Resveratrol Treatment in Different Time-Attenuated Neuronal Apoptosis After Oxygen and Glucose Deprivation/Reoxygenation via Enhancing the Activation of Nrf-2 Signaling Pathway In Vitro

Abstract

Recent studies have indicated that resveratrol has protective effects against cerebral ischemia/reperfusion injury. However, the best therapeutic time for resveratrol treatment after acute ischemic stroke remains unknown. We aim to investigate whether resveratrol, administrated at different times after neuronal oxygen and glucose deprivation/reoxygenation (OGD/R) reduced neuronal injury in vitro. There were six experimental groups: normal, model, resveratrol pretreatment, resveratrol post-treatment, resveratrol OGD-treatment, and resveratrol whole-processing group. We found that resveratrol in a concentration-dependent manner decreased the activity of lactate dehydrogenase (LDH) and increased the activity of superoxide dismutase (SOD). Moreover, resveratrol, administrated at different times, increased neuronal viability, reduced neuronal apoptosis, upregulated the protein expressions of Nuclear factor erythroid 2-related factor 2 (Nrf-2), NAD(P)H: quinone oxidoreductase 1 (NQO-1), heme oxygenase 1 (HO-1), and Bcl-2, downregulated the protein expression of Caspase-3, and promoted Nrf-2 to transfer into the nuclei from the cytoplasm. The most effective treatment group was the whole-processing treatment group. These results suggest that resveratrol treatment at different times increased neuronal viability and inhibited neuronal apoptosis in vitro, at least in part, via enhancing the activation of the Nrf-2 signaling pathway.

Keywords: HO-1; NQO-1; Nrf-2; Resveratrol; apoptosis; neuroprotection; oxidative stress; oxygen and glucose deprivation/reoxygenation.

Fig. 1.

Resveratrol increased SOD activity, decreased LDH activity, and enhanced neuronal viability at 24 h after OGD/R injury. (A) SOD activity was significantly higher in the resveratrol groups than those in the normal and model groups, and peaked in the 40 μmol/L resveratrol group. (B) LDH activity was significantly decreased in the resveratrol groups compared to the model group and lowest in the 40 μmol/L resveratrol group. *P < 0.05 vs. Nor group; ^# P < 0.05 vs. Mod group; ^Δ P < 0.05 vs. 40 μmol/L Res group. (C) Cell viability was significantly increased in the resveratrol groups, especially for the WP and Pre groups, compared to the Mod group.*P < 0.05 vs. Nor group; ^# P < 0.05 vs. Mod group; ^Δ P < 0.01 vs. WP group, ^□ P < 0.05 vs. Pre group.

Fig. 2.

Effect of resveratrol on neuronal apoptosis at 24 h after OGD/R with TUNEL and western blot analysis. Nuclei were labeled with DAPI (blue). (A,B) Few TUNEL-positive cells (red) were identified in the normal group (Nor). There were numerous TUNEL-positive cells in the model (Mod) (C,D) and resveratrol (E–L) groups compared to the normal (A,B) group. However, the TUNEL-positive cells were remarkably decreased in resveratrol groups compared with the model group. (M) Graph showing the number of TUNEL-positive cells in each group. *P < 0.05 vs. Nor group; ^# P < 0.05 vs. Mod group; ^□ P < 0.05 vs. WP and Pre groups; ^Δ P < 0.05 vs. WP group. (N) Protein expression of Bcl-2 and Caspase-3 with western blot analysis. Quantification of data for Bcl-2 (O) and Caspase-3 (P). The protein expression of Bcl-2 and Caspase-3 was significantly upregulated in the Mod and Res groups compared to the Nor group. The Bcl-2 levels were higher in the Res groups than the Mod group, and were highest in the WP resveratrol group. The Caspase-3 levels were lower in the Res groups than in the Mod group, and were lowest in the WP resveratrol group.*P < 0.05 vs. Nor group; ^# P < 0.05 vs. Mod group; ^Δ P < 0.05 vs. WP group; ^□ P < 0.05 vs. Pre group. Scale bar = 40 μm.

Fig. 3.

Resveratrol strengthens activation of the Nrf-2 signaling pathway at 24 h after OGD/R injury in vitro. (A) Neurons were immunostained with antibodies to Nrf-2 (green). Nuclei were labeled with PI (red). Nrf-2 was mainly located in the cytoplasm in the normal group (A,left) . There were a few cells positive for Nrf-2 in the nuclei in the model group (A,middle). Nrf-2 was mainly located in the nucleus in the resveratrol group (A,right). (B) Protein expressions of Nrf-2, HO-1, and NQO-1 with western blot analysis. (C,D) Quantification of data for Nrf2, NQO-1, and HO-1 proteins. The protein expression levels of Nrf2 in the nuclei, and NQO-1 and HO-1 in the cytoplasm, were significantly upregulated in the Mod and Res groups compared to the Nor group, and were highest in the WP group. This suggests that resveratrol increased expression of Nrf2 in the nuclei and NQO-1 and HO-1 in the cytoplasm. *P < 0.05 vs. Nor group; ^#P < 0.05 vs. Mod group; ΔP < 0.05 vs. WP group; □P < 0.05 vs. Pre group. Scale bar = 20 mm