Direct Stimulation of Adult Neural Stem/Progenitor Cells In Vitro and Neurogenesis In Vivo by Salvianolic Acid B Pengwei Zhuang 1 , Yanjun Zhang1*, Guangzhi Cui1, Yuhong Bian 2 , Mixia Zhang1, Jinbao Zhang 1 , Yang Liu1, Xinpeng Yang1, Adejobi Oluwaniyi Isaiah1, Yingxue Lin1, Yongbo Jiang1 1 Tianjin State Key Laboratory of Modern Chinese Medicine, Key Laboratory of Traditional Chinese Medicine Pharmacology, Chinese Materia Medica College, Tianjin University of Traditional Chinese Medicine, Tianjin, China, 2 Chinese Medical College, Tianjin University of Traditional Chinese Medicine, Tianjin, China Abstract Background: Small molecules have been shown to modulate the neurogenesis processes. In search for new therapeutic drugs, the herbs used in traditional medicines for neurogenesis are promising candidates. Methodology and Principal Findings: We selected a total of 45 natural compounds from Traditional Chinese herbal medicines which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on neural stem/progenitor cells (NSPCs). The screening results showed that salvianolic acid B (Sal B) displayed marked effects on the induction of proliferation of NSPCs. We further demonstrated that Sal B promoted NSPCs proliferation in dose- and time-dependent manners. To explore the molecular mechanism, PI3K/Akt, MEK/ERK and Notch signaling pathways were investigated. Cell proliferation assay demonstrated that Ly294002 (PI3K/Akt inhibitor), but neither U0126 (ERK inhibitor) nor DAPT (Notch inhibitor) inhibited the Sal B-induced proliferation of cells. Western Blotting results showed that stimulation of NSPCs with Sal B enhanced the phosphorylation of Akt, and Ly294002 abolished this effect, confirming the role of Akt in Sal B mediated proliferation of NSPCs. Rats exposed to transient cerebral ischemia were treated for 4 weeks with Sal B from the 7th day after stroke. BrdU incorporation assay results showed that exposure Sal B could maintain the proliferation of NSPCs after cerebral ischemia. Morris water maze test showed that delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. Significance: Sal B could maintain the NSPCs self-renew and promote proliferation, which was mediated by PI3K/Akt signal pathway. And delayed post-ischemic treatment with Sal B improved cognitive impairment after stroke in rats. These findings suggested that Sal B may act as a potential drug in treatment of brain injury or neurodegenerative diseases. Citation: Zhuang P, Zhang Y, Cui G, Bian Y, Zhang M, et al. (2012) Direct Stimulation of Adult Neural Stem/Progenitor Cells In Vitro and Neurogenesis In Vivo by Salvianolic Acid B. PLoS ONE 7(4): e35636. doi:10.1371/journal.pone.0035636 Editor: Josef Priller, Charite´-Universita¨tsmedizin Berlin, Germany Received December 2, 2011; Accepted March 19, 2012; Published April 24, 2012 Copyright: 2012 Zhuang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Natural Science Foundation of China (nos. 30873395 and 30472177), National Basic Research Program of China (no. 2011CB505302), National Science & Technology Major Project ‘‘Key New Drug Creation and Manufacturing Program’’ (2012ZX09101202) and the Program for Changjiang Scholars and Innovative Research Team in University (no. IRT0973). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interest exist. * E-mail: zyjsunye@163.com Introduction Ischemic brain damage is one of the most dangerous ailments that lead to learning and memory disability, physical dysfunction and even death. Up to now, no effective treatment has been reported [1]. Neurons as terminally differentiated cells cannot regenerate after injury in traditional view. However, appropriate exercise training can facilitate some neurological function recovery after stroke in clinical practice [2,3], with the evidence that neurogenesis occurs in the adult brain. Neural stem/precursor cells (NSPCs) had been found and confirmed in adult brain in past decades that it can differentiate into neurons or glial cells as a result of neurogenesis [4–7], NSPCs can be stimulated in several pathological conditions, such as neurological diseases, cerebral ischemic in adult brain, and many reports showed that they are an excellent candidate for developing therapeutic strategies to repair the injured CNS [8,9]. Although the NSPCs would be stimulated to proliferation and differentiation during the brain injury, often this response is not sufficient to overcome the damage. It is essential to study the signalling mechanisms that are activated by small molecular materials in the NSPCs to enhance their response pharmacologically. NSPCs proliferation and neurogensis involves a series of intracellular signaling pathways [10,11]. Among these pathways, the activation of Notch, mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are known to play major roles in cell growth and survival responses [12–14]. Numerous studies have shown that small molecular materials such as growth factors [15], retinoic acid [16] and Traditional Chinese Medicine (TCM) active constituent [17,18] can regulate the biological characteristics of neural stem cell and promote neurogenesis. Therefore, regulation of neuro- genesis by NSPCs is anticipated as a noble therapeutic strategy for brain damage. Herbs have been used for treating diseases for centuries, and a lot of natural compounds that with neural beneficial from medicinal plants had been discovered [19]. Treatment of stroke PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35636