从钠钾ATP酶/Src信号通路研究中药紫珠防治心肌肥厚的作用机制及固涩正气的药效物质

The Na/K-ATPase (NKA) is not only an ion pump, but also an important signal transducer. NKA could bind with the nonreceptor tyrosine kinase (Src) to form receptor complex NKA/Src, which starts the signal transduction pathways through binding with cardiotonic steroids (CTS). This is the significant cause leading to myocardial hypertrophy and further heart failure. NKA/Src is the new target for the treatment of heart failure. Medicinal Callicarpa species are traditional Chinese astringent medicines. Our study showed that the effective substances in Callicarpa kuangdong were potential natural CTS antagonists, which could dose-dependently antagonize the NKA/Src pathway and inhibit the abnormal proliferation of myocardial cells. On the basis of the former study, multiple normal and mutant cell lines would be used to study the influence of the effective substance on regulating and expression of Src downstream signal gene. A technology platform based on the ligand-NKA affinity interaction would be established for the search, separation and identification of the astringent components group in Callicarpa species. Wild type and gene knockout animal models would be used to clarify the mechanism of its inhibition on myocardial hypertrophy and heart failure. This research started with mechanism study of effective substance and used the bottom-up strategy to gradually reveal mechanism of the effective components group and fraction on signal regulating, which would help with elucidating the flavor and meridian tropismon of medicinal Callicarpa species on the molecular level and could provide new strategy and theoretical basis for the targeted therapy of cardiovascular disease through CTS antagonists.

钠钾ATP酶(NKA)是重要的信号转导体，它与非受体酪氨酸激酶(Src)形成受体复合物(NKA/Src)，通过与强心甾类固醇(CTS)结合，启动信号级联反应，引起心肌肥厚并进一步导致心衰，因而NKA/Src可作为心衰治疗的新靶点。紫珠为固涩正气药，我们的研究显示其活性成分可剂量依赖地拮抗NKA/Src信号通路，抑制心肌细胞的异常增殖，是潜在的天然CTS拮抗剂。本项目拟采用正常及突变的细胞系阐明活性单体对NKA/Src下游信号的调控及基因表达的影响；基于配体与NKA的亲和作用构建其活性成分群的追踪和表征的技术平台，阐明其固涩正气的药效物质；采用野生型和基因敲除动物模型阐明活性部位抑制心肌肥厚的作用机理。本项目从活性单体的机制研究入手，逐步揭示活性成分群、有效部位调控相关信号的作用机制，有助于从分子水平揭示紫珠的性味归经的生物学内涵，也为CTS拮抗剂对心血管疾病的靶向治疗提供新策略及理论依据。

中药紫珠具有清热解毒，收敛止血的药效，其现代药理学研究集中于抗炎和抗氧化，近年来研究发现中药紫珠亦具有心血管保护作用。Na/K-ATP酶 (NKA)的活性与心血管疾病的发生发展密切相关，是治疗心血管疾病的重要靶点。本课题组前期研究也表明广东紫珠中的苯乙醇苷类具有潜在的NKA特异性结合能力，同时调控NKA与非受体酪氨酸激酶(Src)形成受体复合物(NKA/Src)。本项目在建立了Na/K-ATP酶活性检测平台、工具细胞TCN-YFP-α1、以及多种心肌性疾病细胞模型的基础上研究了紫珠中活性成分Na/K-ATP酶配体作用及其对NKA/Src下游信号的调控及基因表达的影响，阐明了其中的活性单体对靶点 Na/K-ATP/Src 信号通路的调控机制。本项目基于配体与NKA的亲和作用构建结合心肌性疾病细胞模型构建活性成分群及活性成分的定向追踪、制备和鉴定的技术平台，首先从不同种的紫珠中筛选出活性最优的种--广东紫珠，进而继续筛选出其中的活性部位，并对该部位进行定性与定量分析，为后续深入研究提供理论依据和技术支持。同时，对广东紫珠各部位及代表性苯乙醇苷类化合物进行心肌肥厚症的相关研究，结果从广东紫珠中筛选出了具有减缓心肌肥厚症作用的活性部位和活性单体化合物。心肌肥厚发生发展的早期表现是心肌纤维化，同时心肌肥厚是心肌细胞重塑的重要表型，而心肌保护可以减缓心脏重塑和心肌纤维化。故本项目建立了多种体内和体外模型，从多种角度研究总浸提物、活性部位和单体成分通过心肌保护和心肌纤维化等途径达到“多角度多因素协同”减缓心肌肥厚的药效活性及调控机制。本项目的研究对于Na/K-ATP酶的新型配体发现，以及广东紫珠对心肌相关疾病治疗作用的发现和探索具有重要意义，有助于从分子水平揭示其性味归经的生物学内涵，也为CTS调控剂对心血管疾病的靶向治疗提供新策略及理论依据，同时，为临床开发多靶点协同作用抗CVD中药的研发奠定了基础。