Saffron (Crocus sativus) intake provides nutritional preconditioning against myocardial ischemia-reperfusion injury in Wild Type and ApoE(-/-) mice: Involvement of Nrf2 activation

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.

Keywords: ApoE((−/−)) mice; Molecular signaling; Myocardial infarct size; Nrf2; Saffron.