黄蒲通窍胶囊调控钙信号通路网络治疗阿尔茨海默病作用机制研究

The pathogenesis of Alzheimer's disease (AD) is numerous, and a variety of signaling pathways, such as calcium signaling pathway, are involved in pathology of AD. Traditional Chinese medicine consider that the kidney deficiency and the brain malnutrition are the origin of AD, and phlegm resistance and blood stasis are the manifestations of the disease, and the treatment of AD is invigorating qi, nourishing kidney, eliminating phlegm and removing stasis. Huangputongqiao Capsule is used for the treatment of AD with the effect of invigorating Qi, tonifying kidney, eliminating phlegm, and dispersing blood stasis. Our previous study found that Huangpu Tongqiao Capsule had a neuroprotective effect on AD animal model and AD cell model, its mechanism had multiple target effect. To further investigate the mechanism of Huangpu Tongqiao Capsule in the treatment of AD, molecular docking and network pharmacology were used to predict the results. The results showed that The 8 main components containing in Huangpu Tongqiao Capsule had the effect on 14 targets related for AD, and the pathways related to 14 targets consisted of calcium signaling network. Thus, we put forward the hypothesis "Huangputongqiao Capsule could regulate calcium signaling networks play a role in the treatment of AD", adopt in vitro and in vivo experimental methods, utilize iTRAQ quantitative proteomics technology and western blot assay to verify the proposed hypothesis, to further explore the essence of Huangpu Tongqiao Capsule in the treatment of AD and provide experimental basis for clinical application and promotion of Huangpu Tongqiao Capsule.

阿尔茨海默病（AD）的发病机制众多，钙信号通路等多种信号通路参与了AD的发病过程。中医认为，肾精亏虚、髓海失养为AD发病之本，痰阻血瘀为其发病之标，益气补肾、豁痰化瘀为其治疗大法。黄蒲通窍胶囊功能益气补肾、豁痰化瘀，临床用于治疗AD。前期的研究发现，黄蒲通窍胶囊对AD动物模型和细胞模型均具有神经保护作用，其作用机制具有多靶点效应。为深入探讨黄蒲通窍胶囊治疗AD的作用机制，采用分子对接技术及网络药理学方法预测发现：黄蒲通窍胶囊所含的8种主要成分对AD的14个疾病相关靶点有作用，靶点对应的主要通路构成了钙信号通路网络。鉴此，我们提出“黄蒲通窍胶囊可能通过调节钙信号通路网络起到治疗AD的作用”的假说，并采用体内外实验方法，运用iTRAQ定量蛋白质组学技术、Western blot等方法验证所提出的假说，进一步探究黄蒲通窍胶囊治疗AD作用的本质，为黄蒲通窍胶囊的临床应用与推广提供实验依据。

阿尔茨海默病（AD）是一种进行性神经退行性疾病，发病机制复杂。中医认为，益气补肾、豁痰化瘀为其治疗大法。黄蒲通窍胶囊具有益气补肾、豁痰化瘀功效，是治疗AD的特效药物。前期研究发现，黄蒲通窍胶囊在AD模型大鼠及细胞水平均具有神经保护作用，且作用机制具有多靶点效应。为进一步探究黄蒲通窍胶囊的有效成分及发挥作用的具体机制，本研究采用体内外实验方法，运用超高效液相色谱法、网络药理学方法、iTRAQ定量蛋白质组学技术和现代分子生物学技术，进行黄蒲通窍胶囊主要有效成分的检测，治疗AD的作用靶点和信号通路的筛选，并进行体内外实验的验证。UPLC结果表明黄蒲通窍胶囊中的主要有效成分为人参皂苷Rg1、大黄酚、大黄素、芦荟大黄素、β-细辛醚、阿魏酸、二苯乙烯苷7种；iTRAQ定量蛋白质组学技术结果表明黄蒲通窍胶囊干预后AD大鼠海马有86个相关蛋白出现明显的差异表达，富集到钙信号通路网络中的Wnt信号通路、GnRH信号通路、ErbB信号通路和钙离子信号通路；Western blot实验验证了差异比较显著的4个蛋白CAMKV、Pxmp4、ACHE、Rims4，结果与蛋白组学的结果一致。进一步通过体内外实验，采用Western blot、RT-PCR、免疫荧光分别检测钙信号通路网络中Wnt5a/Ca2+信号通路、GnRH信号通路及ErbB信号通路，结果发现黄蒲通窍胶囊对AD动物模型和细胞模型钙信号通路网络具有调控作用。综上，黄蒲通窍胶囊主要有效成分为人参皂苷Rg1、大黄酚、大黄素、芦荟大黄素、β-细辛醚、阿魏酸、二苯乙烯苷，可通过调节钙信号通路网络中Wnt5a/Ca2+信号通路、GnRH信号通路及ErbB信号通路发挥作用。本项目全面观察黄蒲通窍胶囊治疗AD的作用与其通过调节钙信号通路网络、维持钙信号通路网络平衡的关系，深入探讨黄蒲通窍胶囊治疗AD的作用机制，为其临床治疗AD提供实验依据，也为中药复方对疾病的作用机制研究提供一个新的思路和方法。