Zhang Yifan, Zhu Zhihan, Li Zhinuo, Feng Jia, Long Jun, Deng Yushu, Ahmed Waqas, Khan Ahsan Ali, Huang Shiying, Fu Qingling, Chen Lukui
Department of Neurosurgery, Southern Medical University Hospital of Integrated Traditional Chinese and Western Medicine, Southern Medical University, Guangzhou, China.
Department of Neurology, Zhongda Hospital Southeast University, Nanjing, China.
Cell Biosci. 2024 Sep 29;14(1):125. doi: 10.1186/s13578-024-01296-4.
Neural stem cells (NSCs) play a crucial role in the progress of ischemic stroke. Research on zebrafish embryonic demonstrates an association between Strawberry Notch 1 (Sbno1) and central nervous system development. However, the regulation and underlying mechanism of Sbno1 in NSCs have not been studied yet. Here, we investigated the role and the mechanism of Sbno1 in NSCs development and the potential therapeutic value of Sbno1 in ischemic stroke.
Adeno-associated virus (AAV) was used for overexpression or knockdown of Sbno1 in vitro or in vivo. A mouse model of MCAO was established to evaluate the neuroprotective effects of AAV-Sbno1, including balance beam test, rotarod test, and strength evaluation. H&E and immunofluorescence assessed neuronal impairment. Western blot and RT-qPCR were used to detect the expression of Sbno1 and its downstream target genes. RNA-seq and western blot were performed to explore further molecular mechanisms by which Sbno1 promoted endogenous repair of NSCs and macrophages M2 polarization. CCK8 was conducted to assess the effects of Sbno1 on NSCs proliferation. The impact of Sbno1 on NSCs apoptosis was evaluated by flow cytometry. NSCs derived from small extracellular vesicles (sEV) were obtained using ultracentrifugation and identified through nanoparticle tracking analysis (NTA) and western blot analysis.
Our results showed that Sbno1 is highly expressed in the central nervous system, which plays a crucial role in regulating the proliferation of NSCs through the PI3k-Akt-GSK3β-Wnt/β-catenin signaling pathway. In addition, with overexpression of Sbno1 in the hippocampus, post-stroke behavioral scores were superior to the wild-type mice, and immunofluorescence staining revealed an increased number of newly generated neurons. sEV released by NSCs overexpressing Sbno1 inhibited neuroinflammation, which mechanistically impaired the activation of the microglial NF-κB and MAPK signaling pathways.
Our studies indicate that sbno1 promotes the proliferation of NSCs and enhances endogenous repairing through the PI3k-Akt-GSK3β-Wnt/β-catenin signaling pathway. Additionally, NSCs overexpressing sbno1 improve ischemic stroke recovery and inhibit neuroinflammation after ischemia by sEV through the MAPK and NF-κB signaling pathways.
神经干细胞（NSCs）在缺血性中风的进展中起关键作用。对斑马鱼胚胎的研究表明草莓Notch 1（Sbno1）与中枢神经系统发育之间存在关联。然而，尚未研究Sbno1在神经干细胞中的调控及其潜在机制。在此，我们研究了Sbno1在神经干细胞发育中的作用和机制以及Sbno1在缺血性中风中的潜在治疗价值。
腺相关病毒（AAV）用于在体外或体内过表达或敲低Sbno1。建立大脑中动脉闭塞（MCAO）小鼠模型以评估AAV-Sbno1的神经保护作用，包括平衡木试验、转棒试验和力量评估。苏木精-伊红（H&E）染色和免疫荧光评估神经元损伤。蛋白质免疫印迹法（Western blot）和逆转录定量聚合酶链反应（RT-qPCR）用于检测Sbno1及其下游靶基因的表达。进行RNA测序（RNA-seq）和蛋白质免疫印迹法以进一步探索Sbno1促进神经干细胞内源性修复和巨噬细胞M2极化分子机制。采用细胞计数试剂盒-8（CCK8）评估Sbno1对神经干细胞增殖的影响。通过流式细胞术评估Sbno1对神经干细胞凋亡的影响。使用超速离心法获得源自小细胞外囊泡（sEV）的神经干细胞，并通过纳米颗粒跟踪分析（NTA）和蛋白质免疫印迹分析进行鉴定。
我们的结果表明，Sbno1在中枢神经系统中高表达，其通过PI3k-Akt-GSK3β-Wnt/β-连环蛋白信号通路在调节神经干细胞增殖中起关键作用。此外，随着海马中Sbno1的过表达，中风后行为评分优于野生型小鼠，免疫荧光染色显示新生成神经元数量增加。过表达Sbno1的神经干细胞释放的sEV抑制神经炎症，其机制是损害小胶质细胞NF-κB和丝裂原活化蛋白激酶（MAPK）信号通路的激活。
我们的研究表明，Sbno1通过PI3k-Akt-GSK3β-Wnt/β-连环蛋白信号通路促进神经干细胞增殖并增强内源性修复。此外，过表达Sbno1的神经干细胞通过sEV通过MAPK和NF-κB信号通路改善缺血性中风恢复并抑制缺血后神经炎症。
