Resveratrol Preconditioning Protects Against Cerebral Ischemic Injury via Nuclear Erythroid 2-Related Factor 2

Abstract

Background and purpose: Nuclear erythroid 2 related factor 2 (Nrf2) is an astrocyte-enriched transcription factor that has previously been shown to upregulate cellular antioxidant systems in response to ischemia. Although resveratrol preconditioning (RPC) has emerged as a potential neuroprotective therapy, the involvement of Nrf2 in RPC-induced neuroprotection and mitochondrial reactive oxygen species production after cerebral ischemia remains unclear. The goal of our study was to study the contribution of Nrf2 to RPC and its effects on mitochondrial function.

Methods: We used rodent astrocyte cultures and an in vivo stroke model with RPC. An Nrf2 DNA binding ELISA and protein analysis via Western blotting of downstream Nrf2 targets were performed to determine RPC-induced activation of Nrf2 in rat and mouse astrocytes. After RPC, mitochondrial function was determined by measuring reactive oxygen species production and mitochondrial respiration in both wild-type and Nrf2-/- mice. Infarct volume was measured to determine neuroprotection, whereas protein levels were measured by immunoblotting.

Results: We report that Nrf2 is activated by RPC in rodent astrocyte cultures, and that loss of Nrf2 reduced RPC-mediated neuroprotection in a mouse model of focal cerebral ischemia. In addition, we observed that wild-type and Nrf2-/- cortical mitochondria exhibited increased uncoupling and reactive oxygen species production after RPC treatments. Finally, Nrf2-/- astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular antioxidants after RPC treatment.

Conclusions: Nrf2 contributes to RPC-induced neuroprotection through maintaining mitochondrial coupling and antioxidant protein expression.

Keywords: ischemic preconditioning; mitochondria; reactive oxygen species; resveratrol.

Figure 1. RPC-induced neuroprotection is decreased in Nrf2^−/− mice following MCAO injury

A) Representative image of TTC staining of brain slices to assess infarct volume of mice subjected to MCAO injury. Slices are distributed rostrocaudally. VEH: Vehicle. RES: resveratrol preconditioning. B) Quantification of infarct volume. Areas of cortical infarction at seven coronal levels were cumulated and normalized for edema. Number of subjects represented in inset of bar graph. Data are presented as mean ± STDEV; * p≤0.05, (multiple-comparison 1-way ANOVA followed by Bonferroni test).

Figure 2. Reduced RPC-induced neuroprotection in Nrf2^−/− mice is not due to altered cerebral blood flow during ischemia

A) Blood flow measurements during the baseline, ischemic/occlusion, and reperfusion phases of the MCAO injury from the same treatment subjects as in Figure 1. Data represented as a percentage of baseline blood flow, as measured by laser Doppler flowmetry. B) Physiologic variables of WT and Nrf2^−/− mice used for MCAO experiments.

Figure 3. RPC treatment activates Nrf2 in astrocyte cultures

A) ELISA assay was used to measure Nrf2 DNA binding in rat astrocyte nuclear fractions harvested at 1, 24, or 48 hrs after RPC treatment. B) whole-cell astrocyte lysates were probed for NQO-1 expression following RPC treatment. n=4, *p≤0.05, **p≤0.01.

Figure 4. RPC treatment increases mitochondrial uncoupling in Nrf2^−/− and WT mice

A) RCI of WT and Nrf2^−/− non-synaptic mitochondria following RPC and vehicle treatment. n=4, *p≤0.05 (WT vehicle vs. WT RPC); ^#p≤0.05 (WT vehicle vs. Nrf2^−/− vehicle); ^ˆp≤0.05 (Nrf2^−/− vehicle vs. Nrf2^−/− RPC. B) UCP2 and Actin protein levels from WT and Nrf2^−/− astrocyte cultures treated with RPC and Vehicle. (+) positive control: mouse cortex tissue lysate. C) Quantification of UCP2 proteins levels normalized to Actin. n=3 *p≤0.05

Figure 5. RPC increases non-synaptic mitochondrial ROS production and only increases antioxidant expression in WT astrocytes

A) Complex I and Complex III H₂O₂ production rate in WT or Nrf2^−/− non-synaptic mitochondria. n=4–6, *p≤0.05 (WT vehicle vs. WT RPC); ^#p≤0.05 (Nrf2 vehicle vs. Nrf2^−/− RPC). B) Western blots of astrocyte cultures for MnSOD, NQO-1, and actin (loading control) 48 hrs after RPC or Vehicle treatment. C) Quantification of WT astrocyte NQO-1 levels and D) WT and Nrf2^−/− astrocyte MnSOD protein levels, normalized to Actin protein levels. n=3–6. *p<0.05. **p<0.005.

Figure 5. RPC increases non-synaptic mitochondrial ROS production and only increases antioxidant expression in WT astrocytes

A) Complex I and Complex III H₂O₂ production rate in WT or Nrf2^−/− non-synaptic mitochondria. n=4–6, *p≤0.05 (WT vehicle vs. WT RPC); ^#p≤0.05 (Nrf2 vehicle vs. Nrf2^−/− RPC). B) Western blots of astrocyte cultures for MnSOD, NQO-1, and actin (loading control) 48 hrs after RPC or Vehicle treatment. C) Quantification of WT astrocyte NQO-1 levels and D) WT and Nrf2^−/− astrocyte MnSOD protein levels, normalized to Actin protein levels. n=3–6. *p<0.05. **p<0.005.