Neuroserpin在创伤性脊髓损伤中自噬-凋亡的调控作用及机制研究

Neuroserpin (NSP), an axonally secreted serine protease inhibitor, is widely expressed in the adult central nervous system including spinal cord. It plays an important role in regulating migration, remodeling and maintenance of neurons, synapse formation, moreover it has the protective effect with inhibiting apoptosis of nervous cells in brain injury. But the function of NSP after spinal cord injury is still unknown. The treatment of traumatic spinal cord injury (TSCI) is a major challenge to the doctors all around of the world. Apoptosis, in secondary spinal cord injury, is critical for the function protection and recovery of spinal cord. It has been reported that autophagy is closely associated with apoptosis, and regulates the level of apoptosis. Our previous study demonstrated that the expression of NSP increased after TSCI, and the level of Bax, an apoptosis-inducing protein, significantly decreased with the administration of exogenous NSP. Additionally, the gene expression of Beclin-1 changed with time after using NSP in TSCI rat model. Thus, we hypothesize that exogenous NSP has the neuro-protective effect on spinal cord after TSCI, and can reduce apoptotic cell death by regulating autophagy. We will consider the regulatory role of NSP on autophagy after TSCI, and investigate the mechanism and pathway of the balance of autophagy and apoptosis by using some tools reagents. The study will provide the theoretical basis for the treatment of TSCI by NSP.

Neuroserpin（NSP）是一种轴突选择性的神经源性丝氨酸蛋白酶抑制剂，在成人中枢神经系统有广泛表达，调节神经元迁移、突触形成，在神经元重塑与维持上起重要作用。在脑损伤时对神经相关细胞具有抑制凋亡的保护作用。但是其在低位中枢（脊髓）的作用尚无文献报道。外伤性脊髓损伤（TSCI）的治疗是全世界面临的重大难题，细胞凋亡在其中发挥了巨大作用。文献报告，自噬与凋亡密切相关，并可调控凋亡；我们的前期研究也发现NSP在TSCI后表达升高，给予外源性NSP后，损伤部位凋亡诱导蛋白Bax表达明显下降；Beclin-1基因表达随时间发生改变。由此我们提出假设：外源性NSP在TSCI后，对脊髓功能具有保护作用，并调控自噬而减少凋亡性细胞死亡。本研究通过观察NSP对TSCI的作用，并使用相关工作试剂，了解NSP对自噬的调控作用，探讨细胞内自噬－凋亡平衡可能的作用途径与机制，为NSP治疗TSCI提供理论基础

本研究分为体内动物实验和体外细胞实验两个部分。体内实验中，应用大鼠外伤性脊髓损伤模型观察其损伤后自噬凋亡的情况以及外源性NSP干预后的情况。发现脊髓损伤后3天自噬体达到峰值，同时自噬活性下降，而凋亡在脊髓损伤后持续增加并与自噬活性下降明确相关，自噬与凋亡均参与脊髓损伤的病理过程；而早期使用外源性NSP干预后，能够抑制脊髓神经元自噬的表达、减少神经元的死亡，提高脊髓损伤模型的运动功能恢复。在体外神经元细胞实验中，损伤后早期使用NSP干预能够有效诱导神经元细胞的自噬，抑制神经元细胞早期的凋亡，促自噬-凋亡间动态平衡倾向自噬，从而抑制线粒体ROS的水平以及调节线粒体呼吸链复合物II、III，促神经元细胞存活。而在抑制自噬或主动开放mPTP后，NSP的相关作用完全消失，提示外源性NSP主要是通过调控自噬及线粒体级联反应达到保护神经元细胞作用。MiRNA-15b在其中可能起到了重要的作用。