Oxidative stress has been reported to play a causative role in cardiovascular diseases. Although reactive oxygen species (ROS) are known to enhance myocardial ischemia-reperfusion injury (Morihira et al, 2005), it has been demonstrated that a short-timed ischemia, also called preconditioning, may exert a protective role, preventing thus from a subsequent prolonged stress. Several factors have been reported to mediate preconditioning mechanism, such as blockade of reduction in mitochondrial membrane potential or modulation of multiple genes and signalling pathways (Tang et al, 2005). Anyway, the apparent paradox of preconditioning is strictly connected with a biphasic process known as “hormesis”. Hormesis is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition: as a consequence, the same stressor that at higher doses appears toxic, at lower ones leads to beneficial effects (Calabrese and Baldwin, 2002). In this way, preconditioning can be considered as an hormetic response as well. The aim of this work is to investigate the probable protective role of preconditioning on oxidative stress in cultured cardiomyocytes and its effects on antioxidant enzymatic machinery, in order to enlighten the mechanism of protection and the targets involved. In our study we used H2O2 as stressor, as it is well documented that this molecule is capable of adaptative responses similar to those induced by ischemic preconditioning. Materials and methods. Cultured rat neonatal cardiomyocytes were prepared and grown until confluence as previously reported (Hrelia et al., 2002). Preconditiong was simulated exposing the cells to 100 µM H2O2 for ten minutes and, 24 hours later, oxidative stress was induced with 100 µM H2O2 for 30 minutes. Cell viability was evaluated by MTT assay. Apoptosis and necrosis analysis was assessed by cytofluorimetric assay using Guava Nexin Assay (Guava Technologies). Enzymatic activities of glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GPX), thioredoxin reductase (TR), NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT) and superoxide dismutase (SOD) were determined by spectrophotometric methods. Results and discussion. MTT results indicate a significant protective effect of preconditioning against oxidative stress, preconditioning with 100 µM H2O2 led to a complete protection against subsequent peroxide induced-injury. In agreement with MTT data, cytofluorimetric assay showed that preconditioning was able to reduce necrotic and apoptotic populations and increased viable cells in comparison to oxidative stress only. Preconditioning was able to increase GR, NQO1, TR and CAT activities, while it did not influence GPX, GST, and SOD activities. So the preconditioning protective effect may be ascribed to an increased cell ability to detoxify ROS by increasing the activities of fundamental antioxidant/phase II enzymes. As all these enzymes are regulated through the Keap1-Nrf2-antioxidant responsive element signalling pathway, their unlinked regulation underpins the hypothesis that preconditioning could modulate these enzymes through different mechanisms. Supported by Fondazione del Monte di Bologna e Ravenna.

Hormesis and cardioprotection

MOTORI, ELISA;ANGELONI, CRISTINA;HRELIA, SILVANA
2009

Abstract

Oxidative stress has been reported to play a causative role in cardiovascular diseases. Although reactive oxygen species (ROS) are known to enhance myocardial ischemia-reperfusion injury (Morihira et al, 2005), it has been demonstrated that a short-timed ischemia, also called preconditioning, may exert a protective role, preventing thus from a subsequent prolonged stress. Several factors have been reported to mediate preconditioning mechanism, such as blockade of reduction in mitochondrial membrane potential or modulation of multiple genes and signalling pathways (Tang et al, 2005). Anyway, the apparent paradox of preconditioning is strictly connected with a biphasic process known as “hormesis”. Hormesis is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition: as a consequence, the same stressor that at higher doses appears toxic, at lower ones leads to beneficial effects (Calabrese and Baldwin, 2002). In this way, preconditioning can be considered as an hormetic response as well. The aim of this work is to investigate the probable protective role of preconditioning on oxidative stress in cultured cardiomyocytes and its effects on antioxidant enzymatic machinery, in order to enlighten the mechanism of protection and the targets involved. In our study we used H2O2 as stressor, as it is well documented that this molecule is capable of adaptative responses similar to those induced by ischemic preconditioning. Materials and methods. Cultured rat neonatal cardiomyocytes were prepared and grown until confluence as previously reported (Hrelia et al., 2002). Preconditiong was simulated exposing the cells to 100 µM H2O2 for ten minutes and, 24 hours later, oxidative stress was induced with 100 µM H2O2 for 30 minutes. Cell viability was evaluated by MTT assay. Apoptosis and necrosis analysis was assessed by cytofluorimetric assay using Guava Nexin Assay (Guava Technologies). Enzymatic activities of glutathione reductase (GR), glutathione-S-transferase (GST), glutathione peroxidase (GPX), thioredoxin reductase (TR), NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT) and superoxide dismutase (SOD) were determined by spectrophotometric methods. Results and discussion. MTT results indicate a significant protective effect of preconditioning against oxidative stress, preconditioning with 100 µM H2O2 led to a complete protection against subsequent peroxide induced-injury. In agreement with MTT data, cytofluorimetric assay showed that preconditioning was able to reduce necrotic and apoptotic populations and increased viable cells in comparison to oxidative stress only. Preconditioning was able to increase GR, NQO1, TR and CAT activities, while it did not influence GPX, GST, and SOD activities. So the preconditioning protective effect may be ascribed to an increased cell ability to detoxify ROS by increasing the activities of fundamental antioxidant/phase II enzymes. As all these enzymes are regulated through the Keap1-Nrf2-antioxidant responsive element signalling pathway, their unlinked regulation underpins the hypothesis that preconditioning could modulate these enzymes through different mechanisms. Supported by Fondazione del Monte di Bologna e Ravenna.
2009
Atti della 22° Riunione Nazionale "A. Castellani" dei dottorandi di ricerca in discipline biochimiche
B1
B1
E. Motori; C. Angeloni; S. Hrelia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/76827
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