Neuroprotective effects of polydatin against mitochondrial-dependent apoptosis in the rat cerebral cortex following ischemia/reperfusion injury

Abstract

The neuroprotective effect of polydatin (PD) against hemorrhagic shock-induced mitochondrial injury has been described previously, and mitochondrial dysfunction and apoptosis were reportedly involved in ischemic stroke. In the present study the neuroprotective effect of PD in preventing apoptosis was evaluated following induction of focal cerebral ischemia by middle cerebral artery occlusion (MCAO) in rats. PD (30 mg/kg) was administered by caudal vein injection 10 min prior to ischemia/reperfusion (I/R) injury. 24 h following I/R injury, ameliorated modified neurological severity scores (mNSS) and reduced infarct volume were observed in the PD treated group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Annexin V/propidium iodide assays demonstrated the anti-apoptotic effect of PD in the ischemic cortex. In addition, PD improved I/R injury‑induced mitochondrial dysfunction, reflected by morphological observations and measurements of mitochondrial membrane potential and intracellular ATP measurement. Western blot analysis revealed an increase in B‑cell lymphoma 2 apoptosis regulator (Bcl-2) expression, and a decrease in Bcl‑2‑associated protein X apoptosis regulator expression in the PD group in comparison with the vehicle treated group. PD treatment also prevented the release of cytochrome c from mitochondria into the cytoplasm, and blunted the activities of caspase‑9 and caspase‑3. Furthermore, PD treatment decreased the levels of reactive oxygen species in neurons isolated from the ischemic cortex. The findings of this study, therefore, suggest that PD has a dual effect, ameliorating both oxidative stress and mitochondria‑dependent apoptosis, making it a promising new therapy for the treatment of ischemic stroke.

Figure 1.

PD is neuroprotective following middle cerebral artery occlusion. (A) Evaluation of neurological deficit by modified neurological severity score. (B) Representative brain sections with 2,3,5-triphenyltetrazolium chloride staining and quantification of infarct volume. Data are presented as the mean + standard deviation (n=6 in each group). *P<0.05, vs. sham group; ^#P<0.05, vs. vehicle group. PD, polydatin.

Figure 2.

PD treatment reduces apoptosis in the cortex following middle cerebral artery occlusion. (A) TUNEL staining in the cortex (white arrows indicate the TUNEL-positive cells). Original magnification, ×200 magnification. (B) Quantification of TUNEL-positive cells averaged over 10 microscopic fields per animal. (C) Isolated neuronal apoptosis as evaluated by the Annexin V/PI double stain assay. Data are presented as the mean + standard deviation (n=6 in each group). *P<0.05 vs. sham group; ^#P<0.05 vs. vehicle group. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 3.

PD protects against middle cerebral artery occlusion induced morphological changes in mitochondria. Mitochondrial morphology was examined by transmission electron microscopy. Swollen mitochondria are indicated by arrows. PD; polydatin.

Figure 4.

PD protects against mitochondrial dysfunction in isolated neurons following middle cerebral artery occlusion. (A) ∆Ψ_m was measured using the fluorescent probe JC-1 and analyzed by flow cytometry. (B) Neuronal ATP levels were determined by a luciferase-based assay. Data are presented as the mean + standard deviation (n=6 in each group). *P<0.05, vs. sham group; ^#P<0.05, vs. vehicle group. PD, polydatin.

Figure 5.

PD treatment reduces Bax upregulation, Bcl-2 downregulation and cytochrome c release in cortex following middle cerebral artery occlusion. (A) Bax and Bcl-2 protein expression levels in the cortex were detected by western blot. (B) Quantification of Bax protein expression levels relative to GAPDH. (C) Quantification of Bcl-2 protein expression levels relative to GAPDH. (D) Cytosolic cytochrome c levels were determined using a cytochrome c ELISA kit. Data are presented as the mean + standard deviation (n=6 in each group). *P<0.05 vs. sham group; ^#P<0.05 vs. vehicle group. PD, polydatin; Bax, Bcl-associated protein X apoptosis regulator; Bcl-2, B-cell lymphoma 2 apoptosis regulator.

Figure 6.

PD protects against caspase-3 and caspase-9 activation in the cortex following middle cerebral artery occlusion. (A) Caspase-3 and (B) caspase-9 activity were measured using fluorometric assay kits. Data are presented as the mean + standard deviation (n = 6 in each group). *P<0.05 vs. sham group; ^#P<0.05 vs. vehicle group. PD, polydatin.

Figure 7.

PD reduces mitochondrial ROS production in isolated neurons following middle cerebral artery occlusion. (A) Intracellular ROS were detected by DCFH-DA and observed by fluorescent microscopy. Original magnification, ×400. (B) Quantification of MitoSOX Red fluorescence by flow cytometry. Data are presented as the mean + SD (n=6 in each group). *P<0.05 vs. sham group; ^#P<0.05 vs. vehicle group. PD, polydatin; ROS, reactive oxygen species.