基于VEGF相关信号通路的糖网明目颗粒治疗糖尿病视网膜病变物质基础及作用机制研究

Tangwang Mingmu Granule is a VI type Chinese herbal medicine for the treatment of non-proliferative diabetic retinopathy based on a clinical experience prescription. After more than 20 years of clinical practice, it has been proved that the drug has a significant effect on improving the visual acuity of the patients with diabetic retinopathy. Diabetic retinopathy, a devastating ocular microvascular complication of diabetes mellitus, is one of the leading causes of blindness among working-age adults. These events are regulated by numerous mediators, including vascular endothelial growth factor (VEGF), what is physiologically required for regulating function endothelial cells. However, various pathological conditions are induced in the body during diabetes which deviates VEGF from its physiological role and lead to various pathological demonstrations such as angiogenesis, increased permeability of endothelium, and so on. According to previous pharmacological work, our team speculated that the main mechanism of Tangwang Mingmu Granule for the improvment of diabetic retinopathy is probably based on VEGF related signaling pathways. After the analysis of VEGF related signaling pathways, our team chooses the key proteins of PKC-MAPK/ERK pathway, PKC-Ras-Raf1-MEK-MAPK/ERT pathway, and PIK3-PKB/Akt pathway for further research object, and devotes to expatiate the active components and mechanism of Tangwang Minmu granule for the treatment of diabetic retinopathy based on the VEGF related signaling pathways by combination of multiple technology in different fields. Our work will provide an experimental basis for the development of modern traditional Chinese medicine for Tangwang Mingmu Granule.

“糖网明目颗粒”是基于我国著名中医眼科专家高健生教授的临床经验处方（密蒙花方）开发的中药6类新药，主治糖尿病视网膜病变。糖尿病视网膜病变是糖尿病最为常见和严重的微血管并发症，已成为成年人主要的视力下降和致盲的病因之一。血管内皮生长因子（VEGF）作为诱导和维持血管内皮细胞功能的关键调节因子，在该病变发生发展过程中存在多因素导致的功能失调。结合前期药理学工作基础，课题组推测糖网明目颗粒治疗糖尿病视网膜病变的主要机制很可能来源于VEGF相关的信号通路。通过对VEGF相关信号通路的梳理，课题组拟从PKC-MAPK/ERK通路，PKC-Ras-Raf1-MEK-MAPK/ERT通路，以及PIK3-PKB/Akt通路中的关键节点蛋白出发，运用虚拟筛选技术，化学分离及合成技术，分子生物学技术等研究手段，阐述糖网明目颗粒治疗糖尿病视网膜病变的物质基础及作用机制，为该复方现代组分中药的开发提供实验基础。

糖尿病视网膜病变是糖尿病最为常见和严重的微血管并发症，已成为成年人主要的视力下降和致盲的病因之一。课题组以糖网明目颗粒为研究对象，从血管内皮生长因子（VEGF）相关信号通路中的关键节点蛋白出发，进行计算机虚拟计算及体外活性的研究，探寻糖网明目颗粒治疗糖尿病视网膜病变的物质基础及作用机制。课题组首先建立了糖网明目颗粒各药味的3D化合物库，共包含410个天然产物分子及其多种衍生构象。同时，建立了血管内皮生长因子（VEGF）介导通路中的关键节点蛋白，VEGFR2，PKCbeta-II，ERK，PI3K及AKT1共5种靶点蛋白的结合口袋模型。虚拟筛选结果表明，黄芪黄酮类成分，密蒙花黄酮类成分，益母草黄酮类成分和女贞子环烯醚萜类成分普遍与VEGFR2存在较高的相互作用，女贞子环烯醚萜类成分，女贞子三萜类成分，乌梅有机酸类的新绿原酸，以小檗碱为代表的异喹啉类黄连生物碱及木兰花碱对MAPK/ERK靶点具有较高作用，女贞子环烯醚萜类成分和三萜类成分对PKC相互作用的评分都较高，益母草碱和以小檗碱为代表的异喹啉类黄连生物碱及木兰花碱对AKT具有较高的得分。采用LC-MS分析方法，对糖网明目颗粒非挥发性成分进行全面系统的表征，共鉴定出93个化合物（正离子模式下38个，负离子模式下52个）。将这些化合物与虚拟筛选的命中化合物进行交集分析，确认潜在的活性成分19个，其中，乌梅有机酸类成分1个，女贞子环烯醚萜类成分4个，密蒙花黄酮类成分4个，益母草生物碱类成分1个，益母草黄酮类成分6个，黄连生物碱类成分3个，并通过提取分离的方式对其中各个结构的代表性分子进行了制备。通过活性验证表明，芹菜素，木樨草素，槲皮素为代表的黄芪黄酮类成分及密蒙花黄酮类成分，小檗碱为代表的黄连季铵盐生物碱类成分对VEGFR2具有抑制活性，小檗碱为代表的黄连季铵盐生物碱类成分和益母草碱具有一定的AKT激酶抑制活性；女贞子环烯醚萜类成分及三萜类成分，小檗碱为代表的黄连季铵盐生物碱类成分，乌梅中的新绿原酸和密蒙花中的红景天苷对MAPK/EKR表现出良好的抑制活性，益母草碱也显示出了中等的MAPK/EKR抑制活性。通过总结分析数据，发现了成方各组方药味中的代表性成分/组分改善糖尿病视网膜微血管环境的物质规律和作用机制。