以YAP为靶向的复方血栓通治疗糖尿病视网膜病的物质基础与药理机制研究

Diabetic retinopathy (DR) is a complication in the late stage of various types of diabetes with high incidence and great risk. Fufang Xueshuantong (FXST), which can activate blood, remove stasis, nourish qi and Yin, is one of the Protection of Varieties of Traditional Chinese Medicines. FXST is effective in treatment of DR, but its material basis and pharmacological mechanisms are not clarified. This leads to the constraint on the clinical use of FXST. Literature indicates that angiogenesis is the key characteristics of DR, and YAP is the core component of angiogenesis. Moreover FXST inhibited angiogenesis and treated DR through targeting YAP. Thus the aim of this study is to choose YAP as the target protein and screen the effective constituents by ultrafiltration mass spectrometry. On the basis of this, the network pharmacological technology will be used to illustrate the target proteins which interact with YAP. In combination with the cell biology and molecular biology, the network which includes effect components - the target molecules - signalling pathway - disease will be constructed. In the end these results will bring light to the material basis and pharmacological mechanisms of FXST. This project will develop a new paradigm for the material basis and pharmacological mechanism research of traditional Chinese medicine. And it is of great value for the development of new drugs for other diseases with angiogenesis.

糖尿病视网膜病（DR）是各型糖尿病的晚期并发症，患病率高，危害大。复方血栓通作为国家二级中药保护品种，活血化瘀，益气养阴，治疗DR疗效显著，但是其具有中药复方的典型局限性，即物质基础与药理机制不清，导致其临床应用价值被严重制约。文献调研发现血管增生是DR的标志性事件，YAP作为转录辅助因子，是血管增生的核心机制。而且课题组证实，复方血栓通通过靶向YAP调控血管增生，从而达到治疗DR的效果。本课题拟以YAP为靶蛋白，采用超滤质谱技术筛选复方血栓通效应分子（群）；以此为基础，利用网络药理学技术阐明效应分子（群）以YAP为核心的靶标；结合经典的细胞生物学及分子生物学实验验证，构建效应成分-靶标分子-信号通路-疾病网络，最终明晰复方血栓通以YAP为靶向的治疗DR的物质基础及药理机制。本课题不仅为研究中药复方的物质基础和药理机制开发了新颖的研究范式，而且对于其他血管增生性疾病的药物开发具有重大意义。

过度血管生成是DR的关键病理过程；YAP作为转录辅助因子，能够调控血管新生，且在视网膜血管发育及疾病过程起关键作用。复方血栓通由四味中药组成，包含三七、丹参、黄芪和玄参，对于DR疗效显著，而且显著抑制血管新生。课题组已证实YAP是复方血栓通改善DR的关键环节，但是复方血栓通以YAP为靶向的调控作用及其效应成分尚未阐明。本课题应用超滤质谱技术筛选复方血栓通与YAP结合的效应成分，采用葡萄糖诱导的视网膜内皮细胞评价效应成分的药效；在体内体外进行药效观察；利用网络药理学方法揭示复方血栓通效应成分防治DR的药物靶点；应用荧光定量PCR和全自动蛋白质毛细管电泳实验揭示复方血栓通效应成分的药理机制。结果表明①超滤质谱实验指纹图谱共解析出17个化合物；②解析出的化合物中人参皂苷Rg2药效最优，能够显著减少葡萄糖刺激的视网膜内皮细胞细胞迁移和成管；人参皂苷Rg2抑制VEGF诱导的内皮细胞迁移等；③网络药理学结果提示人参皂苷Rg2对多条信号通路多个靶点具有影响；④人参皂苷Rg2可以抑制CAM和OIR小鼠模型诱导的血管新生;⑤人参皂苷Rg2与YAP结合，当YAP沉默时，人参皂苷Rg2抑制RF/6A细胞迁移和内皮管道形成的能力均减弱；⑥YAP敲除后，TLR通路发生显著改变；⑦人参皂苷Rg2抑制YAP1及TLR通路TLR2、TLR4、TRAF6、IRAK1和NF-κB的mRNA表达水平，调控 YAP1、p-YAP1、TLR4、TRAF6和NF-κB的蛋白表达水平；⑧在自发型糖尿病小鼠中，Rg2改善糖尿病视网膜病变。结论：人参皂苷Rg2通过YAP调控TLRs信号通路，从而抑制细胞迁移及管道形成，最终限制血管增生，缓解DR病变。本课题为防治DR提供了新的策略，为揭示DR的新的发病机制增添了助力；并为复方血栓通的二次开发打下了基础。