共同调控PirB和NgR1的microRNAs参与电针促进脑缺血后轴突再生的作用及其表观遗传学机制

Acupuncture has definite effect of treating sequela of apoplexy whereas the mechanism remains unclear. Recent studies have demonstrated microRNAs (miRNAs) are beneficial to neuronal survival and axonal regeneration in the penumbra by simultaneously regulating the expression of many genes. Our previous studies have found that electroacupuncture (EA) can enhance neurologic rehabilitation after stroke by regulating the expression of Nogo-66 receptor (NgR1) and paired immunoglobulin-like receptor B (PirB) as myelin-associated inhibitory receptors. We also forecasted that five miRNAs acted on NgR1 and PriB gene by miRNA microarray and bioinformatics screening. Moreover, the latest researches have demonstrated that it can enhance neuronal expression of orphan nuclear receptor tailless homolog (TLX) and regulate the miRNA level by inhibiting glycogen synthase kinase 3 beta (GSK-3β) activity, which plays a crucial role in EA exerting neuroprotection after ischemic injury. Therefore, we proposed the hypothesis that EA regulating both PirB and NgR1 expression by targeting GSK-3β/TLX/miRNA to enhance neuronal survival and axonal outgrowth after cerebral ischemia. This project tries to screen and investigate miRNAs which simultaneously regulate the expression of NgR1 and PriB by epigenetics method. We will explore the effect and mechanism of miRNAs in the EA exerting neuroprotection after ischemic injury. This project will provide novel theoretical and experimental basis in the mechanism of EA therapy against stroke. It also would be helpful for searching the novel future intervention targets and strategies for this major public health burden.

针灸治疗中风后遗症疗效肯定但机制尚不清楚。研究发现miRNA能同时对多个基因进行调控，对促进脑缺血区神经元存活和轴突再生具有重要作用。申请者前期发现电针通过抑制脑缺血后髓磷脂抑制因子受体NgR1和PirB表达促进神经功能恢复，通过基因芯片技术和生物信息学筛查预测5个miRNA可同时作用于NgR1和PirB基因。最新研究显示通过抑制GSK-3β的活性可增加神经元TLX表达从而调节miRNA水平，而GSK-3β是电针发挥脑保护作用的关键分子。据此提出“电针通过GSK-3β/TLX/miRNA共同调控PirB和NgR1减轻脑缺血后神经元损伤，促进轴突再生和神经功能恢复”的假设。本研究将以表观遗传调控基因表达为切入点筛选共同调控PirB和NgR1的miRNAs，研究miRNAs在电针促进脑缺血后神经功能恢复中的作用及机制，为针灸治疗中风提供新的理论和实验依据，为脑卒中临床干预提供新的靶点和策略。

中枢神经系统损伤后轴突再生困难是神经功能难以恢复的重要原因。microRNA（ miRNA）是表观遗传调控基因表达的重要方式，可调控多个靶基因，包括配对免疫球蛋白样受体B（paired immunoglobulin-like receptor B, PirB），因此可能是中枢神经系统损伤潜在的治疗靶点。本项目采用行为学、形态和分子生物学等综合方法，研究microRNA及关键分子PirB在中枢神经系统脑损伤中的作用及机制。研究结果如下：（1）miR-132在脑缺血再灌注后明显下调，而针刺可以上调miR-132的表达，进而抑制SOX2的表达，改善神经行为功能恢复；在原代神经元氧糖剥夺模型中，上调miR-132的表达可以抑制SOX2，促进神经轴突再生，具有神经保护作用。（2）24周龄糖尿病小鼠海马组织中PirB蛋白表达上调，敲低PirB的表达，可挽救高糖诱导的轴突生长抑制、突触相关蛋白丢失，恢复树突形态异常及树突棘密度的降低，改善糖尿病小鼠空间学习记忆功能。（3）睡眠剥夺7天后海马CA1区PirB蛋白表达上调，敲除PirB促进睡眠剥夺引起的工作记忆损伤恢复，其机制主要通过作用于RhoA/ROCK2/LIMK1/Cofilin信号通路，改善睡眠剥夺引起的肌动蛋白失调，调节锥体神经元结构和功能可塑性。上述结果揭示了电针促进脑缺血损伤后神经功能恢复的表观遗传学机制，证实了PirB在轴突再生、调节突触可塑性及学习记忆损伤中的关键作用，为针灸治疗中风后遗症提供新的见解和理论依据，为靶向PirB蛋白治疗认知功能障碍提供新的策略。