Direct Stimulation of Adult Neural Stem/Progenitor Cells In Vitro and Neurogenesis In Vivo by Salvianolic Acid B

Some actual TCM research.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035636

Introduction Top

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 neurogenesis 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 by TCM has a wealth of clinical experience and theoretical basis, and a large number of effective clinical prescriptions have been accumulated. In recent years a large number of studies have shown that TCM prescription and its active ingredient can improve cerebral ischemic injury in experimental animal [20], [21]. Ginsenoside Rb1 and Rg1, for example, improved spatial learning and increase hippocampal synaptophysin level in mice [22]. Curcumin had been demonstrated to stimulate developmental and adult hippocampal neurogenesis, and a biological activity that may enhance neural plasticity and repair [23]. A recent report has shown that NeuroAid (MLC601 and MLC901), a Traditional Chinese Medicine is used in China for patients after stroke, reduced the increase in escape latency and in swim distance induced by ischemia [24]. With an extensive clinical experience, there are ample opportunities to discover natural compounds that effectively promote the proliferation of NSPCs and neurogenesis from TCM.

Results Top

Salvianolic acid B induced the proliferation of cultured NSPCs in vitro

Forty-five herbal compounds, which are extensively used clinically for treating stroke in China, were screened in an in vitro proliferation assay to identify compounds that could induce proliferation of NSPCs. As shown in Fig. 1, among these natural compounds screened, berberine and Sal B displayed marked activity promoting NSPCs proliferation. In the following study, the proliferative effect of berberine and Sal B were systematically investigated but the action of berberine was proved to be an 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 µM dose exposure for 24 hours, and at 20 µM 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 µM of Sal B (Figure 2C–D). These results suggested that Sal B significantly increased the viability of NSPCs in dose- and time- dependent manners.