A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes. Sulforaphane (SF), a naturally occurring isothiocyanate from Brassicaceae, demonstrated a strong chemopreventive effect mediated by Nuclear factor E2-related factor-2 (Nrf2), a transcription factor that binds to the Antioxidatn responsive Element orchestrating antioxidant and cytoprotective responses. No data are available to support a similar role of SF in cardioprotection. Using cultured rat cardiomyocytes we have characterized the time-dependent gene induction of phase 2 enzymes and Nrf2 elicited by SF, the corresponding protein expression and Nrf2 translocation to the nucleus. Methods: Cultured rat cardiomyocytes were supplemented with 5 mM SF for different times (6-48 h). The gene induction of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), NAD(P)H-quinone reductase 1 (NQO1), thioredoxine reductase (TR) and Nrf2 were determined by RT-PCR. Western blot analyses of GR, GST, GPx, TR, NQO1 and Nrf2 were performed using specific antibodies. Nrf2 translocation to the nucleus was evaluated by laser confocal microscopy. Results: Both the mRNA and protein levels of GR, GST, NQO1 and TR significantly increased after SF supplementation in a time-dependent manner. GR, GST, NQO1 mRNA increased after 3h treatment, while all protein expressions appear significantly different from unsupplemented cells only at longer exposure times (24, 48h) apart of NQO1 and TR which expressions increased after 6h and 12h respectively. This is probably due to the delay between gene induction and active protein synthesis and confirm our previous data on the time course of phase 2 enzymes activities (1). GPx was not affected by SF at any time point. SF caused an increase of Nrf2 mRNA and laser confocal microscopy evidenced its nuclear translocation. In this study we have demonstrated that SF modulates phase 2 enzymes and Nrf2 induction in cardiomyocytes, eliciting an indirect antioxidant activity to counteract oxidative stress that characterizes many cardiovascular diseases. Supported by MIUR (PRIN 2005) and Fondazione del Monte di Bologna e Ravenna 1. E. Leoncini et al., Nutrition, Oxygen Biology and Medicine (P.Cillard, L.Packer et al eds)2007,55
E. Leoncini, C. Angeloni, M. Malaguti, N. Calonghi, S. Angelini, P. Biagi, et al. (2007). Sulforaphane modulates phase 2 enzyme and Nrf2 expression in cultured rat cardiomyocytes..
Sulforaphane modulates phase 2 enzyme and Nrf2 expression in cultured rat cardiomyocytes.
LEONCINI, EMANUELA;ANGELONI, CRISTINA;MALAGUTI, MARCO;CALONGHI, NATALIA;ANGELINI, SABRINA;BIAGI, PIERLUIGI;HRELIA, SILVANA
2007
Abstract
A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes. Sulforaphane (SF), a naturally occurring isothiocyanate from Brassicaceae, demonstrated a strong chemopreventive effect mediated by Nuclear factor E2-related factor-2 (Nrf2), a transcription factor that binds to the Antioxidatn responsive Element orchestrating antioxidant and cytoprotective responses. No data are available to support a similar role of SF in cardioprotection. Using cultured rat cardiomyocytes we have characterized the time-dependent gene induction of phase 2 enzymes and Nrf2 elicited by SF, the corresponding protein expression and Nrf2 translocation to the nucleus. Methods: Cultured rat cardiomyocytes were supplemented with 5 mM SF for different times (6-48 h). The gene induction of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), NAD(P)H-quinone reductase 1 (NQO1), thioredoxine reductase (TR) and Nrf2 were determined by RT-PCR. Western blot analyses of GR, GST, GPx, TR, NQO1 and Nrf2 were performed using specific antibodies. Nrf2 translocation to the nucleus was evaluated by laser confocal microscopy. Results: Both the mRNA and protein levels of GR, GST, NQO1 and TR significantly increased after SF supplementation in a time-dependent manner. GR, GST, NQO1 mRNA increased after 3h treatment, while all protein expressions appear significantly different from unsupplemented cells only at longer exposure times (24, 48h) apart of NQO1 and TR which expressions increased after 6h and 12h respectively. This is probably due to the delay between gene induction and active protein synthesis and confirm our previous data on the time course of phase 2 enzymes activities (1). GPx was not affected by SF at any time point. SF caused an increase of Nrf2 mRNA and laser confocal microscopy evidenced its nuclear translocation. In this study we have demonstrated that SF modulates phase 2 enzymes and Nrf2 induction in cardiomyocytes, eliciting an indirect antioxidant activity to counteract oxidative stress that characterizes many cardiovascular diseases. Supported by MIUR (PRIN 2005) and Fondazione del Monte di Bologna e Ravenna 1. E. Leoncini et al., Nutrition, Oxygen Biology and Medicine (P.Cillard, L.Packer et al eds)2007,55I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
