基于颅内嗅觉通路的电针刺激介导PI3K/AKT及Wnt/β-Catenin信号通路对 SAH CI海马神经元凋亡/增殖的作用

How to reduce the cognitive impairment (CI) caused by subarachnoid hemorrhage (SAH) is regarded as a difficult medical problem. Hippocampus neurons apoptosis and proliferation disorder is the key cause of the CI. Our team found that stimulate the “ying-xiang” and "yin-tang" can improve CI symptoms，and summed up them as "olfactory three-needle",and “olfactory three-needle” has a good effect on cognitive function and hippocampus neurons morphology and hippocampus tissue protein molecule in our experiment，but the mechanism remains unclear. Then the experimental results show that“olfactory three-needle” can improve the olfactory nerve Bcl-2 and hippocampus tissue bFGF expression which is associated with neural apoptosis and proliferation, Whereas both are associated with apoptosis proliferation regulated by the PI3K/AKT and the Wnt/β-Catenin signaling pathway. According to our hypothesis, this mechanism works based on “lateral olfactory stria” intracranial anatomy. The "olfactory three-needle” can improve the bFGF expression which can regulate the apoptosis and proliferation by activate PI3K/AKT and Wnt/β-Catenin signal pathways. We present the hypothesis that “olfactory three-needle” may regulate apoptosis and proliferation in hippocampus neurons by mediating bFGF and GSK-3β and PI3K/AKT and Wnt/β-Catenin signaling pathway based on intracranial olfactory pathways. Tissue,cell and molecular multilayers detection technique are used to confirm the hypothesis. The implementation of this study can provide theoretical basis for electroacupuncture intervention CI.

如何减轻蛛网膜下腔出血（SAH）认知障碍（CI）是医学难题。海马神经元凋亡/增殖调控失常是CI核心原因。本课题组发现电针刺激迎香及印堂穴可改善CI症状，将其总结为“嗅三针”，并通过前期实验证实“嗅三针”基于颅外嗅神经完整性对大鼠认知功能、海马神经元形态、海马组织蛋白分子均有良性干预作用，但机制未明。而“嗅三针”机制学实验证实其可提高嗅神经bFGF及海马Bcl-2表达，鉴于二者与PI3K/AKT及Wnt/β-Catenin信号通路调控的细胞凋亡增殖有关，我们提出“嗅三针”基于“外嗅纹”颅内嗅觉通路介导PI3K/AKT及Wnt/β-Catenin信号通路调控海马神经元凋亡与增殖平衡进而缓解神经元损伤以改善SAH CI的假说，并从神经电生理、组织、细胞、分子多层面证实此假说。本研究的实施可为电针干预CI提供理论依据。

本项目按计划进行了“基于颅内嗅觉通路的电针刺激介导PI3K/AKT及Wnt/β-Catenin信号通路对SAH CI海马神经元凋亡/增殖的作用”研究。项目将bFGF-PI3K/AKT-GSK3β-Wnt/β-Catenin-SAH CI相联系，基于凋亡/增殖这对平衡机制，以切断嗅球的SAH大鼠为实验对象，通过分子生物学等技术，对“电针刺激-bFGF介导-PI3K/AKT激活-GSK3β抑制-Wnt/β-Catenin活化-海马神经元凋亡/增殖”的级联过程进行系统研究。第一，应用Morris水迷宫，开展嗅三针对SAH大鼠认知能力的效应学研究，结果证实嗅三针能够显著提高SAH CI大鼠学习能力和空间记忆能力；第二，采用DAPI染色、TNUEL标记等技术，开展了嗅三针对SAH CI大鼠海马神经元形态学研究，结果表明嗅三针能够抑制模型大鼠海马神经元凋亡，减轻凋亡反应的泛化和持续，降低SAH刺激引发的凋亡性坏死反应；第三，采用Western blot、RT-PCR 、AKT干扰技术，开展凋亡相关因子bFGF、PI3K、AKT、GSK-3β，caspase3蛋白水平及其 mRNA 变化的效应学研究，证实嗅三针调控 bFGF、GSK-3β、PI3K/AKT信号发挥减缓海马神经元凋亡的作用；第四，采用RT-PCR、Western blot、免疫荧光双标配合Wnt干扰技术检测GSK-3β、Wnt1、β-Catenin、LMNB1、Nestin、BrdU标记蛋白及mRNA 表达的效应研究，证实嗅三针具有介导GSK-3β、Wnt/β-Catenin通路调控海马神经元增殖的作用，证实以上干预效应均有赖于嗅觉通路完整性。初步阐明了嗅三针通过嗅觉通路改善SAH大鼠认知障碍，提高学习能力和空间记忆能力，此机制与bFGF表达水平增强，调控海马神经元PI3K/AKT活性，上调抗凋亡细胞因子，同时抑制GSK-3β水平，进而活化Wnt/β-Catenin，增强增殖分子蛋白表达，减少海马神经元损伤具有一定相关性。通过本项目研究，解决了嗅三针影响SAH CI的作用途径和干预机制这一关键科学问题，为针刺治疗血管性认知障碍的临床应用提供了理论和实验依据。