从血管内皮祖细胞VEGF/KDR环路研究滋阴补肾活血法治疗糖尿病视网膜病变的作用机制

Diabetic retinopathy ( DR ) is an important vascular complication of diabetes, and the main cause of adults' Ablepsia. Our preliminary studies found that "kidney deficiency combined with blood stasis" contributed to the basic pathogenesis of DR, and it is certain that the method of traditional Chinese medicine called Ziyin Bushen Huoxue and its prescription were effective in clinical treatment of diabetes. In occurrence and development of DR, retinal blood vessel neogenesis is an important component element. however, inhibition of angiogenesis is a key means of prevention and treatment of DR .On the basis of preliminary study on the level of VEGF which restrain retinal neovascularization regulated by the method of Ziyin Bushen Huoxue, along with the latest research progress of VEGF and its receptor KDR loop and endothelial progenitor cells to initiate angiogenesis theory, our research group propose a hypothesis that the method of Ziyin Bushen Huoxue and its prescription may inhabit the neogenesis of retinal blood vessel by regulation of endothelial progenitor cells VEGF/KDR loop ,and therefore is a treatment for diabetes. TThe model of animals and primary cells are adopted in this issue, and the experiment methods such as histological observstion, immunohistochemisty and RT-PCR are used in the models for the study that the method of Ziyin Bushen Huoxue and its prescription have an influence on the VEGF/KDR loop and its signaling pathway, which can explain the mechanism of the method of Ziyin Bushen Huoxue in the treatment of diabetes and reveal the characteristics and regularity of VEGF/KDR loop and its signaling pathway. The theory of traditional Chinese medicine is expected to be improved from the regulation level of inhibition of retinal blood vessel neogenesis.

糖尿病视网膜病变（DR）是糖尿病重要的血管并发症，也是成人致盲的主要原因。课题组前期研究发现DR以"肾虚血瘀"为基本病机，采用滋阴补肾活血治法组方，临床疗效确切。视网膜血管新生是DR发生发展的关键环节，而抑制血管新生是防治DR的关键手段。课题组基于前期对滋阴补肾活血法调控血管内皮生长因子（VEGF）表达而抑制视网膜血管新生的研究，结合内皮祖细胞启动血管新生理论及VEGF及其受体KDR环路的最新研究进展，提出滋阴补肾活血法及其组方通过调控内皮祖细胞VEGF/KDR环路抑制视网膜血管新生从而治疗DR的假说。本课题采用动物和原代细胞模型，通过组织学观察、免疫组化、RT-PCR等方法，研究滋阴补肾活血法及其组方对VEGF/KDR环路及其后信号通路的影响，从而阐明滋阴补肾活血法治疗DR的作用机理，揭示VEGF/ KDR环路及其信号通路的特点和规律。有望从视网膜新生血管的抑制调控水平提升中医理论。

背景及主要研究内容：本研究是在“滋阴补肾活血法论治DR”的学术思想指导下，把中医的理论思维和前沿技术有机结合，对滋阴补肾活血法及组方双丹明目胶囊防治DR的具体作用机制进行了深入研究。在该药取得显著临床疗效的基础上，本研究在双丹明目胶囊经过多中心临床研究验证其临床疗效的基础上，重点围绕VEGF/KDR环及其通路与双丹明目胶囊之间的关系展开研究，从细胞增殖、上下游信号调控影响、视网膜结构改变等方面探讨双丹明目胶囊防治DR的具体作用机制。.结果：（1）体外细胞实验研究，本实验成功分离了脐带血来源的EPCs；双丹明目胶囊含药血清干预后，EPCs的增殖受到明显抑制，通过多浓度多时间点分析发现，以1.12 mg/ml、干预24 小时为最佳；双丹明目胶囊能调控VEGF/KDR环路相关蛋白的表达，降低其下游因子Ras、Raf-1、MEK、ERK蛋白及基因的表达；双丹明目胶囊能与VEGF/KDR环路特异性阻断剂SU1498发挥协同增效作用，说明其确实能通过调控VEGF/KDR环路而发挥作用。（2）体内动物实验研究：双丹明目胶囊能明显改善糖尿病视网膜病变大鼠模型视网膜组织形态和超微组织结构，抑制微血管生成；能抑制DR大鼠视网膜VEGF/KDR环路的表达，能明显降低VEGF-a、VEGF-b、VEGF-c及其受体Flk-1在视网膜中的表达，并调控其上游信号HIF-1/NF-κB/Spred-1及下游信号Akt, eNOS mRNA、Ras-Raf-1-MEK-ERK而发挥抑制微血管生成、治疗DR的作用。 .科学意义：本研究重点探讨了双丹明目胶囊对血管内皮祖细胞及VEGF-VEGFR上下游信号转导的影响，结果表明双丹明目胶囊能明显抑制EPCs增殖，其作用与调控VEGF/KDR环路有关；能抑制DR大鼠视网膜VEGF/KDR环路的表达，能明显降低VEGF-a、VEGF-b、VEGF-c及其受体Flk-1在视网膜中的表达，并调控其上游信号HIF-1/NF-κB/Spred-1及下游信号Akt、eNOS、Ras-Raf-1-MEK-ERK而发挥抑制微血管生成、进而起到治疗DR的作用。这对为DR寻求新的药物治疗靶点，为中医药防治DR的研究提供新思路，为中医药较低DR的致盲理论依据，对提高DR患者的生活质量和减少医疗财政支出，推动中医治法理论发展，促进中医治法研究的转化医学新模式具有重要意义。