基于VEGF调控通路研究清肝明目中药治疗视网膜退行性疾病的活性成分与作用机制

Retinal degenerative disease is a common and serious disease that causes blindness without effective drug to cure the disease, which is classified as “Shizhan Hunmiao” by traditional Chinese medicine (TCM). The research of retinal degenerative disease pathological mechanism revealed that VEGF signal pathway is one of the essential mechanisms on retinal neovascularization, closely related to the onset of retinal degenerative disease. And the VEGFR antibody drugs have been used for treating retinal degenerative disease in clinical, which could alleviate the condition of the diseases. Based on the theory of TCM that “eyes diseases is usually caused by the fiery evil”, we performed phytochemistry study and successfully identified two natural products as potent VEGFR inhibitors from Glechoma longituba (Nakai) Kupr. In addition, their in vivo activities have also been confirmed by mice model. This study suggested that the natural products from Chinese herbal medicine, which regulates the liver meridian to treat eyes disease for a long history, may achieve its function via the regulation of VEGF signal pathway through inhibiting retinal neovascularization to prevent and treat retinal degenerative disease. Based on our previous study results and with the basic theory of TCM, this project was designed to explore the active components of traditional Chinese herbs (Eriocaulon buergerianum Koern, Buddleja officinalis, et al.) with the therapeutic function of “regulating liver and improving eyesight”, by using techniques of modern isolation & purification, molecular docking and in vitro & in vivo pharmacological technologies, for the purpose of better understanding the mechanism of the herbs with the effect to cure retinal degenerative disease. Moreover, we hope to get natural VEGFR inhibitors by oral administration and provide lead compounds for the drug research and development of retinal degenerative diseases.

视网膜退行性疾病是常见的致盲性眼部疾病，但无有效根治药物上市，属中医“视瞻昏渺”等范畴。研究发现，血管内皮生长因子（VEGF）信号通路与该类疾病的发生发展密切相关，而VEGFR的抗体药物已用于该类疾病的临床治疗，可延缓疾病的进展。项目组前期基于“目不因火则不病”等中医理论从中药连钱草中发现了两个VEGFR的天然抑制剂，在动物体内具有显著的保护视网膜光感受器细胞损伤的活性，表明清肝明目中药中的天然产物可通过调控VEGF信号通路抑制新生血管形成，发挥治疗视网膜退行性疾病的作用。基于前期研究成果并结合传统中医理论，本项目将采用现代分离纯化技术、计算机虚拟筛选和体内外药效评价相结合的方式就谷精草、密蒙花等典型清肝明目中药开展药效物质基础研究，在揭示清肝明目中药防治视网膜退行性疾病的物质基础和作用机制的同时，希望获得口服有效的VEGFR天然抑制剂，为视网膜退行性疾病药物研发提供成药性高的先导化合物。

目的：视网膜退行性疾病是常见的致盲性眼部疾病，但无有效根治药物上市，属中医“视瞻昏渺”等范畴。研究发现，血管内皮生长因子（VEGF）信号通路与该疾病的发生发展密切相关，而VEGFR的抗体药物已用于该疾病的临床治疗，可延缓疾病进展。密蒙花、谷精草、野菊花是常见的具有清肝明目功效的中药，本研究采用现代分离纯化技术、计算机虚拟筛选和体内外药效评价相结合的方式对上述中药开展了药效物质基础研究，目的在于揭示上述药物防治视网膜退行性疾病的物质基础和作用机制，为该疾病药物研发提供成药性高的先导化合物。方法：（1）采用大孔树脂、MCI GEL CHP20P、ODS以及Sephadex LH20等柱色谱方法对密蒙花、谷精草、野菊花传统汤剂中主成分进行分离和纯化，通过理化性质和多种波谱数据鉴定化合物结构；（2）采用本课题组自主开发的虚拟筛选软件Docking Data Bank对化合物的VEGFR抑制活性进行预测；（3）选择打分较好的化合物开展体外VEGFR1和VEGFR2抑制活性评价；（4）采用BALB/c小鼠，分为正常对照组、模型组以及密蒙花中VEGFR天然抑制剂治疗组。模型组和治疗组接收白炽光光照30min[光照强度为（10000±10）lx]建立视网膜光损伤模型。治疗组连续给药7d，光照前暗适应24h，治疗组分别在光照前30min腹腔注射给予100mg/kg药物。光照7d通过光学相干断层扫描（OCT）检测小鼠视网膜形态结构。结果：（1）确定了三味中药汤剂中45个化合物结构，包括6个新化合物；（2）Docking Data Bank打分结果显示：密蒙花中7个化合物、谷精草中3个化合物、野菊花中1个化合物对VEGFR2具有潜在的抑制活性；体外酶活测试结果证实其中5个化合物为强效的VEGFR2天然抑制剂，其IC50值分别为1.21、0.32、0.51、0.42、0.38 μM；（3）动物体内活性测试显示，上述5个化合物（100mg/kg，i.p.）对小鼠视网膜光感受器细胞外核层（ONL）损伤具有显著保护效应。结论：常用清肝明目中药汤剂中5个特征性天然小分子化合物能显著减轻光损伤引起的光感受器细胞凋亡，对光损伤小鼠视网膜功能具有保护作用，具有开发成为治疗视网膜退行性病变药物的潜力。