illusion by BrdU incorporation assay (See Figure S1 in the Supporting Information). To study the proliferation-inducing effect of Sal B in detail, we investigated effects of Sal B on the viability of NSPCs in vitro using the MTS assay, NSPCs were treated with Sal B at different concentrations and for different durations. We investigated Sal B at 5, 10, 20, 30, 40, 50 mM dose exposure for 24 hours, and at 20 mM dose incubated for 24, 48, 72 hours on promoting NSPCs proliferation. The results showed that the viability of NSPCs significantly increased as the dose (P,0.01, F(6, 35) = 103.06) and time increases (P,0.01, Fig. 2A–B). The number and size of neurospheres were increased by addition of 20 mM of Sal B (Figure 2C–D). These results suggested that Sal B significantly increased the viability of NSPCs in dose- and time- dependent manners. The MTS assay is a good indicator of cell viability. However, it does not give explicit information on cell proliferation. To provide further evidence that Sal B promotes NSPCs proliferation, a BrdU-incorporation assay was performed. BrdU incorporated cells were detected by immunocytochemistry with anti-BrdU antibody. As shown in Fig. 3, Sal B significantly increased BrdU-labeled cells compared with untreated control (P,0.01, F(3, 40) = 6.63, Fig. 3). Effect of Salvianolic acid B on self-renew genes of NSPCs To confirm the effects of Sal B on maintaining the self-renew cell fate of NSPCs, we further investigated whether treatment of the NSPCs with Sal B could influence their self-renew potential. We studied the effect of Sal B on cell fate specification using RT- PCR method. NSPCs were treated with Sal B (5, 10, 20 mM) for 3 days in proliferation condition, and then the total RNA was extracted. The effect of Sal B on mRNA expression of the self- renew genes, Nestin and Notch-1, was evaluated. The results showed that Sal B up-regulated the expression of Nestin (P,0.01, F(5, 24) = 6.92) and Notch-1 (P,0.01, F(5, 24) = 6.10) mRNA of NSPCs (Fig. 4). These observations suggested that Sal B can maintain self-renewal of NSPCs. Salvianolic Acid B Promote Neurogenesis NSPCs proliferation mediated by Akt activation To investigate the molecular mechanism of Sal B on promoting proliferation of NSPCs, we checked the PI3K/Akt, MEK/ERK and Notch signaling pathways, which were closely related to the proliferation and differentiation of NSPCs [12–14]. The Akt pathway inhibitor Ly294002, ERK pathway inhibitor U0126, and Notch pathway inhibitor DAPT were used. NSPCs were maintained in the presence or absence of Sal B (20 mg/mL), Ly294002 (20 mM), U0126 (10 mM) and DAPT (10 mM) for 2 days. The proliferative response of Sal B/inhibitors was studied using the cell proliferation assay. MTS results showed that Sal B (20 mg/mL) increased the cell survival (Control 0.1760.01 vs Sal 0.3060.01, n = 5, P,0.01). Inhibition of Akt activity by Ly294002 significantly reduced the number of NSPCs, the effects of Sal B on promoting NSPCs proliferation were abolished by the PI3K inhibitor Ly294002, Notch and ERK1/2 had no significant effect on the Sal B - mediated cell survival (P,0.01, F(7, 40) = 158.48, Fig. 5). These results suggested that PI3K/Akt pathway is involved in Sal B- induced proliferation of NSPCs. We further investigated the phosphorylation of Akt in Sal B- treated cultures. NSPCs were stimulated with Sal B (20 mM) for 15, 30, 60, 120 min and the phosphorylation of Akt was detected using phospho-specific antibodies. Western blotting results showed that Sal B could increase the level of Akt phosphorylation at all of the time tested compared with control (Fig. 6A). The effect of Ly294002 on the blocking of Akt phosphorylation induced by Sal B was investigated by treated NSCPs with Ly294002 (20 mM) and/or Sal B for 30 min. As a result, Ly294002 (PI-3K inhibitor) abolished the phosphorylation of Akt induced by Sal B (Fig. 6B). These results suggested that PI3K/Akt signaling was involved in Sal B mediated proliferation of NSPCs. Figure 2. The promotive effect of salvianolic acid B on NSPCs proliferation. (A) Sal B dose-dependently promoted NSPCs proliferation. (B) Sal B time-dependently promoted NSPCs proliferation. Data represent mean 6 S.D. from three independent experiments. **Significant difference from the control group at P,0.01. (C–D) Representative microphotographs of formed neurospheres in the absence (C) or presence (D) of Sal B (20 mM). Sal B increased the size of formed neurospheres. Scale bars: 200 mm. doi:10.1371/journal.pone.0035636.g002 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35636