/Akt and p38 MAPK activation partially reduced the cardioprotection elicited by H2O2preconditioning and the induction and activity of phase II enzymes. These findings demonstrate, for the first time, a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes triggered by H2O2preconditioning.

Ischemic preconditioning is a cardioprotective phenomenon that involves adaptive changes in cells and molecules. This adaptation occurs in a biphasic pattern: an early phase (1-2h), and a late phase (12-24h). While it is widely accepted that reactive oxygen species (ROS) are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H2O2 induced cardioprotection in cardiac cells focusing on the induction of phase II enzymes and the involvement of Nuclear factor E2-related factor 1 (Nrf1) and Nrf2. Rat neonatal cardiomyocytes were grown until confluence. Preconditioning was simulated with 100 µM H2O2 for 10 min and after different times oxidative stress was generated by 100 µM H2O2 for 30 min. Cell viability was evaluated by MTT, and ROS production by 2′,7′-dichlorofluorescein-diacetate. Phase II enzyme activities were determined spectrophotometrically, and Nrf1 and Nrf2 nuclear translocation by immunoblotting. To evaluate the involvement in phase II enzyme induction, Nrf1 and Nrf2 were silenced with Nrf1-siRNA and Nrf2-siRNA. H2O2-preconditioning was more effective against oxidative stress in the late than in the early phase of adaptation. Surprisingly, H2O2-preconditioning led to a decrease in intracellular ROS production consistent with the induction of glutathione reductase, catalase, thioredoxine reductase and NADPH quinone oxidoreductuase 1. H2O2-preconditioning induced the nuclear translocation of both Nrf1 and Nrf2. The down-regulation of Nrf1 and Nrf2 by Nrf1-siRNA and Nrf2-siRNA partially inhibited the enzyme induction. These findings demonstrate a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes following H2O2-preconditioning.

Epidemiological studies have correlated the intake of whole grains with a reduced incidence of chronic disease (1). Among health-promoting phytochemicals in whole grains, phenolic compounds have gained attention in many research areas as they have strong antioxidant properties and can protect against many degenerative human diseases (2). Aim of this study was to characterize the phytochemical composition of new and old wheat varieties and to evaluate their potential cytoprotective/antiproliferative effect in different cell culture systems. Methodologies The study was performed on 6 common wheat genotypes (1 modern and 5 old cultivars). Polyphenols, flavonoids, FRAP test and HPLC/ESI-TOF-MS determinations were conducted according to (3). Cultured rat cardiomyocytes, grown as reported in (4), and human promyelocytic leukemia cell line HL60 were supplemented with different extracts. Cell viability was evaluated by MTT test. ROS production was measured using 2’-7’-dichlorofluorescein diacetate. Results and Discussion In the old cultivars the mean content of total polyphenols was significantly higher than in the modern variety (165±8 and 133±8 mg GAE/100g, respectively). Within the experimental set the mean total content of flavonoids was 39.9±16.7 mg CE/100g. The old cultivars Verna and Gentil Rosso exhibited the highest values, while the lowest content was found in the modern cultivar Palesio. A significant correlation was observed between antioxidant activity, total polyphenol and total flavonoid content. The HPLC-TOF-MS analyses allowed the identification of aproximately 60 different phenolic compounds. MTT test and intracellular ROS production were carried out in in vitro models to highlight differences in cyotoprotective/antiproliferative properties between cultivars and to correlate them to phytochemical/nutraceutical profile of the extracts. References (1) Fardet A. (2010) Nutr Res Rev. 23:65-134. (2) L. Dykes and L.W. Rooney (2007) Cereal Foods World 52, 105 (3) Dinelli G. et al. (2009) J Chromatogr. 1216, 7229-40 (4) Hrelia S. et al. (2002) Biochim. Biophys. Acta 1567, 150-156.

Sulforaphane is a natural dietary isothiocyanate produced by the enzymatic action of the myrosinase on glucopharanin contained in cruciferous vegetables of the genus Brassica such as broccoli, brussel sprouts, and cabbage. It has been found that SF has significant pharmacological properties, such as antiinflammatory and chemiopreventive. In addition, studies have demonstrated protective roles for SF on oxidative stress and cardio protective activity. Recent studies have demonstrated that sulforaphane increases gene transcription, protein expression, and enzyme activity of phase II enzymes including GR, GST, NQO1, thioredoxin reductase in cultured rat neonatal cardiomyocytes model. Also physical exercise, if moderate and constant, can lead to a higher antioxidant and detoxifying enzymes expression, while exhaustive exercise leads to oxidative stress. Our aim was to assess the capacity of a diet of broccoli extract to modifying the redox state of the cell, modulating the expression of phase 2 detoxifying enzymes and modifying GSH levels. We used female adult wistar rats divided into 3 groups (16 rats each): 1) controls, 2) exercised and 3) broccoli supplemented (250 mg/100gdiet so that they introduce 0,15 mg SF/ die). For each group, 8 rats were sacrified before and 8 after, forced intense exercise on a treadmill for 30 min. Our data show that both physical exercise and broccoli supplementation prevent the lactate dehydrogenase (LDH) level raise observed in control animals. Furthermore we observed modification in some antioxidant activity that resemble, altough with minor magnitude, the modification in antioxidant activity observed following acute tratement with purified sulforaphane, as previously reported.

An impaired inflammatory response represents the initial starting point for many neurodegenerative diseases. For their capability of releasing and being target as well of pro-inflammatory molecules and growth factors, astrocytes can be considered key players participating in the development of these neu-rodegenerative processes. Moreover, astroglial cells cover unique functions in the adult brain parenchyma by regulating synaptic transmission and, therefore, contributing to the maintenance of neuronal homeostasis. To investigate how astrocytes react to the initial phases of inflammation, we cultured rat cortical astrocytes and exposed them to pro-inflammatory molecules such as lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). We observed that stimulated cells rapidly (8-24h) acquire typical features of reactive atrocytes, by increasing the expression of markers such as glial fibrillary acidic pro-tein (GFAP) and inducible nitric oxide synthase (iNOS) evaluated by immunocytochemistry and im-munoblotting, respectively. Surprisingly, LPS/IFN-gamma treatment did not influence cell viability, meas-ured by MTT test, even at later time-points, but inflammation altered astrocyte morphology inducing the formation of vacuoles characteristic of autophagic processes. We analyzed the expression of the au-tophagic marker Microtubule Associated Protein 1 Light Chain (LC3B) over time and correlated it with an altered morphology of mitochondria in these cells by time-lapse microscopy. Our data demonstrate that astrocytes rapidly respond to inflammation by undergoing autophagy. Further experiments will reveal the molecular pathways involved in this reaction and what role these events have in controlling the inflammatory response.

Oxidative stress plays an important role in the pathogenesis of the Alzheimer disease, many epidemiological studies have shown an improvement in cognitive activity and a slowing of disease progression following the administration of antioxidant molecules. Furthermore a previous work has shown that α-tocopherol enriched human lipoproteins enhance the survival of developing rat neurons in vitro [1]. It has recently been shown that when the brain is rich in DHA (an omega-3 fatty acid that can also be biosynthesized starting from α-linolenic acid), production of amyloid plaques is lower, with a decrease of characteristic symptoms of the disease [2]. The purpose of this work was the formulation of liposomes for nasal administration of tacrine-HCl. This route has many advantages; in particular is possible to convey the drug directly to the Central Nervous System (CNS), through the olfactory bulb[3] [4] [5]. Part of Tacrine administered can also be absorbed into the systemic circulation avoiding hepatic first pass effect. Liposomes were formulated with ingredients that have a beneficial effect against the disease. In particular, the classical excipients cholesterol (Cho) and phosphatidylcholine (Phc) were enriched with α-tocopherol (Toc) and polyunsaturated fatty acid EPA and DHA (Ω3). This formulative approach allowed us to obtain 100% active liposomes against symptoms of Alzheimer's disease.

Gli isotiocianati sono metaboliti secondari derivanti dall’idrolisi dei glucosinolati, una classe di composti presente nei vegetali della famiglia delle Brassicaceae. Il sulforafane (SF), in particolare, è un isotiocianato che viene prodotto in seguito all’idrolisi del composto glucorafanina da parte dell’enzima mirosinasi, una tioglucosidasi presente in questi vegetali. SF è un composto a riconosciuta azione chemiopreventiva, antiinfiammatoria e neuroprotettiva. Da un precedente studio condotto presso il nostro laboratorio è emerso che SF protegge la cellula cardiaca dallo stress ossidativo inducendo una serie di enzimi antiossidanti/detossificanti di fase II. Per meglio chiarire i meccanismi sottesi alla cardioprotezione indotta da SF, obiettivo di questo studio è stato l’identificazione di nuovi target molecolari del composto nutraceutico SF, mediante analisi proteomica. Come modello di studio sono state utilizzate colture primarie di cardiomiociti neonatali di ratto. Le cellule sono state trattate con SF 5 µM per tempi differenti (1-48 h). Gli estratti proteici sono stati analizzati mediante elettoforesi 2D-PAGE combinata con digestione enzimatica degli spot di interesse, MALDI/MS dei digeriti ed individuazione delle proteine mediante ricerca bioinformatica. Nell'analisi sono state identificate circa 60 proteine differentemente espresse di cui 4 potenzialmente coinvolte nei meccanismi cardioprotettivi precedentemente osservati. In particolare, è emersa una significativa modulazione di “macrophage migration inhibitory factor” (MIF), gliossalasi I, elfina e “heat shock protein” 60 (HSP60). I dati relativi a queste proteine sono stati confermati tramite analisi immunoblotting utilizzando anticorpi specifici. I risultati ottenuti suggeriscono che la cardioprotezione esercitata da SF è un meccanismo estremamente complesso che coinvolge, non solo l’induzione degli enzimi di fase II, ma, inaspettatamente, proteine con un ruolo antiapototico e che agiscono come adattatori tra proteine chinasi e il citoscheletro.

Isothiocyanates are metabolites derived from hydrolysis of glucosinolates, a class of compounds present in plants of the Brassicaceae family. The sulforaphane (SF), in particular, is an isothiocyanate produced after hydrolysis of the glucoraphanin by the enzyme myrosinase, a tioglucosidase present in these plants. SF is known for its chemopreventive, anti-inflammatory a nd neuroprotective action (1, 2). A previous study conducted in our laboratory has shown that SF protects cardiomyocytes by oxidative stress inducing a pattern of antioxidant/phase II detoxifying enzymes (3). In order to clarify the mechanisms underlying the cardioprotection induced by SF, the purpose of this study was the identification of new molecular targets of the compound SF, by proteomic analysis. As model system we used primary cultures of rat neonatal cardiomyocytes. Cells were treated with 5 μM SF for different times (1-48 h). The protein extracts were analyzed by 2D-PAGE electrophoresis combined with enzymatic digestion of the spots of interest, MALDI/MS of digested and identification of proteins by bioinformatics analysis. About 60 differently expressed proteins were identified, 4 of which are potentially involved in the cardioprotective mechanisms previously observed. In particular, there was a significant up-regulation of macrophage migration inhibitory factor (MIF), gliossalase I, elfin and a down-regulation of heat shock protein 60 (HSP60). The data for these proteins were confirmed by immunoblotting analysis using specific antibodies. These results suggest that the cardioprotection elicited by SF is a complex mechanism that involves the modulation of proteins with different cardioprotective role.

Oxidative damage plays an important role in the pathogenesis of colorectal (CR) cancer. This study investigated the activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione-S-transferase (GST) in plasma of 82 participants of a screening program for CR cancer prevention (30 females and 52 males; age 50-70 years). All subjects resulted positive to fecal occult blood test and were subsequently classified, according to the colonoscopy and histological findings, in patients with CR cancer, patients with colorectal polyps or controls. Furthermore, the activity of clastogenic factors (CFs) in plasma from study population was measured as the ability of inducing micronuclei (MN) in vitro in peripheral of a healthy donor. CAT and GR activities were significantly lower in CR cancer patients compared to controls (P< 0.05) and polyps groups (P< 0.05). SOD activity was significantly higher in patients with CR cancer than in polyp (P

It has been demonstrated that a short-timed ischemia, also called preconditioning (PC), may exert a protective role against a subsequent prolonged stress. This phenomenon is the strongest form of in vivo protection against myocardial ischemic injury. The ischemic PC concept has been extended to PC triggered by non-ischemic stress, such as stretch, some chemicals, hypoxia, and reactive oxygen radicals. The present study was designed to investigate the mechanisms behind hydrogen peroxide preconditioning cardioprotection focusing on antioxidant and phase II enzyme induction and their modulation by mitogen activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)/Akt.

Cardioprotective activity of sulforaphane (SF) has been predominantly associated with its ability to induce phase II detoxification enzymes. In the present study, novel targets of SF were identified and characterized using a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to produce protein profiles of rat cardiomyocytes treated with 5 µM SF for 1-48 h. Gel comparisons showed the regulation of nearly 60 proteins. In particular we observed a strong modulation of macrophage migration inhibitory factor, lactoylglutathione lyase, elfina and HSP60. Moreover, proteins were analyzed using western blotting. Results suggested that SF cardioprotection is a complex mechanism involving not only the induction of phase II enzymes1, but also unexpected proteins with antiapoptotic role2 or acting as adapters between kinases and cytoskeleton3. This study contributed to clarify the mechanisms behind SF cardioprotection.