中西医均认为血管内皮细胞损伤在慢性肾脏病（CKD）进展中起着重要作用。肾脏微血管内皮细胞表层糖萼、细胞间连接或细胞内骨架蛋白遭到破坏时，内皮细胞通透性增高可导致蛋白尿的发生及肾组织炎症反应和缺氧。黄芪甲苷、阿魏酸的组合物能减轻蛋白尿和延缓CKD进展；还可抑制细胞内游离钙和细胞骨架重排，有可能改善内皮细胞屏障功能。本研究将利用5/6肾切除和糖尿病肾病两种模型确定黄芪甲苷和阿魏酸改善肾功能的最佳配伍方案，动态观察该配伍剂量的组合物对内皮细胞通透性（葡聚糖通透性、细胞表层厚度、细胞连接蛋白和细胞骨架蛋白表达）的影响与蛋白尿和肾脏炎症及缺氧的关系；利用体外培养的肾脏微血管内皮细胞，从促进细胞连接聚合和解离两方面因素（PKA、Rac1、Rap1及PKCα、RhoA）着手，探讨其影响内皮细胞通透性的作用靶点和环节，以期阐明两有效组分在延缓CKD进展中的协同作用机制，为复方中药应用提供现代医学理论依据。

The impairment of vascular endothelial cell is regarded as an important factor in the progression of chronic kidney diseases (CKD) by Chinese and western medicine. Dysfunction of any component of capillary barrier, including the endothelial glycocalyx, intracellular junction or intercellular cytoskeleton protein, can disrupt normal microvascular permeability, which results in proteinuria, interstitial inflammation and hypoxia. Many kinds of Chinese herbs, including Astragali and Angelica, have been used to treat kidney diseases in China for hundreds of years. Astragloside IV and Ferlic acid were two ingredients from the decoction of Astragali and Angelica (A&A). The combination of Astragloside IV and Ferlic acid (AF) was proved to be the main active fraction of A&A due to its similar antifibrotic effects to A&A. AF was found to improve endothelial cell dysfunction characterized by enhancement of nitric oxide production. Recently it was found to reduce urinary protein excretion. And several research works supported that Astragloside IV or Ferlic acid could inhibit the intercellular calcium influx and disassembly of cytoskeleton, which related to improvement of endothelial cell barrier. Thus to the least extent, the renal protective effects of AF partly depends on the improvement of endothelial cell function. In this study, 5/6 nephrectomy and diabetic nephropathy are employed to determine the optimal compatibility program of Astragloside IV and Ferlic acid to improve renal function. The impact of the compatibility-dose combination of Astragloside IV and Ferlic acid on the endothelial permeability,including dextran permeability, thickness of the capillary endothelial surface layer, the expression of cell junction proteins and cytoskeletal protein, will be observed in the dynamic animal experiments to explore the relationship between endothelial permeability and proteinuria,inflammation and hypoxia in kidney. In vitro, renal microvascular endothelial cells will be used to explore the target of AF in improving endothelial permeability via two kinds of cellular signal pathways, including the factors to increase cell-cell adhesion and stabilize endothelial barrier (PKA, Rac1, Rap1) and the factors to disrupt cell-cell adhesion and break endothelial barrier (PKCα, RhoA). The study is designed to clarify the synergy mechanisms of Astragaloside IV and Ferulic acid in retarding the progression of CKD, in order to provide the modern medicine theoretical basis to the future clinical practice of traditional Chinese herbs.

血管内皮细胞损伤在慢性肾脏病（CKD）进展中起着重要作用。内皮细胞通透性增高可导致蛋白尿的发生及肾组织炎症反应和缺氧，而中药单体组分黄芪甲苷和阿魏酸的组合物（AF）能改善内皮细胞功能。本研究在动物模型和体外培养内皮细胞两个层面上，探讨AF降低内皮细胞通透性与延缓肾病进展中的作用及其机制。本课题在糖尿病肾病（DN）大鼠后第2周、第3周和第4周时微量白蛋白/肌酐比值（ACR）显著增高，AF治疗组的ACR明显降低于DN组。第4周时DN组大鼠肾小球体积增大, 部分肾小球系膜基质增多，在AF治疗组未见系膜基质增殖。同期DN组肾小球中几乎未见VE-钙粘素表达，而在AF治疗组中VE-钙粘素表达显著高于DN组。以DN模型组大鼠APP表达量占视野面积的百分比计算，在模型制备后的第2周始， DN模型组大鼠肾脏中的APP表达较对照组明显减少，AF治疗组较DN组显著增加。在第1周时还发现DN组肾脏中Claudin-1表达减少，在AFi治疗组明显增加，该作用持续到第3周。各组间CD68表达无明显差异。因此AF减少蛋白尿的作用与其降低内皮细胞通透性及抑制内皮细胞减少有密切关系。经硝酸镧进行肾脏灌注，在电镜下发现在模型制备后第1周时，DN模型组大鼠肾小球内皮细胞表面糖萼（ESL）表达明显减少，在部分区域呈现节段性完全脱落。同期AF组ESL显著高于DN组。此后各时间点均呈相同趋势。因此在糖尿病肾病的早期即出现肾小球内皮细胞ESL减低而导致尿蛋白，AF通过改善内皮细胞功能而减轻糖尿病引起的肾脏损伤。在肾小球内皮细胞及足细胞共培养体系中证实AF治疗组内皮细胞糖萼成分（syndecan-1）、VE-钙粘蛋白和CKD31较TNF-α刺激组表达增加。在脐静脉内皮细胞和肾小管上皮细胞共培养体系中，加入TNF-α后内皮细胞紧密连接（VE-钙粘蛋白）表达下调，α-SMA明显增高， AF治疗后均得到部分改善。表明AF通过影响内皮细胞连接蛋白改善内皮细胞通透性，减轻内皮细胞的表型转化。因此AF通过改善肾脏内皮细胞功能起到肾脏保护作用，为今后的中药开发和利用提供理论依据。