黄蒲通窍胶囊干预Ca2+-CaMKIV信号通路抑制tau蛋白过度磷酸化在AD治疗中的研究

Alzheimer's disease (AD) is the commonest type of senile dementia，endangering severely the health of old people. Neurofibrillary tangles (NFT) is AD’s main pathological feature, and NFT formation is directly associated with over-phosphorylation of tau protein, which is regulated by Ca2+-CaMKⅣ signal pathway. Traditional Chinese medical science considers the pathogenesis of AD as marrow depletion, brain deficit and mind malajustment and makes the kidney-jing deficiency as a principal part and nourishing kidney to produce marrow as an important way. Huangpu Tongqiao Capsule is an effective nosocomial agents based on our many years’ clinical experiences, invigorating Qi, tonifying kidney, eliminating phlegm, and dispersing blood stasis. Our previous experiments showed that Huangpu Tongqiao Capsule could improve study and memory abilities, reduce the loss of hippocampal neurons and the over-phosphorylation of tau protein. Therefore, this program targets Ca2+-CaMKⅣ signal pathway-mediated over-phosphorylation of tau protein, adopts in vitro and in vivo experimental methods, utilize modern morphological and molecular biology technology to investigate the effect essence of Huangpu Tongqiao Capsule, to further clarify the target of Huangpu Tongqiao Capsule to treat AD, providing experimental proof for its clinical application and propagation, and also providing new method for clinical treatment of AD.

阿尔茨海默病(AD)是老年期痴呆最常见的类型，严重威胁着老年人的健康。神经元纤维缠结（NFT）是AD的主要病理特征，其形成和tau蛋白过度磷酸化直接相关，而tau蛋白过度磷酸化受到Ca2+-CaMKⅣ信号通路的调控。中医认为AD基本病机为髓减脑消、神机失用，以肾精亏虚为本、痰阻血瘀为标，益气补肾、豁痰化瘀为其治疗大法。黄蒲通窍胶囊是我们基于多年临床经验而成的治疗痴呆的有效院内制剂，功能益气补肾、豁痰化瘀。我们前期的实验研究发现，黄浦通窍胶囊可改善AD大鼠的学习记忆能力，减少海马神经元丢失，减少tau蛋白的过度磷酸化。鉴此，本课题以Ca2+-CaMKⅣ信号通路介导的tau蛋白过度磷酸化为靶点，采用体内外实验方法，运用现代形态学和分子生物学实验技术，探究黄浦通窍胶囊发挥作用的本质，进一步明确黄蒲通窍胶囊治疗AD的作用靶点，为其临床应用和推广提供实验依据，为临床治疗AD提供新方法。

神经元纤维缠结是阿尔茨海默病（AD）的主要病理特征，其形成和tau蛋白过度磷酸化直接相关，而tau蛋白过度磷酸化受到Ca2+-CaMKⅣ信号通路的调控。黄蒲通窍胶囊（HPTQ）功能益气补肾、豁痰化瘀，在AD治疗上有潜力。因此，本课题以Ca2+-CaMKⅣ信号通路介导的tau蛋白过度磷酸化为靶点，采用体内外实验方法，研究HPTQ治疗AD的作用机制。体内实验时，建立AD大鼠模型，采用Morris水迷宫实验检测大鼠行为学改变，HE染色观测大鼠海马形态学改变，透射电镜观察大鼠海马超微结构的变化；体外实验采用血清药理学的方法，原代培养海马神经元，MTT法筛选Aβ造模浓度和HPTQ含药血清安全浓度及对AD细胞模型细胞存活率的影响，Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡率，透射电镜观察神经元超微结构的变化。Western blot法和RT-PCR法检测大鼠、原代培养海马神经元、PC12细胞中Ca2+-CaMKIV信号通路相关蛋白表达和mRNA含量。结果发现：（1）Morris水迷宫实验显示HPTQ可明显提高AD大鼠的学习记忆能力；（2）HE染色显示HPTQ可减少AD大鼠海马神经元的丢失；（3）透射电镜显示HPTQ可减少AD动物和细胞模型海马神经元超微结构损伤；（4）MTT法显示HPTQ可提高AD细胞模型的细胞存活率；（5）流式细胞术显示HPTQ可减少AD细胞模型神经元凋亡；（6）激光共聚焦实验显示HPTQ可减少AD细胞模型钙离子浓度；（7）Western blot法显示HPTQ可减少AD动物模型和细胞模型中CaM、CaMKK、p-CaMKⅣ、p-tau蛋白表达；（8）RT-PCR法显示HPTQ可减少AD动物模型和细胞模型中CaM、CaMKK、CaMKⅣ、tau的mRNA含量。结论：（1）HPTQ可显著提高AD大鼠学习记忆能力，具有神经保护作用；（2）HPTQ的神经保护可能通过调控Ca2+-CaMKIV信号通路抑制tau蛋白过度磷酸化起作用。本课题的顺利完成，揭示了黄蒲通窍胶囊治疗AD、改善学习记忆能力的部分作用机制，为临床防治AD提供新的思路和方法。