Resveratrol protects against experimental stroke: putative neuroprotective role of heme oxygenase 1

Abstract

Epidemiological and experimental reports have linked mild-to-moderate wine and/or grape consumption to a lowered incidence of cardiovascular, cerebrovascular, and peripheral vascular risk. This study revealed that resveratrol, an enriched bioactive polyphenol in red wine, selectively induces heme oxygenase 1 (HO1) in a dose- and time-dependent manner in cultured mouse cortical neuronal cells and provides neuroprotection from free-radical or excitotoxicity damage. This protection was lost when cells were treated with a protein synthesis or heme oxygenase inhibitor, suggesting that HO1 induction is at least partially required for resveratrol's prophylactic properties. Furthermore, resveratrol pretreatment dose-dependently protected mice subjected to an optimized ischemic-reperfusion stroke model. Mice in which HO1 was selectively deleted lost most, if not all, of the beneficial effects. Together, the data suggest a potential intracellular pathway by which resveratrol can provide cell/organ resistance against neuropathological conditions.

Fig 1. Resveratrol induces HO1 and protects against excitotoxicity in primary neuronal cultures

(A) Effect of different concentrations of resveratrol and treatment periods on HO1 and HO2 protein expression in neuronal cultures. Mouse cortical neurons cultured in serum-free conditions for 10–14 days were harvested at different times after resveratrol treatment; proteins were analyzed by Western blot. Resveratrol induced HO1 expression dose and time dependently but did not affect HO2. Actin was used to confirm equal loading. (B) Effect of resveratrol (RV) and the protein synthesis inhibitor cycloheximide (CHX) on HO1 protein expression in neuronal cultures. Cells were treated with vehicle (control), resveratrol (25 µM) or resveratrol together with CHX for the first 4 h, then with resveratrol, CHX (10 µg/ml) or both together for the remaining 4 h before being harvested and analyzed. CHX blocked the increase in HO1 expression. (C) The protective effect of resveratrol pretreatment against excitotoxicity induced by glutamate in primary cultured neurons was significantly reduced by treatment with CHX. Neuronal cultures were first pretreated for 4 h with CHX or vehicle, rinsed once, and then treated with resveratrol for 6 h. Then, cells were rinsed and incubated with fresh medium containing 40 µM glutamate or vehicle (control). After 24 h, cell survival was estimated by MTT assay, which is an indicator of mitochondrial function. (D) The protective effect of resveratrol pretreatment against excitotoxicity induced by glutamate in cultured neurons was significantly reduced by HO inhibitor. Cells were pretreated with 25 µM resveratrol for 6 h; then cells were rinsed and incubated with fresh medium containing 30 µM glutamate or vehicle (control), with or without 5 µM of the HO inhibitor SnPPIX. After 24 h the cell survival was estimated by MTT assay. Experimental conditions were carried out in quadruplicate, and the experiment was reproduced four times with different primary culture batches.

Fig 2. Acute and chronic pretreatment with resveratrol protects mice subjected to ischemic-reperfusion injury

(A) Resveratrol dose-dependently reduced infarct volume compared to vehicle when given orally 2 h before 90-min MCA occlusion and 24-h reperfusion. (B) Pretreatment with oral resveratrol once daily for 7 days reduced infarct volume caused by the transient ischemia protocol. (C) Representative 2,3,5-triphenyltetrazolium chloride-stained brain sections of mice treated with vehicle or resveratrol before being subjected to MCA occlusion. (D) The significant acute protective effect of resveratrol in WT animals (left) was lost in HO1^−/− mice. (E) Acute pretreatment with resveratrol did not alter the intraischemic blood flow distribution. WT mice were treated orally with vehicle (n = 7) or 20 mg/kg of resveratrol (n = 5) 2 h before MCA occlusion. The [¹⁴C]-iodoantipyrine (4 µCi) was infused intravenously over 45 s while arterial blood was sampled to obtain the arterial input function. Brains were immediately harvested after the infusion and sectioned for autoradiographic analysis at 1-mm coronal increments from +2 through −3 mm from bregma. No significant differences were observed between vehicle and resveratrol treatment in cortical or subcortical regions ipsilateral or contralateral to the occlusion.