Salvianolic Acid B Promote Neurogenesis Figure 6. Salvianolic acid B activated PI3K/Akt in NSPCs. Cell lysates from NSPCs treated or untreated with Sal B (20 mM) were subjected to Western blot analysis with antibodies against both total and phosphorylated forms of ERK1/2, and Akt. Actin was used as loading control. (A) Sal B increased the phosphorylation of Akt. Akt phosphorylation in Sal B-treated runners (15 min, 30 min, 60 min, 120 min) were significantly greater than control runners. Histograms represent the change in the phosphorylation of Akt normalized to anti-actin antibodies (n = 4). **Significant difference from the control group at P,0.01. (B) PI3K/Akt inhibitors Ly294002 regulated the Sal B-mediated phosphorylation of Akt. Ly294002 abolished the phosphorylation of Akt induced by Sal B. Histograms represent the change in the phosphorylation of Akt normalized to anti-actin antibodies (n = 4). *P,0.05 as compared with control, **P,0.01 as compared with control, ##P,0.01 as compared with Sal B-treated cells. No change in total Akt was observed. doi:10.1371/journal.pone.0035636.g006 latency during the learning process in the three groups clearly reflected memory for the escape platform place. Repeated measures ANCOVA revealed no interaction between training days and groups (F(2,30) = 0.90, P.0.05). However, differences were observed between the three groups (F(2,30) = 9.96, P,0.01, Repeated measures ANCOVA), sham-operated animals memo- rized the position of the hidden platform very quickly, reaching it in a shorter latency during the training. In contrast, the untreated ischemic animals showed a severe deficiency compared to the control. Although they were gradually trained and showed better performance, their escape latencies were still much longer than those of the control animals. And there are signifitient difference between sham and model of the escape latency in the day 4 (Sham 32.76618.54 vs Model 54.55626.05, n = 10, P,0.05) and day 5 (Sham 16.3866.56 vs Model 48.89627.39, n = 10, P,0.01). Administration of Sal B (50 mg/kg) significantly ameliorated such deficiencies. After 5 days of studying the acquisition of place learning, rats had learned the escape platform position. To determine the degree of memory, on the sixth day the animal was placed in the pool for Figure 7. Salvianolic acid B increased the number of BrdU-positive cells in vivo. (A) Representative photomicrographs showing BrdU- positive cells in the hippocampus of sham, ischemia and ischemia+Sal B (50 mg/kg)-treated rats. BrdU-labeled cells were indicated by arrows. Scale bar: 200 mm. (B) Quantification of BrdU-positive cells in the hippocampus. Each column represents the represent the mean 6 S.D. (n = 10). **Significant difference from the sham group at P,0.01. doi:10.1371/journal.pone.0035636.g007 PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35636