Netrin-1/UNC5H2对脑梗死后自噬调节和神经保护的实验研究

Increased autophagy activity has been reported to play an important role in cell death and neural recovery after cerebral ischemia, but the underlying mechanisms of which remains unclear. In our previous studies supported by the National Natural Science Foundation of China (No.30973108), UNC5H2, which is a pro-apoptotic receptor when unbinding to its ligand netrin-1, was found to be distinctly up-regulated in neurons in peri-infarct area and remote thalamus nucleus in hypertensive rats 1 and 2 weeks after cerebral infarction, which was temporally and spatially correlated to neuron autophagy. In addition, netrin-1/UNC5H2 not only can be modulated by p53 which induced by cerebral ischemia, but also can reversely inhibit the activity of p53 via PI3K-AKT, a known signal pathway of autophagy. Thus, the present study is taken to testify the hypothesis that netrin-1/UNC5H2 is able to modulate autophagy after cerebral ischemia. First, we plan to administer in vivo intracerebroventricular infusion of UNC5H2/Fc fusion protein to interference with the function of intrinsic UNC5H2 in hypertensive rats after cerebral ischemia. The immuno-adhesion tactic with fusion proteins has been successfully practiced in our previous studies. Secondly, UNC5H2 siRNA will be used to inhibit the function of UNC5H2 and LY294002 be used to inhibit PI3K-AKT signal pathway in vitro. The activity of autophagy (LC3 and p62), the cell viability, the expressions of neural plastic proteins (MAP2 and GAP43), AKT, p-AKT and p53 will be detected by electron microscope, immunofluorescence, flow cytometry, RT-PCR and Western Blot. The study will be much helpful to reveal the modulating mechanisms of autophagy, and to improve neural recovery after cerebral ischemia.

新近研究发现脑缺血后自噬增强，并对神经细胞死亡和神经功能恢复有重要影响。在前期国家自然科学基金（30973108）资助下，我们已发现依赖配体netrin-1的条件凋亡受体UNC5H2在脑缺血后呈优势表达，与自噬和凋亡存在时间和部位的一致，外源性netrin-1可改善这种细胞死亡和神经功能缺损；同时，已证实缺血诱导的p53既可直接调控netrin-1/UNC5H2，又可被后者经自噬信号通路PI3K-AKT反向抑制，进一步提示netrin-1/UNC5H2可能参与脑缺血后的自噬调节。本课题在前期基础上，体内应用UNC5H2/Fc融合蛋白的"免疫粘附"策略，体外应用UNC5H2 siRNA，干扰内源性UNC5H2功能；同时，应用LY294002抑制PI3K-AKT信号，观察缺血损伤后netrin-1/UNC5H2对自噬调节和神经保护的作用和机制。研究结果对临床促进卒中后的神经功能恢复有重要意义。

脑卒中后产生一系列神经和血管可塑性反应，但其机制未明。新近研究发现脑缺血后自噬增强，并对神经细胞死亡、血管再生和神经功能恢复有重要影响。在前期国家自然科学基金资助下，我们已发现依赖配体netrin-1的条件凋亡受体UNC5H2在脑缺血后呈优势表达，与自噬和凋亡存在时间和部位的一致，外源性netrin-1可改善这种细胞死亡和神经功能缺损；同时，已证实缺血诱导的p53既可直接调控netrin-1/UNC5H2，又可被后者经自噬信号通路PI3K-AKT反向抑制，因此，我们假设netrin-1/UNC5H2可能参与脑缺血后的自噬调节。. 本课题体内观察netrin-1对大鼠脑梗死后远隔丘脑血脑屏障的保护作用和对神经功能的影响；体外观察netrin-1对氧糖剥夺后大鼠脑微血管内皮细胞和神经母细胞瘤细胞存活的作用，以及对自噬活性的影响。同时，作为脑卒中后神经和血管可塑性的一部分，我们也观察了软骨素酶ABC对脑梗死后神经保护，以及自噬相关蛋白beclin-1在血管新生内膜中的作用和机制。. 结果发现：（1）Netrin-1侧脑室注射（600ng/d，持续7天）可减轻脑卒中后远隔丘脑的继发性血脑屏障损害并改善神经功能，促进内皮紧密连接功能和内皮细胞生存是其主要机制，且这种保护作用可能经由受体UNC5H2介导的PI3K自噬激活而实现。（2）Netrin-1在体外促进氧糖剥夺后人神经母细胞瘤细胞的存活，这种作用可能经其受体UNC5H2介导的自噬降低而实现。（3）软骨素酶ABC脑梗死灶内注射可减轻病灶局部和远隔丘脑部位的损害，这种作用可能经由抑制脑梗死后形成的神经抑制性环境而实现。（4）自噬调节蛋白beclin-1敲低加重大鼠颈动脉损害后的新生内膜形成，由于细胞自噬抑制和凋亡增强而导致的再内皮化延迟可能是其机制之一。. 本项目共发表相关论文9篇，其中，SCI论文4篇，SCI论文摘要1篇，国内核心期刊4篇，参与国际会议交流1次，参与国内会议交流5次，共培养硕士研究生8人。研究结果对临床促进卒中后神经功能恢复和改善血管可塑性有重要意义。