Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway

Abstract

Aim: To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI).

Methods: Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg(-1)·d(-1), ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg(-1)·d(-1), ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting.

Results: Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg(-1)·d(-1)) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor LY294002. Treatment with Rd (25, 50, and 100 μmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of LY294002.

Conclusion: Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.

Figures

Figure 1

Contents of VEGF/BDNF in CM of PC12 cells. (A) Quantitative data from each group. ANOVA was used for statistical analysis. (B) Quantitative data from each group. ANOVA was used for statistical analysis. Data are mean±SD. n=6. bP<0.05, cP<0.01 vs OGD.

Figure 2

Effects of Rd on phosphorylation of Akt and ERK after OGD. Increased phosphorylation of Akt and ERK proteins in PC12 cells treated by OGD+Rd. An antibody for GAPDH was used to show equal protein loading. (A) Western blot analysis of the phosphorylated forms of Akt (p-Akt-ser473); total Akt; phosphorylated ERK1/2 (p-ERK1/2-tyr204) or total ERK1/2. (B) Bar graphs show quantitative evaluation. Data are mean±SD. n=3. bP<0.05 vs OGD.

Figure 3

Effects of LY294002 on the PI3K/Akt pathway. (A) PC12 cells were treated with Rd (50 μmol/L) or LY294002 (10 μmol/L) for 6 h after OGD, and the expression of Akt was verified by Western blotting. (B) Bar graphs show quantitative evaluation. Data are the mean±SD. n=3. bP<0.05 vs OGD. eP<0.05 vs OGD+Rd.

Figure 4

Effects of Rd on apoptosis of PC12 cells after OGD. (A–D) Apoptosis was analyzed by flow cytometry following PI/Annexin V-FITC double-staining. (E) The percentages of apoptotic cells are indicated. n=6. bP<0.05 vs OGD. eP<0.05 vs OGD+Rd.

Figure 5

Infarct volume and neurological scores. (A) Representative TTC staining of the cerebral infarct in comparable sections of rat brain, 7 d after ischemia. (B) Quantification of infarct volume. (C) Neurological scores. Mean±SD. n=6. bP<0.05, cP<0.01 vs I/R group.

Figure 6

Akt phosphorylation in ipsilateral hemisphere. (A) Western blot analysis of Akt expression in the ipsilateral hemisphere 24 h after reperfusion. (B) Bar graphs show quantitative evaluation of phospho-Akt/Akt expression. Means±SD. n=3. bP<0.05 compared with I/R group.

Figure 7

Accumulation of newborn neurons. (A) The merged confocal images confirmed the presence of BrdU (red), GFAP (red), DCX (green), and Nestin (green) expressing cells in ipsilateral brain sections. Scale bar=20 μmol/L. (B) Quantitative data of each cell type. Means±SD. n=10. bP<0.05, cP<0.01 vs I/R. eP<0.05, fP<0.01 vs I/R+Rd. Str, Striatum; DG, Dentate gyrus.