开心散调控LRP6/Wnt/β-catenin通路介导突触可塑性改善痴呆小鼠认知功能的机制研究

Synaptic plasticity injury is an important cause of cognitive impairment in Alzheimer's disease (AD). LRP6/Wnt/β-catenin-mediated synaptic dysfunction plays a key role in this process. Kai Xin San has the functions of regulating synaptic-related protein expression, anti-oxidation and anti-apoptosis, but the mechanism has not yet been elucidated. This project intends to carry out the following research:①The APP/PS1 double transgenic mice were used as the research object. Transmission electron microscopy, immunofluorescence, Real time-PCR and other techniques were used to observe dendritic spine density, synaptic morphology and ultrastructure, synapse-associated protein (MAP-2, SYP, PSD95), signaling pathway indicators (LRP6, Wnt7a, β-catenin, etc.); ② Using LRP6 gene silencing method, SH-SY5Y cells were injured by Aβ1-42. The ultrastructure, synapse-associated protein and signal pathway were observed by transmission electron microscopy, Real time-PCR and Western-blot.The mechanism of KXS treatment of AD synaptic plasticity injury was explored around the inactivation and regulation of LRP6/Wnt/β-catenin pathway, providing a new method for the treatment of AD.

突触可塑性损伤是阿尔茨海默病（AD）认知障碍的重要原因，LRP6/Wnt/β-catenin介导的突触功能障碍在此过程中起关键作用。前期研究证实开心散具有调节突触相关蛋白表达、抗氧化及抗凋亡等作用，但机制尚未阐明。本课题拟从如下方面开展研究：①以APP/PS1双转基因小鼠为研究对象，采用透射电镜、免疫荧光法、Real time-PCR等技术，观察树突棘密度、突触形态及超微结构、突触相关蛋白（MAP-2、SYP、PSD95）、信号通路指标（LRP6、Wnt7a、β-catenin等）；②采用LRP6基因沉默方法，以Aβ1-42损伤SH-SY5Y细胞为研究对象，应用透射电镜、Real time-PCR、Western-blot技术，观察突触超微结构、突触相关蛋白、信号通路指标。围绕LRP6/Wnt/β-catenin通路失活与调控，探讨开心散治疗AD突触可塑性损伤的机制，为AD治疗提供新方法。

突触可塑性损伤是阿尔茨海默病（Aizheimer's disease, AD）认知障碍的重要发病机制之一，LRP6/Wnt/β-catenin信号通路在维持突触可塑性方面起关键作用。本研究提出“开心散通过调控LRP6/Wnt/β-catenin信号通路介导突触可塑性，进而改善痴呆小鼠认知功能”的假说，分别从体内、体外实验开展研究。一、动物实验方面：运用APP/PS1小鼠构建AD动物模型，Morris水迷宫、Y迷宫和新物体识别实验检测学习记忆能力，透射电镜观察海马神经元超微结构，Western blot、Real-time PCR检测LRP6/Wnt/β-catenin及IRE1/ XBP1s信号通路相关基因及蛋白表达，比色法检测胆碱能神经递质、氧化应激因子，ELISA法检测促炎因子。结果表明开心散具有：① 提高学习记忆及新物体识别能力；② 减轻线粒体肿胀、变形及损伤，改善神经元超微结构；③ 激活LRP6/Wnt/β-catenin信号通路，促进Wnt7a、β-catenin、LRP6表达，抑制GSK-3β、NF-kB活性，提高PSD95、MAP-2、Bdnf、Ngfb、Dlg2、Dlg4、Syp 蛋白和mRNA水平；④ 促进PDI表达，降低p-IRE1/IRE1比值，减少XBP1s、BIP表达水平，减轻内质网应激损伤。⑤ 改善胆碱能神经递质紊乱，减轻氧化应激损伤，抑制血清促炎因子水平。二、细胞实验方面：运用Aβ1-42诱导SH-SY5Y构建AD细胞模型，采用siRNA转染技术沉默LRP6基因，MTT法检测SH-SY5Y细胞活力，ELISA法检测细胞上清液中促炎因子TNF-α、IL-1β和IL-6水平；Real-time PCR检测LRP6/Wnt/ β-catenin信号通路及突触相关蛋白mRNA表达。结果表明开心散含药血清具有：①提高SH-SY5Y细胞活力；②抑制促炎因子TNF-α、IL-1β和IL-6表达；③激活LRP6/Wnt/β-catenin信号通路，促进LRP6、Wnt7a、β-catenin mRNA表达；④提高Bdnf、Ngfb、Dlg2、Dlg4、Syp mRNA表达，改善突触功能。综合上述结果表明，开心散通过激活LRP6/Wnt/ β-catenin信号通路减轻突触可塑性损伤，提高学习记忆能力，为改善AD认知障碍提供新的实验依据。