自噬性神经保护中基于Notch调控内源性神经干细胞参与脊髓损伤修复及补肾活血法干预研究

During spinal cord injury (SCI), the spinal dysfunction are directly linked to neuron death, oligodendrocyte damage, demyelination, and axon disruption. SCI is generally considered incurable because axonal regeneration in the central nervous system is extremely challenging, due to the unfavorable conditions, which include harsh microenvironment, the poor neuron viability, glial scar formation, and restraint of axon regeneration. The key to neural regeneration and repair after SCI is to promote effective neurogenesis, which could be achieved by properly activation of endogenous neural stem cells (ENSCs). Studies have demonstrated that Notch signaling play a crucial role in regulating the fate of stem cells. However, the precise mechanism of how Notch regulate ENSCs is still unclear. Our previous research have found that autophagy can induce neuroprotective functions and Bushen Huoxue Decoction can help repair damage after spinal cord injury through upregulating autophagy. Moreover, Bushen Huoxue Decoction can regulate exogenous (transplanted) mesenchymal stem cells to treat knee osteoarthritis through Notch signaling. Therefore, we propose that (1) we would establish the mice model of conditional knocknout/gain-of-function of Notch, and build the hypoxia model of spinal motor neurons(SMNs), to explore the mechanisms of Notch signaling in regulating the behavior of ENSCs; (2) we would establish Allen's model of mice and hypoxia SMNs with or without autophagy agonist/ inhibitor, to explore the mechanism of autophagy in mediating neuroprotection via crosstalking with Notch signaling; (3) we would establish Allen's model of mice and hypoxia model of ENSCs and SMNs with or without Bushen Huoxue Decoction, to explore the effects of the drugs on affecting neural function and the underlying mechanism, thus providing novel therapeutic targets and theoretical support for clinical treatment of SCI.

脊髓损伤后，改善微环境，调控内源性神经干细胞，促进其增殖、定向分化和迁移，使神经轴突再生，建立起新的神经网络是神经再生的关键。研究表明Notch在干细胞活化过程中扮演着重要角色，但其对内源性神经干细胞的精准调控机制尚不明确。本课题组前期研究发现，补肾活血汤可减轻脊髓损伤，且与上调自噬相关。此外，其可促进体外培养的骨髓间充质干细胞向神经表型稳定；也能通过调节Notch信号促进移植的骨髓间充质干细胞参与膝骨关节炎的损伤修复。因此，本课题组拟通过（1）建立基因敲除和过表达小鼠及脊髓运动神经元损伤模型来探讨脊髓损伤后Notch对内源性神经干细胞的调控作用以及对神经元的影响;（2）通过建立脊髓损伤模型及脊髓运动神经元损伤模型探讨脊髓损伤后自噬与Notch之间的交互作用；（3）探讨补肾活血汤调控自噬和Notch信号对神经干细胞及脊髓损伤的干预作用，为临床治疗提供新的治疗靶点和理论支持。

脊髓损伤（SCI）后，改善微环境、调控内源性神经干细胞，促进其增殖、定向分化和迁移，使神经轴突再生，建立起新的神经网络是神经功能修复的关键。但在此过程中，参与改善微环境的自噬与调控干细胞的Notch之间的交互作用未明。本课题通过体内外实验，利用野生型与转基因小鼠、原代脊髓运动神经元（SMNs）与脊髓神经干细胞（NSCs），从动物、细胞、分子三个层面，较为全面地探讨了调控自噬与Notch对SCI后NSCs的作用、二者交互关系与初步机制，以及补肾活血汤的干预作用。体内研究显示：SCI后，自噬被激活，在7d内呈先升高、后回落的变化规律，且于3d时达到自噬高峰；Notch亦被激活，在7d内其通路相关蛋白及配体蛋白表达量持续升高；在自噬激动剂与抑制剂作用下，我们发现抑制自噬后Notch相关蛋白上调，反之亦然；利用条件性敲除与过表达Notch的转基因小鼠，发现调控Notch可影响自噬，亦能调控NSCs的增殖与分化；补肾活血汤可通过影响自噬和Notch信号通路发挥神经保护作用。体外研究进一步证实：糖氧剥夺条件下细胞自噬被显著激活，且自噬对糖氧剥夺条件下的SMNs起到保护作用；在体外培养的NSCs中，调控自噬可影响干细胞的增殖、迁移和分化；而补肾活血汤中的活性单体成分（虎杖苷）可以促进体外培养的干细胞向神经分化，其机制与上调Nrf2相关，而Nrf2与自噬之间通过p62/keap1/Nrf2存在通路串扰，拟于后续研究中进一步解析p62/keap1/Nrf2与Notch之间的调控关系，以更好地阐释复方中活性单体的药效机制。本项目研究结果初步揭示了自噬性神经保护中Notch调控神经干细胞的规律与交互作用机制，及补肾活血汤促神经再生、发挥神经保护的机理。本研究将“补肾活血”与“调控干细胞命运”相结合，为中医骨伤“补肾活血”法临床治疗脊髓损伤提供了实验依据与全新的现代科学诠释。