Resveratrol pretreatment protects rat brain from cerebral ischemic damage via a sirtuin 1-uncoupling protein 2 pathway

Abstract

Resveratrol is a natural polyphenol found in grapes and wine and has been associated with protective effects against cardiovascular diseases. In vitro, both resveratrol preconditioning (RPC) and ischemic preconditioning (IPC) require activation of sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, to induce neuroprotection against cerebral ischemia. In the present study, we tested two hypotheses: (a) that neuroprotection against cerebral ischemia can be induced by RPC in vivo; and (b) that RPC neuroprotection involves alterations in mitochondrial function via the SIRT1 target mitochondrial uncoupling protein 2 (UCP2). IPC was induced by 2 min of global ischemia (temporary bilateral carotid artery occlusion with hypotension), and RPC, by i.p. injection of resveratrol at 10, 50 and 100 mg/kg dosages. Forty-eight hours later, we compared the neuroprotective efficacy of RPC and IPC in vulnerable cornu ammonis 1 hippocampal pyramidal neurons using a rat model of asphyxial cardiac arrest (ACA). SIRT1 activity was measured using a SIRT1-specific fluorescent enzyme activity assay. In hippocampal mitochondria isolated 48 h after IPC or RPC, we measured UCP2 levels, membrane potential, respiration, and the mitochondrial ATP synthesis efficiency (ADP/O ratio). Both IPC and RPC induced tolerance against brain injury induced by cardiac arrest in this in vivo model. IPC increased SIRT1 activity at 48 h, while RPC increased SIRT1 activity at 1 h but not 48 h after treatment in hippocampus. Resveratrol significantly decreased UCP2 levels by 35% compared to sham-treated rats. The SIRT1-specific inhibitor sirtinol abolished the neuroprotection afforded by RPC and the decrease in UCP2 levels. Finally, RPC significantly increased the ADP/O ratio in hippocampal mitochondria reflecting enhanced ATP synthesis efficiency. In conclusion, in vivo resveratrol pretreatment confers neuroprotection similar to IPC via the SIRT1-UCP2 pathway.

Figure 1. Schematic diagram of pathways activated by resveratrol induced SIRT1 activation

Resveratrol leads to SIRT1 activation which in turn decreases UCP2 levels. Subsequently the decrease in UCP2 level in mitochondria is coupled with increased mitochondrial ATP producing capacity.

Figure 2. Experimental design

Figure 3. In vivo pretreatment with resveratrol mimics ischemic preconditioning in CA1 region of hippocampus

(A) Representative histological images and (B) number of normal neurons in the CA1 region of hippocampus are presented. Scale bar indicates 30 μm. *P<0.05 as compared to vehicle, and resveratrol 100 mg/Kg of body weight. ^#P<0.05 as compared to IPC; 10, and 50 mg/Kg of resveratrol.

Figure 4. SIRT1 activity increases in rat hippocampus after IPC and after resveratrol pretreatment. Intracerebral Ventricular (ICV) injection of sirtinol blocks SIRT1 activation following resveratrol pre-treatment

(A) SIRT1 enzyme activity was measured in the rat hippocampus at 1 and 48 hours after IPC or treatment with 10 mg/Kg of body weight of resveratrol. *P<0.02 as compared to sham group. (B) SIRT1 enzyme activity was measured in the rat hippocampus at 1 hour after DMSO ICV, treatment with 10 mg/Kg of body weight of resveratrol + sirtinol ICV, and sirtinol ICV alone. *P<0.02 as compared to control group.

Figure 5. Sirtinol blocks resveratrol induced neuroprotection

(A) Representative histological images and (B) number of normal neurons in the CA1 region of hippocampus are presented. Scale bar indicates 30 μm. *P<0.05 as compared to sirtinol + ACA, DMSO +ACA; and resveratrol 10 mg/Kg + sirtinol groups. ^#P<0.05 as compared to sirtinol ICV; and resveratrol 10 mg/Kg + DMSO groups.

Figure 6. Resveratrol pretreatment decreases UCP2 levels after 48 hours in mitochondria isolated from rat hippocampus

(A) UCP2 levels were measured in mitochondria isolated from rat brain hippocampus at 48 hours after treatment with vehicle and resveratrol (10 mg/Kg). (B) Sirtinol blocks the resveratrol-induced decrease in UCP2. The following treatments were used: resveratrol (10 mg/Kg) + intracerebral ventricular injection (ICV) of DMSO (sirtinol vehicle), and resveratrol (10 mg/Kg) + 3.94 μg sirtinol ICV. *P<0.02 as compared with sham.

Figure 7. Effect of resveratrol on mitochondrial physiology

(A) ΔΨ_m, (B) state 3 and 4 respiration rates, (C) respiratory coefficient index (RCI), (D) typical polarographic traces. Arrows A and B indicate addition of pyuvate + malate, and adenosine diphosphate, respectively. (E) ADP/O ratio (ratio of rates of ATP synthesis and oxygen consumption) were measured in hippocampal mitochondria isolated at 48 hours after RPC (10 mg/Kg), vehicle treatment, and RPC + sirtinol ICV. *P<0.02 as compared with vehicle; and *P<0.05 as compared with RPC + sirtinol ICV.