Emanuela Leoncini

Modulation of Phase II Enzymes by Sulforaphane: Implications for Its Cardioprotective Potential

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.

H2O2 preconditioning modulates phase II enzymes through p38 MAPK and PI3K/Akt activation.

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 H(2)O(2)-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-E(2)-related factor-1 (Nrf1) and Nrf2. H(2)O(2) preconditioning was able to counteract oxidative stress more effectively in the late than in the early phase of adaptation. In particular, H(2)O(2) preconditioning 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 H(2)O(2) preconditioning 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 H(2)O(2) preconditioning.

Sulforaphane treatment protects skeletal muscle against damage induced by exhaustive exercise in rats

Sulforaphane (SF), one of the most important isothiocyanates in the human diet, present in cruciferous vegetables, is known to have chemopreventive activities in different tissues. No data are available on its effects in the prevention of skeletal muscle damage. In this study, we investigated the potential protective effects of SF treatment on muscle damage and oxidative stress induced by an acute bout of exhaustive exercise in rats. Male Wistar rats were treated with SF (25 mg/kg body wt ip) for 3 days before undergoing an acute exhaustive exercise protocol in a treadmill (+7% slope and 24 m/min). Acute exercise resulted in a significant increase in plasma lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) activities. It also resulted in a significant increase in thiobarbituric acid-reactive substances, in a significant decrease in tissue total antioxidant capacity, and in a significant decrease in NAD(P)H:quinone oxidoreductase 1 (NQO1) expression and activity in vastus lateralis muscle. SF treatment significantly increased muscle NQO1, glutathione-S-transferase, and glutathione reductase expression and activity, with no effect on glutathione peroxidase and thioredoxin reductase. The observed SF-induced upregulation of phase II enzymes was accompanied by a significant increase in nuclear erythroid 2 p45-related factor 2 expression and correlated with a significant increase in total antioxidant capacity and a decrease in plasma LDH and CPK activities. Our data demonstrate that SF acts as an indirect antioxidant in skeletal muscle and could play a critical role in the modulation of the muscle redox environment, leading to the prevention of exhaustive exercise-induced muscle damage.

Bioactive peptides in cereals and legumes: agronomical, biochemical and clinical aspects.

Cereals and legumes are key components of a healthy and balanced diet. Accordingly, many national nutritional guidelines emphasize their health promoting properties by placing them at the base of nutritional food pyramids. This concept is further validated by the observed correlation between a lower risk and occurrence of chronic diseases and the adherence to dietary patterns, like the Mediterranean diet, in which cereal grains, legumes and derived products represent a staple food. In the search for a dietary approach to control/prevent chronic degenerative diseases, protein derived bioactive peptides may represent one such source of health-enhancing components. These peptides may already be present in foods as natural components or may derive from hydrolysis by chemical or enzymatic treatments (digestion, hydrolysis or fermentation). Many reports are present in the literature regarding the bioactivity of peptides in vitro and a wide range of activities has been described, including antimicrobial properties, blood pressure-lowering (ACE inhibitory) effects, cholesterol-lowering ability, antithrombotic and antioxidant activities, enhancement of mineral absorption/bioavailability, cyto- or immunomodulatory effects, and opioid-like activities. However it is difficult to translate these observed effects to human. In fact, the active peptide may be degraded during digestion, or may not be absorbed or reach the target tissues at a concentration necessary to exert its function. This review will focus on bioactive peptides identified in cereals and legumes, from an agronomical and biochemical point of view, including considerations about requirements for the design of appropriate clinical trials necessary for the assessment of their nutraceutical effect in vivo.

Role of quercetin in modulating rat cardiomyocyte gene expression profile

Despite extensive studies, the fundamental mechanisms responsible for the development and progression of cardiovascular diseases have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species play a major pathological role. Oxidative stress reduction induced by flavonoids has been regarded by many as the most likely mechanism in the protective effects of these compounds; however, there is an emerging view that flavonoids may also exert modulatory actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid present in the human diet, has been widely studied, and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. The main goal of this study was to examine the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin using DNA microarrays and to relate these data to functional effects. Results showed distinct temporal changes in gene expression induced by quercetin and a strong upregulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect antioxidant mechanism.

Phytochemical profile and nutraceutical value of old and modern common wheat cultivars.

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.

Impact of personalized diet and probiotic supplementation on inflammation, nutritional parameters and intestinal microbiota – The “RISTOMED project”: Randomized controlled trial in healthy older people

OBJECTIVES To assess the impact of a personalized diet, with or without addition of VSL#3 preparation, on biomarkers of inflammation, nutrition, oxidative stress and intestinal microbiota in 62 healthy persons aged 65-85 years. DESIGN Open label, randomized, multicenter study. PRIMARY ENDPOINT High-sensitivity C-reactive protein. SETTING Community. INTERVENTIONS Eight week web-based dietary advice (RISTOMED platform) alone or with supplementation of VSL#3 (2 capsules per day). The RISTOMED diet was optimized to reduce inflammation and oxidative stress. MEASUREMENTS Blood and stool samples were collected on days 1 and 56. RESULTS Diet alone reduced ESR (p = 0.02), plasma levels of cholesterol (p < 0.01) and glucose (p = 0.03). Addition of VSL#3 reduced ESR (p = 0.05) and improved folate (p = 0.007), vitamin B12 (p = 0.001) and homocysteine (p < 0.001) plasma levels. Neither intervention demonstrated any further effects on inflammation. Subgroup analysis showed 40 participants without signs of low-grade inflammation (hsCRP<3 mg/l, subgroup 1) and 21 participants with low-grade inflammation at baseline (hsCRP≥3 mg/l, subgroup 2). In subgroup 2 addition of VSL#3 increased bifidobacteria (p = 0.005) in more participants and improved both folate (p = 0.015) and vitamin B12 (p = 0.035) levels compared with subgroup 1. The increases were positively correlated to the change in the bifidobacteria concentration for folate (p = 0.023) and vitamin B12 (p = 0.001). As expected change in homocysteine correlated negatively to change in folate (r = -0.629, p = 0.002) and vitamin B12 (r = -0.482, p = 0.026). CONCLUSIONS Addition of VSL#3 increased bifidobacteria and supported adequate folate and vitamin B12 concentrations in subjects with low-grade inflammation. Decrease in homocysteine with VSL#3 was clinically relevant. suggesting protective potentials for aging-associated conditions, e.g. cardiovascular or neurological diseases. ClinicalTrials.gov: NCT01069445-NCT01179789.

Cruciferous Vegetable Phytochemical Sulforaphane Affects Phase II Enzyme Expression and Activity in Rat Cardiomyocytes through Modulation of Akt Signaling Pathway

The isothiocyanate sulforaphane (SF), abundant in Cruciferous vegetables, is known to induce antioxidant/detoxification enzymes in many cancer cell lines, but studies focused on its cytoprotective action in nontransformed cells are just at the beginning. Since we previously demonstrated that SF elicits cardioprotection through an indirect antioxidative mechanism, the aim of this study was to analyze the signaling pathways through which SF exerts its protective effects. Using cultured rat cardiomyocytes, we investigated the ability of SF to activate Akt/protein kinase B (PKB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways, which are implicated in cardiac cell survival, and to increase the phosphorylation of Nuclear factor E2-related factor 2 (Nrf2) and its binding to the antioxidant response element. By means of specific inhibitors, we demonstrated that the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway represents a mechanism through which SF influences both expression and activity of glutathione reductase, glutathione-S-transferase, thioredoxin reductase, and NAD(P)H:quinone oxidoreductase-1, analyzed by western immunoblotting and spectrophotometric assay, respectively, and modulates Nrf2 binding and phosphorylation resulting in a cytoprotective action against oxidative damage. Results of this study confirm the importance of phase II enzymes modulation as cytoprotective mechanism and support the nutritional assumption of Cruciferous vegetables as source of nutraceutical cardioprotective agents.

Synthesis and Antiproliferative Activity of Some Thiazolylbenzimidazole-4,7-diones

Some thiazolylbenzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Compounds 2d and 2e show a very good activity on K562 cells, whereas compounds 2a and 2b are active on SW620 cells. The importance of the methoxy group on the quinone moiety is confirmed and the function at 4-position of the thiazole ring plays a determining role for the activity.

Steviol Glycosides Modulate Glucose Transport in Different Cell Types

Extracts from Stevia rebaudiana Bertoni, a plant native to Central and South America, have been used as a sweetener since ancient times. Currently, Stevia extracts are largely used as a noncaloric high-potency biosweetener alternative to sugar, due to the growing incidence of type 2 diabetes mellitus, obesity, and metabolic disorders worldwide. Despite the large number of studies on Stevia and steviol glycosides in vivo, little is reported concerning the cellular and molecular mechanisms underpinning the beneficial effects on human health. The effect of four commercial Stevia extracts on glucose transport activity was evaluated in HL-60 human leukaemia and in SH-SY5Y human neuroblastoma cells. The extracts were able to enhance glucose uptake in both cellular lines, as efficiently as insulin. Our data suggest that steviol glycosides could act by modulating GLUT translocation through the PI3K/Akt pathway since treatments with both insulin and Stevia extracts increased the phosphorylation of PI3K and Akt. Furthermore, Stevia extracts were able to revert the effect of the reduction of glucose uptake caused by methylglyoxal, an inhibitor of the insulin receptor/PI3K/Akt pathway. These results corroborate the hypothesis that Stevia extracts could mimic insulin effects modulating PI3K/Akt pathway.