藏药"勇哇西汤"调控DN血管内皮网络稳态的配伍机制研究

Microvascular endothelial injury is the central link in diabetic nephropathy (DN), early prevention is the key, effective of single-target was limitations. Prophase NSFC projects research had found the manifest protection effective for diabetes microvascular endothelial using Berberis Cotex, associated with vascular endothelial network regulation by multiple-targets (such as PKC and HIF and VEGF and etc.) with multiple-components. Based on the tibetan medicine theory that cause of DN disease was growth of the Phlegm and the Fat can not be converted, detailed treatment was using "Siwei Jianghuang Soup " (prescription with Berberis Cotex and other three herba drugs) orally, which can inhibit capillary permeability and etc., but in the classics of tibetan medicine theory, The Four Medical Classics, was not record the dosage for each drug, and the relationship between doseage and effect of the modern preparation "Siwei jianghuang Soup" was not clear yet, on this basis, this project put forward the hypothesis that "An amount-effective compatibilities should present for Siwei Jianghuang Soup to treatment DN, associated with multi-components and multi-targets regulation of vascular endothelial network stability", intends to adopt Uniform Design(UD)-Partial Least Squares Analysis (PLSA) and Grey Correlation AnalysisI(GCA) and etc., application STZ-induced DN model and spontaneity DN model and high-sugar foster DN cell model, UFLC-UV/MS and other Methods to analysis the serum fingerprinting and its variation characteristics before and after administration, medical testing methods such as high-resolution colored ultrasound technology and Full automatic biochemical analyzer to accurately reflect the vascular endothelial function and renal function (UAER, UA), biological technologies such as RT-PCR and Western blot to detect vascular cytokines, such as PKC and HIF and VEGF and Ang and NO and TGF and etc., exploring the multiple-targets regulatory mechanisms for vascular endothelial networks stability with multiple-compatibility-components associated PKC and HIF and VEGF and Ang and NO and etc., in order to increase scientific evidence for predominance and characteristics of diabetes treatment with Tibetan medicine.

微血管内皮损伤是糖尿病肾病（DN）的中心环节，前期基金研究发现小檗皮对糖尿病微血管内皮有保护作用，调控相关的PKC、HIF、VEGF等多靶点网络。藏医认为DN病缘为培根、脂肪增长不能转化，治法用"勇哇西汤"（小檗皮等四味藏药组方）内服，有抑制微血管通透性增高等作用，而《四部医典》"有药无量"、现代制剂量效关系不明确，在此基础上，本项目提出假说"勇哇西汤防治DN存在量效配伍，与多靶点调控血管内皮网络稳态有关"，拟采用均匀设计-偏最小二乘及灰色关联等分析，应用STZ诱导DN模型、自发性DN模型、高糖DN微血管细胞内皮模型，UFLC-UV/MS等手段分析血清指纹图谱及变化特征，高分辨彩超及全自动生化测定等技术检测血管内皮功能、肾功能，与RT-PCR、Western免疫印迹及免疫组化等生物技术结合，探索多药味量效配伍对PKC、HIF、VEGF、Ang、TGF等DN血管内皮网络的多靶点调控机制，以发挥藏医药防治糖尿病的优势。

本项目在藏医药“整体观”的指导下，从整体、细胞、分子三个层次，在体动物试验、离体细胞试验两个方面，筛选并确定了四味姜黄方（SWJH）不同剂量配比调控DN血管内皮网络稳态的量效关系及其作用机制，为藏医临床用药提供了科学证据。采用链脲霉素诱导SD大鼠糖尿病肾病（DN）模型进行筛选试验，采用8水平均匀设计表建立SWJH剂量配比矩阵，各给药组的药效学指标均有不同程度的改善，肾指数、空腹血糖水平（FBG）、血管内皮生长因子（VEGF）、肾小球基底膜厚度（GBM）、血尿素氮（BUN）、血尿酸（UA）、血肌酐（Scr）等7指标与剂量配比存在多元一次线性关系或多元二次非线性关系，改善DN大鼠模型的优化剂量配比为姜黄：小檗皮：余甘子：蒺藜＝1:2:1:2；LC-MS非靶向代谢组学的探索研究表明SWJH可能主要影响了精氨酸-脯氨酸代谢通路，其潜在的生物标志物可能是鸟氨酸、黄嘌呤核苷，从而改善了BUN、UA等指标，因此，作为显著性的代谢通路及差异代谢物具有药理学意义及临床价值，有必要深入研究。在此基础上，采用db/db自发性DN小鼠模型进行验证试验，SWJH优化剂量配比显著降低FBG、转化生长因子（TGF-β1）及尿液白蛋白排泄率（UAE）、微量白蛋白（UAlb）水平，显著降低血清Scr、BUN、UA水平，明显减少GBM，显著降低肾组织肿胀程度，有效地改善肾组织病变；免疫组化（IHC）、蛋白印迹（WB）及基因扩增（RT-PCR）试验结果表明，SWJH优化剂量配比可能通过多点调控机制显著降低肾组织缺氧诱导因子（HIF-1α）、TGF-β1、VEGF的表达水平；高糖培养293T肾小球内皮细胞损伤模型也显示具有降低HIF-1α、TGF-β1、VEGF蛋白表达的趋势；主成分分析（PCA）及偏最小二乘分析（PLS）显示SWJH优化剂量配比组与模型组有良好的区分度（分别属于两个类别），与空白组及阳性药组区分不明显（属于一个类别），显示SWJH与二甲双胍等阳性药属于改善DN症状的有效药物，值得深入研究；原核微生物16S rRNA基因测序结果表明，SWJH优化剂量配比不会引起药源性的肠道菌群失调，不会降低肠道乳酸杆菌属菌落丰度，增强了肠道菌群的自我调节机制，有可能通过肠道菌群的反馈机制，改善了DN的相关指标，有待深入研究。