P Efentakis1, A Rizakou1, E Christodoulou2, A Chatzianastasiou1, M G López3, R León4, E Balafas5, N P E Kadoglou2, I Tseti6, H Skaltsa7, N Kostomitsopoulos5, E K Iliodromitis8, G Valsami2, I Andreadou9. 1. National and Kapodistrian University of Athens, Laboratory of Pharmacology, Faculty of Pharmacy, Athens, Greece. 2. National and Kapodistrian University of Athens, Laboratory of Biopharmaceutics, Faculty of Pharmacy, Athens, Greece. 3. Departamento de Farmacología y Terapéutica, Instituto Téofilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain. 4. Departamento de Farmacología y Terapéutica, Instituto Téofilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain; Instituto de Investigación Sanitaria del Hospital Universitario la Princesa, Madrid, Spain. 5. Academy of Athens Biomedical Research Foundation, Centre of Clinical Experimental Surgery and Translational Research, Biomedical Research Foundation, Athens, Greece. 6. Uni-Pharma S.A., Athens, Greece. 7. National and Kapodistrian University of Athens, Department of Pharmacognocy and Chemistry of Natural Products, Faculty of Pharmacy, Athens, Greece. 8. National and Kapodistrian University of Athens, Medical School, Second University Department of Cardiology, Athens, Greece. 9. National and Kapodistrian University of Athens, Laboratory of Pharmacology, Faculty of Pharmacy, Athens, Greece. Electronic address: jandread@pharm.uoa.gr.
Abstract
BACKGROUND AND AIMS: Saffron is an antioxidant herbal derivative; however, its efficacy as a nutritional cardioprotective agent has not been fully elucidated. We investigated the cardioprotective properties of a standardized saffron aqueous extract (SFE) against ischemia/reperfusion (I/R) injury in Wild-Type (WT) and ApoE(-/-) mice and the underlying molecular mechanisms. METHODS AND RESULTS: WT and ApoE(-/-) mice were subjected to 30 min I and 2 h R, with the following per os interventions for 4 weeks: 1) WT Control Group, receiving Water for Injection (WFI); 2) WT Crocus Group, receiving SFE at a dose of 60 mg/kg/day; 3) WT Crocus + Wort group, receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus 15 min before R; 4) ApoE(-/-) Control Group, receiving WFI; 5) ApoE(-/-) Crocus Group, receiving SFE at a dose of 60 mg/kg/day and 6) ApoE(-/-) Crocus + Wort: receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus, 15 min before R. Ischemic area/area at risk (I/R%) ratio was measured. Blood samples and ischemic myocardial tissue were collected at the 10th min of reperfusion for assessment of troponin I, malondialdehyde (MDA), nitrotyrosine (NT), p-eNOS, eNOS, p-Akt, Akt, p-p42/p-p44, p-GSK3β, GSK3β, IL-6, Nrf2, HO-1 and MnSOD expression. The effect of SFE on Nrf2 expression was also evaluated in vitro. SFE reduced infarct size in WT (16.15 ± 3.7% vs 41.57 ± 2.48%, ***p < 0.001) and in ApoE(-/-) mice (16.14 ± 1.47% vs 45.57 ± 1.73%, ***p < 0.001). The administration of wortmannin resulted in partial inhibition of the infarct size limitation efficacy of SFE (in both WT and Apo-E(-/-) mice). Mice receiving SFE showed increased levels of eNOS, p-Akt, p-ERK1/2, p-44/p-42 and p-GSK3β-Ser9 and reduced expression of IL-6 and iNOS; furthermore, SFE reduced the levels of MDA and NT. SFE induced Nrf2 expression and its downstream targets, HO-1 and MnSOD in the myocardium of the treated animals, and induced Nrf2 expression in vitro in a dose-dependent manner. CONCLUSIONS: SFE limits myocardial infarction in Wild-Type and ApoE(-/-) mice in a multifaceted manner including activation of Akt/eNOS/ERK1/2/GSK3-β and through Nrf2 pathway, bestowing antioxidant protection against I/R.
BACKGROUND AND AIMS: Saffron is an antioxidant herbal derivative; however, its efficacy as a nutritional cardioprotective agent has not been fully elucidated. We investigated the cardioprotective properties of a standardized saffron aqueous extract (SFE) against ischemia/reperfusion (I/R) injury in Wild-Type (WT) and ApoE(-/-) mice and the underlying molecular mechanisms. METHODS AND RESULTS: WT and ApoE(-/-) mice were subjected to 30 min I and 2 h R, with the following per os interventions for 4 weeks: 1) WT Control Group, receiving Water for Injection (WFI); 2) WT Crocus Group, receiving SFE at a dose of 60 mg/kg/day; 3) WT Crocus + Wort group, receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus 15 min before R; 4) ApoE(-/-) Control Group, receiving WFI; 5) ApoE(-/-) Crocus Group, receiving SFE at a dose of 60 mg/kg/day and 6) ApoE(-/-) Crocus + Wort: receiving SFE as described above and wortmannin at a dose of 60 μg/kg bolus, 15 min before R. Ischemic area/area at risk (I/R%) ratio was measured. Blood samples and ischemic myocardial tissue were collected at the 10th min of reperfusion for assessment of troponin I, malondialdehyde (MDA), nitrotyrosine (NT), p-eNOS, eNOS, p-Akt, Akt, p-p42/p-p44, p-GSK3β, GSK3β, IL-6, Nrf2, HO-1 and MnSOD expression. The effect of SFE on Nrf2 expression was also evaluated in vitro. SFE reduced infarct size in WT (16.15 ± 3.7% vs 41.57 ± 2.48%, ***p < 0.001) and in ApoE(-/-) mice (16.14 ± 1.47% vs 45.57 ± 1.73%, ***p < 0.001). The administration of wortmannin resulted in partial inhibition of the infarct size limitation efficacy of SFE (in both WT and Apo-E(-/-) mice). Mice receiving SFE showed increased levels of eNOS, p-Akt, p-ERK1/2, p-44/p-42 and p-GSK3β-Ser9 and reduced expression of IL-6 and iNOS; furthermore, SFE reduced the levels of MDA and NT. SFE induced Nrf2 expression and its downstream targets, HO-1 and MnSOD in the myocardium of the treated animals, and induced Nrf2 expression in vitro in a dose-dependent manner. CONCLUSIONS: SFE limits myocardial infarction in Wild-Type and ApoE(-/-) mice in a multifaceted manner including activation of Akt/eNOS/ERK1/2/GSK3-β and through Nrf2 pathway, bestowing antioxidant protection against I/R.