基于TGF-β1/Smads通路调控PAI-1表达及谱效关系的黄芪三七配伍组分治疗慢性肾小球肾炎质量标志物研究

Radix Astragali and Radix Notoginseng, promoting blood circulation and attending qi deficiency and blood stasis syndrome, is widely used in the treatment of Chronic Glomerulonephritis (CGN) in clinical practice. The high coagulation state of chronic nephritis is the main factor for its deterioration, and the high expression of fibrinolytic factor PAI-1 leads to deterioration of blood coagulation and fibrinolysis, which accelerates renal fibrosis.In prophase stage, our research group has systematically studied in vitro the chemical constituents of the prescription, and preliminary confirmed the constituents in rat serum were notoginseng saponins and astragalus flavonoids and saponins, respectively. . This project intends to adopt the LC-MS technology combining with pharmacology methods. The component library compositions and metabolites in blood of rats after the oral administration of their compatibility components will be established.Based on the influence of serum containing compatibility components on the expression of PAI-1 through the TGF-βl/Smads pathway, we will explore the potential mechanism of regulating of fibrinolytic system and improving of glomerular hypercoagulability from the cellular level by the prescription. The network pharmacology method will be used to carry out molecular docking and component target corresponding analysis, and the potential Q-Marker components will be screened. Study on the spectral effect relationship between the serum fingerprints of different time points and the changes of the expression of PAI-1 will be furtherly investigated. Furthermore, the correlation between the serum fingerprint and the inhibition rate and index changes will be investigated. The CGN Q-marker will be determined through the traceability and structural confirmation of the compositions and metabolites in blood.. The results will provide important in vivo information for a better understanding of the affective substance foundation and compatibility connotation for Radix Astragali and Radix Notoginseng, which also provide strong support for secondary development and offer a novel way for pharmaceutical research of new drugs for the treatment of CGN.

黄芪、三七主治气虚血瘀证，临床合用广泛应用于治疗慢性肾炎(CGN)。高凝状态是该病发展恶化的主要因素，其中纤溶因子PAI-1的高表达导致凝血亢进和纤溶减退，加速肾脏纤维化。项目前期开展组方化学成分研究，并确认黄芪黄酮、皂苷及三七皂苷类为入血成分。本项目拟采用LC-MS数据挖掘策略多技术联合方法建立黄芪、三七配伍组分入血成分及代谢物成分库；采用网络药理学方法进行分子对接及成分-靶标对应性分析，筛选潜在Q-Marker成分；并基于血清药理学方法观察其对TGF-βl/Smads通路抑制PAI-1表达的影响，从细胞水平探讨组分对纤溶系统调控、改善肾小球高凝血态的的潜在机制；进一步考察不同时间点含药血清指纹图谱与PAI-1表达指标变化之间的谱效关系，通过入血成分及代谢物溯源、结构确证，确定组方治疗CGN的Q-markers。本项目有利于阐明组方的药效物质基础及配伍内涵，为拓宽CGN新药研究提供依据。

本课题通过黄芪、三七配伍组分（APCC）含药血清特征图谱建立，以及入血成分、代谢产物鉴别和成分库的建立，主要鉴定黄芪黄酮类成分25个，主要以原型或葡萄糖醛酸形式检出；三七皂苷类成分20种，主要以原型及次级的皂苷类形式存在，黄芪皂苷类未检出。. 网络药理学研究表明TGF-β1/Smad信号通路的上游通路RAS信号通路、趋化因子信号通路、Rap1信号通路、PI3K/Akt信号通路可能是黄芪-三七配伍方治疗慢性肾炎更重要的信号通路，AKT1、SRC、CASP3、EGFR、NOS2可视为治疗慢性肾炎、肾纤维化的潜在靶点，它们均可调控下游的TGF-β1/Smad信号通路。三七皂苷类成分与核心靶点进行分子对接，显示出良好的结合活性。. 随着APCC浓度的增加，对大鼠GMCs增殖的抑制作用逐渐增强，不同浓度APCC大鼠GMCs的PAI-1、TGF-βl、Smad3、Smad4、TβR-I等蛋白表达有降低的趋势，但是在统计学上没有显著性差异，应考虑APCC具有多蛋白表达抑制多重叠加的药效作用。此外，模型组对比空白组，PAI-1 mRNA呈现降低结果，且PAI-1含量和PAI-1 mRNA表达变化趋势相反，无法判定APCC对PAI-1表达的实际作用。免疫荧光法可发现Smad4蛋白的表达抑制且具有量效关系。. 采集不同时间点APCC含药血清进行图谱测定及药效评价，筛选鼠GMCs增殖活性抑制和PAI-1蛋白表达作为主要的药效学指标，对数据进行规格化处理后，分别采用趋势比较、灰色关联度和偏最小二乘法等统计方法。综合数据判定黄芪、三七配伍方治疗慢性肾炎的质量标志物为三七中的人参皂苷Rb1、Rg1、R1、Re、Rd、Rf、Rg2、Re、Rc、Rb2、Rb3，以及黄芪中的毛蕊异黄酮、毛蕊异黄酮葡萄糖苷、芒柄花苷。这些成分即是组方中的主要成分，也是潜在的活性成分或前体成分。. 本课题的研究，有助于揭示黄芪-三七配伍方治疗慢性肾炎的药效物质基础及可能机制，为医院制剂的新药研发提供实验依据，通过网络药理学及谱效关系的研究也为慢性肾小球疾病的治疗研究提供新思路和方法。