“通督调神固本法”调控PTEN/PI3K/Akt/mTOR信号转导改善VD模型大鼠学习记忆的机制研究

Neuronal apoptosis is closely related to the pathogenesis of Vascular Dementia （short for VD）, PTEN/PI3K/Akt/mTOR signaling pathway plays a key role in maintaining cell survival and apoptosis inhibition. The latest researches show thatPTEN/PI3K/Akt/mTOR signaling pathway is related to learning and cognition functions; The method of " Dredging Governor Vessel and Regulating spirit for firming of origin" may improve the ability of learning and memory capabilities of the VD rats. Currently, there is no relationship between PTEN/PI3K/Akt/mTOR signal pathway and this treatment. Based on the previous progress of animal researches, our group proposed hypothesis is " Dredging Governor Vessel and Regulating spirit for firming of origin ",which means the therapy affects apoptosis , thus improving cognitive function deficits through activating PTEN/PI3K/Akt/mTOR cell signaling pathway in VD rats. The method of Four-Vessel Occlusion (4-VO) is applied to establish rats' VD model, using Morris water maze test combined with Western bloting, TUNEL and other modern scientific methods to detect the hippocampus pathological morphology, ultrastructure of neurons, apoptosis and related proteins from the impact , as a result of which, exploring the regulating way of using " Dredging Governor Vessel and Regulating spirit for firming of origin " on cell apoptosis , thus may improve learning and memory functions in rats through PTEN/PI3K/Akt/mTOR signal transduction pathways by Electro-Acupuncture.

神经细胞凋亡与VD的发病密切相关，PTEN/PI3K/Akt/mTOR信号转导通路在维持细胞生存和抑制细胞凋亡中起关键作用，研究表明：此信号转导通路与学习和认知功能有关；“通督调神固本法”能有效提高VD模型大鼠学习记忆能力。目前，关于两者的关系国内外暂无报道。结合前期研究，本课题组提出“通督调神固本法”靶向PTEN/PI3K/Akt/mTOR信号转导通路治疗VD的假说，即此法可能通过调控PTEN/PI3K/Akt/mTOR信号转导通路、细胞凋亡，保护海马神经元，从而改善VD模型大鼠的学习记忆功能。采用4-VO法制备VD大鼠模型，利用Morris 水迷宫实验结合Western bloting、TUNEL等方法，检测此法对VD模型大鼠的学习记忆功能、海马病理形态、神经元超微结构、细胞凋亡及相关蛋白的影响，探寻VD发病的新机制和防治新策略。

本研究以四血管阻断法（4-VO法）制备的VD模型SD大鼠作为实验对象，以通督调神固本法作为干预措施，观察通督调神固本法对VD模型大鼠学习记忆能力及PTEN/PI3K/Akt/mTOR信号转导通路的影响，探讨通督调神固本法对VD模型大鼠学习记忆能力的影响极其作用机制，为今后的电针治疗VD的机理研究提供新的依据。 本研究将60只SPF级SD大鼠分为假手术组、血管性痴呆模型组（模型组）、通督调神固本电针组（电针组）、侧脑室注射LY294002组（LY模型组）、侧脑室注射LY294002+电针组（LY电针组），每组12只。采用四血管阻断法（4-VO）制作VD模型，在VD模型制备后立即对LY模型组及LY电针组大鼠侧脑室注射PI3K/AKt信号转导通路阻断剂LY294002，造模完毕饲养一周后，对电针组、LY电针组大鼠进行电针干预治疗，取穴为“百会”、“大椎”、“脾俞”、“肾俞”，每日治疗25min，模型组、LY模型组不针刺，假手术组只进行手术创伤处理。治疗两周后，进行Morris水迷宫测试。水迷宫测试后立即取材，运用Western Blot法观察各组大鼠海马组织中PTEN/PI3K/Akt/mTOR信号通路相关蛋白表达情况，检测凋亡相关蛋白Bcl-2、Bcl-XL、BAX的表达情况，并观察大鼠海马区凋亡情况。经过论证，通督调神固本法能改善血管性痴呆模型大鼠学习记忆能力，促进血管性痴呆模型大鼠学习记忆能力的恢复；通督调神固本法能调节PTEN/PI3K/Akt/mTOR信号转导通路中相关蛋白表达，抑制细胞凋亡，减轻缺血对大鼠海马组织的损伤，发挥神经细胞保护作用，这可能是通督调神固本法治疗血管性痴呆的机制之一。