Kuwanon V Inhibits Proliferation, Promotes Cell Survival and Increases Neurogenesis of Neural Stem Cells

Neural stem cells (NSCs) have the ability to proliferate and differentiate into neurons and glia. Regulation of NSC fate by small molecules is important for the generation of a certain type of cell. The identification of small molecules that can induce new neurons from NSCs could facilitate regenerative medicine and drug development for neurodegenerative diseases. In this study, we screened natural compounds to identify molecules that are effective on NSC cell fate determination. We found that Kuwanon V (KWV), which was isolated from the mulberry tree (Morus bombycis) root, increased neurogenesis in rat NSCs. In addition, during NSC differentiation, KWV increased cell survival and inhibited cell proliferation as shown by 5-bromo-2-deoxyuridine pulse experiments, Ki67 immunostaining and neurosphere forming assays. Interestingly, KWV enhanced neuronal differentiation and decreased NSC proliferation even in the presence of mitogens such as epidermal growth factor and fibroblast growth factor 2. KWV treatment of NSCs reduced the phosphorylation of extracellular signal-regulated kinase 1/2, increased mRNA expression levels of the cyclin-dependent kinase inhibitor p21, down-regulated Notch/Hairy expression levels and up-regulated microRNA miR-9, miR-29a and miR-181a. Taken together, our data suggest that KWV modulates NSC fate to induce neurogenesis, and it may be considered as a new drug candidate that can regenerate or protect neurons in neurodegenerative diseases.

神经干细胞（Neural stem cells，NSCs）具备增殖并分化为神经元与胶质细胞的能力。小分子物质对神经干细胞命运的调控，对于特定功能细胞的生成具有关键意义。筛选可从神经干细胞中诱导新生神经元的小分子，将有助于神经退行性疾病的再生医学研究与药物开发。本研究通过筛选天然化合物，旨在发掘可有效调控神经干细胞命运的活性分子。我们发现，从桑树（Morus bombycis）根部分离得到的桑酮V（Kuwanon V，KWV），可增强大鼠神经干细胞的神经发生能力。此外，在神经干细胞分化阶段，通过5-溴-2-脱氧尿苷（5-bromo-2-deoxyuridine）脉冲实验、Ki67免疫染色及神经球形成实验可证实，桑酮V可提升细胞存活率并抑制细胞增殖。值得注意的是，即便在表皮生长因子（epidermal growth factor）与成纤维细胞生长因子2（fibroblast growth factor 2）等有丝分裂原存在的环境中，桑酮V仍可促进神经元分化并抑制神经干细胞增殖。对神经干细胞施加桑酮V处理，可降低细胞外信号调节激酶1/2（extracellular signal-regulated kinase 1/2）的磷酸化水平，上调细胞周期蛋白依赖性激酶抑制剂p21的mRNA表达量，下调Notch/Hairy通路的基因表达，并上调miR-9、miR-29a及miR-181a的表达。综上，本研究结果表明，桑酮V可通过调控神经干细胞命运以促进神经发生，有望成为治疗神经退行性疾病、实现神经元再生或保护的新型候选药物。