基于Hippo通路的复方血栓通治疗糖尿病视网膜病的药理机制与物质基础研究

Diabetic retinopathy is one of the most prominent complications of diabetes and the effect of anti-vascular endothelial growth factor drug is limited. FufangXueshuantong has been described for its medicinal effect against diabetic retinopathy, but the underlying mechanism and active substance have not been clarified. Hippo pathway is reported to regulate vascular endothelial growth factor. In previous results, we showed that FufangXueshuantong inhibited vascular pathological changes, suppressed vascular endothelial growth factor expression and improved activity or content of Hippo pathway. Furthermore, we discovered the potential substances using Hippo structure based virtual screening..The purpose of present study is to evaluate if Hippo pathway plays key roles in the medical effect of FufangXueshuantong, calculate and verify the bioactive molecules from FufangXueshuantong when Hippo pathway is perceived as targets. In the following study, we will measure the cell viability, cell migration and tube formation of retinal vascular endothelial cells after the Hippo pathway has been overexpressed or inhibited. And then we can analyze the role of the Hippo pathway in the therapeutic effect of FufangXueshuantong. Moreover, we will examine the cell function of retinal vascular endothelial cells using high content screening, then investigate the changes of retinal vascular morphology in diabetic mice after FufangXueshuantong is administered. In the end, we can determine the active substances of FufangXueshuantong by combining the efficacy and the Hippo changes. This project will contribute to reveal a new regulator for diabetic retinopathy, provide a new mode to elucidate the mechanism and active substrate for Chinese medicine and at last lay a foundation for development of FufangXueshuantong.

糖尿病视网膜病是成人致盲的首要原因，现有药物对部分患者无效。复方血栓通治疗糖尿病视网膜病疗效显著，但其药理机制与物质基础不明,不利于其临床广泛使用。我们已经证实复方血栓通不但可以抑制血管内皮生长因子在大鼠视网膜组织中的表达，而且影响该生长因子上游Hippo通路蛋白的活性；我们进一步应用分子对接技术筛选了复方血栓通调控Hippo通路的可能的活性成分。后期我们将在视网膜血管内皮细胞中，过表达或敲除Hippo通路核心基因，观察复方血栓通对细胞增殖、迁移、血管形成等指标的影响，揭示基于Hippo通路的复方血栓通的药理机制；然后运用高内涵系统分析复方血栓通可能的活性成分（组合）对细胞功能及Hippo通路活性的影响，最终通过检测糖尿病动物病理指标的变化，确认复方血栓通治疗糖尿病视网膜病的物质基础。本课题不仅为研究中药复方的药理机制及物质基础提供新的研究模式，而且为防治糖尿病视网膜病提供新的靶标。

DR的主要危害是新生血管形成；Hippo通路一旦活性异常，即可造成非控制性的血管细胞的增殖，进而导致血管增生。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，而且显著抑制血管增生。但是复方血栓通针对Hippo信号通路的调控作用及其效应成分尚未阐明。课题组应用质粒转染或者抑制剂等手段，观察Hippo信号通路对内皮细胞的影响；采用葡萄糖诱导的视网膜内皮细胞，筛选复方血栓通的有效部位，并进一步以YAP为核心，探讨复方血栓通对YAP及其靶蛋白的调控作用；采用分子对接，筛选复方血栓通的效应成分，并在体外体内进行药效观察。结果表明①YAP敲除后，细胞迁移减少，管道长度变短；②复方血栓通水提取物效果最佳，能够显著减少视网膜内皮细胞迁移、成管，并且抑制YAP的表达，当YAP被敲除或者抑制时，复方血栓通的药效减弱；③以YAP为靶点，筛选复方血栓通的效应成分，并对其中的部分成分进行体外药效研究，结果表明Rb3、叶酸、丹酚酸b均能有效减少细胞迁移、增殖等，Rb3与叶酸减少YAP的表达；④在自发性糖尿病小鼠中，Rb3对视网膜厚度、视网膜电生理有一定的改善作用。结论：YAP促进视网膜血管形成；复方血栓通与效应成分可以有效防治葡萄糖诱导内皮细胞损伤，其中YAP发挥了关键作用。本课题将有助于揭示DR的新的发病机制，为防治DR提供新的策略和干预环节，并为复方血栓通的二次开发奠定基础。