School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China.
School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong 510006, China.
J Stroke Cerebrovasc Dis. 2022 Feb;31(2):106221. doi: 10.1016/j.jstrokecerebrovasdis.2021.106221. Epub 2021 Nov 24.
To assess the potential effect of dl-3-N-butylphthalide (dl-NBP) for the proliferation and differentiation of neural stem cells (NSCs) against hypoxia and the underlying mechanism.
Hippocampal NSCs were obtained from fetal rats. NSCs combined with dl-NBP and single NSCs were cultured. The impact of siRNA-mediated hypoxia-inducible factor-1alpha (HIF-1α) knockdown on NSCs was detected with western blotting (WB) and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). Cell-counting kit-8 assay was used for evaluating the viability of NSCs. Levels of HIF-1α protein were measured using WB, and vascular endothelial growth factor (VEGF) expression was quantified using RT-qPCR and enzyme-linked immunosorbent assay.
Compared with 7 different concentrations of dl-NBP, 0.25 g/L was determined as the optimal concentration to significantly increase the viability of NSCs (p < 0.001). Dl-NBP can significantly increase the viability of hypoxic NSCs (p < 0.001) and improve the differentiation of hypoxic NSCs into astrocytes (p = 0.001) and oligodendrocytes (p < 0.001). Meanwhile, Dl-NBP can significantly elevate levels of HIF-1α protein (p < 0.001) and VEGF mRNA (p = 0.001) / protein (p < 0.001) in NSCs in the hypoxic environment. However, after transfection with HIF-1α siRNA in NSCs, the viability and differentiation of NSCs was not recovered using dl-NBP under the hypoxic condition, as well as levels of HIF-1α and VEGF.
Dl-NBP can reverse the weaker proliferation and differentiation power of NSCs in the hypoxic environment. The HIF-1α - VEGF pathway may be implicated in this protective effect of dl-NBP.
评估 dl-3-N-正丁基邻苯二甲酸（dl-NBP）对缺氧条件下神经干细胞（NSCs）增殖和分化的潜在作用及其潜在机制。
从小鼠胚胎海马区分离 NSCs，将 NSCs 与 dl-NBP 共培养，或单独培养，用 Western blot（WB）和实时定量逆转录聚合酶链反应（RT-qPCR）检测 siRNA 介导的缺氧诱导因子-1α（HIF-1α）敲低对 NSCs 的影响。用细胞计数试剂盒-8（CCK-8）检测 NSCs 的活力。用 WB 检测 HIF-1α 蛋白水平，用 RT-qPCR 和酶联免疫吸附测定（ELISA）检测血管内皮生长因子（VEGF）的表达。
与 7 种不同浓度的 dl-NBP 相比，0.25 g/L 被确定为显著提高 NSCs 活力的最佳浓度（p<0.001）。dl-NBP 能显著提高缺氧 NSCs 的活力（p<0.001），并改善缺氧 NSCs 向星形胶质细胞（p=0.001）和少突胶质细胞（p<0.001）的分化。同时，dl-NBP 能显著提高缺氧环境下 NSCs 中 HIF-1α 蛋白（p<0.001）和 VEGF mRNA（p=0.001）/蛋白（p<0.001）的水平。然而，在 NSCs 中转染 HIF-1α siRNA 后，dl-NBP 不能恢复缺氧条件下 NSCs 的活力和分化，以及 HIF-1α 和 VEGF 的水平。
dl-NBP 能逆转缺氧环境下 NSCs 增殖和分化能力减弱的现象。HIF-1α-VEGF 通路可能参与了 dl-NBP 的这种保护作用。
