化瘀通络中药不同配伍对糖尿病肾病基于足细胞滤过屏障相关蛋白表达的效应机制

Proteinuria is an iconic characteristic of diabetic nephropathy (DN) in early and clinical period. The aggravateion of proteinuria and development could interact with each other, So that the control of proteinuria plays a significant role in treating DN. However, there is no targeting therapy to decrease urine albumin in modern medicine. The damage of pydocyte filtration barrier is believed to be the key factor for proteinuria and glomerulosclerosis of DN. On the other hand, "stasis blocking collaterals" has been accepted to be the main pathogenesis of DN. And we have established the DN "stasis blocking collaterals" model. In addition, definite effect of different combinations of Chinese recipe of stasis removing and collaterals dredging on DN proteinuria, which is a multi-target, multi-channel effect, has been confirmed by our previous clinical observation and experimental research. But so far, there is no report on whether the effect has relevance with pydocyte filtration barrier related proteins. This project is guided by collaterals theory of Chinese medicine, aimed to explore the effective mechanism of Chinese recipe of stasis removing and collaterals dredging on DN proteinuria from the angle of expression of pydocyte filtration barrier related proteins, by establishing DN "stasis blocking collaterals" model, in order to explicit whether the target is the pydocyte filtration barrier. And meanwhile, we will also observe the targeting effect of different combinations of Chinese recipe of stasis removing and collaterals dredging on abnormal expression of filtration barrier related proteins of cultured pydocyte induced by high glucose, in order to provide scientific experimental basis for sifting best combinations of Chinese recipe of stasis removing and collaterals dredging in clinical treatment.

蛋白尿是糖尿病肾病（DN）早期和临床期的标志性症状，蛋白尿加重与DN进展可相互作用，有效控制蛋白尿对治疗DN具有重要意义，然而现代医学尚无靶向性降低尿蛋白的方法。足细胞滤过屏障损伤被认为是DN蛋白尿和肾小球硬化的关键因素。另一方面，中医界已普遍认可"瘀血阻络"是DN的主要病机，我们已成功建立DN瘀血阻络模型，并通过前期临床观察和实验研究证实，化瘀通络中药对DN蛋白尿具有确切疗效，为多靶点多途径的综合效应，但此疗效是否与足细胞滤过屏障相关蛋白有关，尚未见报道。本项目拟以中医络病理论为指导，建立DN瘀血阻络模型，从足细胞滤过屏障相关蛋白表达的角度，探索化瘀通络中药不同配伍对DN瘀血阻络模型蛋白尿的效应机制，以明确足细胞滤过屏障是否其作用靶点；并通过高糖诱导体外培养的足细胞滤过屏障相关蛋白的异常表达，观察化瘀通络中药不同配伍干预的靶向性作用，为临床化瘀通络法最佳药物组合的筛选提供科学的实验依据。

糖尿病肾病（DN）发生发展与足细胞滤过屏障相关蛋白的表达失常密切相关。DN的主要表现为蛋白尿和肾小球硬化，而足细胞损伤被认为是引起DN蛋白尿和肾小球硬化的关键因素。“瘀血阻络”是DN的主要病机，且化瘀通络中药及其拆方干预对DN模型有显著的肾保护作用，可明显降低DN模型动物的蛋白尿。本项目基于现代医学与中医学对DN不同角度的认识，旨在探索化瘀通络中药及其拆方改善DN蛋白尿的作用途径和靶点是否与足细胞滤过屏障有关。①瘀血阻络DN模型的建立：高糖高脂联合小剂量链脲佐菌素（STZ）腹腔注射制备大鼠糖尿病（DM）模型。16周末对肾皮质进行大体及光镜、电镜观察，与正常组比较，模型组（M组）肾脏体积增大，肾小球肿胀肥大，基底膜均质性增厚且结构杂乱，系膜基质增多，足突节段性融合，呈现DN典型病理改变；加之24h尿蛋白定量升高，表明DN模型制备成功。检测血液粘稠度的主要指标（血脂、血小板相关指标、血凝及其相关指标），均提示血液存在高粘、高凝状态，结合爪心、尾静脉紫暗的瘀血表现，与瘀血阻络中医证候特征相符，表明DN模型存在瘀血阻络证候。②化瘀通络中药对瘀血阻络DN模型的干预作用：对模型大鼠灌胃化瘀通络中药颗粒混悬液16周。M组大鼠肾小球体积增大，基底膜结构欠清晰，均匀弥漫增厚，系膜区增宽，肾小囊腔变窄，呈裂隙状；足突增宽，广泛融合。与M组相比，各治疗组均有不同程度改善。24h尿蛋白定量、糖化血红蛋白（HBA1c）、肾功能及肾脏指数均有显著改善，提示化瘀通络中药对DN具有肾保护作用，也反证了“瘀血阻络”证型的存在。比较大鼠外在瘀血表现及血液相关指标，均提示化瘀通络中药能改善DN大鼠瘀血阻络证候。③化瘀通络中药对DN瘀血阻络大鼠模型足细胞相关蛋白的影响：化瘀通络中药干预模型大鼠16w，留取肾皮质，检测足细胞各相关蛋白。M组Nephrin、NEPH1、Podocin、CD2AP、ZO-1、PCX和GLEPP-1等蛋白表达均下调，化瘀通络中药干预后各指标蛋白表达均上调，且其拆方对部分靶点（PCX）作用明显优于阳性对照药厄贝沙坦。④化瘀通络中药对高糖刺激下足细胞的干预作用：高糖干预可以下调PCX、GLEPP1、podocin、CD2AP的表达，加化瘀通络中药含药血清干预能上调PCX、GLEPP1、podocin、CD2AP的表达。