基于“毒损肾络”理论研究TGF-β信号通路在足细胞EMT的肾小球硬化中的响应及稳心草干预作用

The incidence of Chronic kidney disease (CKD) is increasing with years in recent years as a big threaten to human health, and brings a heavy burden to the family and the society because of its costly treatment fee and the fact that many people have to receive the replacement therapy. As we know the change of the profile of glomerular sclerosis mechanism and the clinical practice that Chinese traditional medicine could delay end-stage renal disease, and according to the theory that turbid bane causes kidney disease in our previous studies. The present study aims to detect the involvement of TGF-β signaling in the glomerular sclerosis of epithelial-mesenchymal transition (EMT) and how metacentric grass flavones affect this process. Glomerular podocytes cultivated in vitro and adriamycin administrated rats were used as the experimental objects. The techniques of quantitative real-time PCR, western blotting, cyto-immunofluorescene and confocal microscopy were carried out to investigate the dynamic expression characteristics of the key signal molecular of the TGF-β signaling mediating podocytes EMT as the prototypic inducer. Metacentric grass flavones and decocta were administered and correlation analysis was used to disclose the mechanism of glomerular sclerosis occurrence and the scientific implications of the method that "invigorating the circulation blood, infusing the turbid bane and detoxification" in the perspective that TGF-β signaling mediated the EMT, and also lay a theoretical support for clinical practice and develop a better Chinese traditional medicine for prevention and treatment of CKD.

目前慢性肾脏病发病率呈逐年增长趋势,接受替代治疗者甚多,治疗费用昂贵,给社会和家庭带来沉重的负担,严重威胁着人类健康。本研究基于中医药干预可有效延缓终末期肾病发生的临床实践和肾小球硬化病机变化规律，以"毒损肾络"理论为基础结合前期工作，建立"TGF-β通路在足细胞间充质转分化(EMT)的肾小球硬化中的响应及稳心草及其黄酮干预的可能作用"工作假说，以体外培养的足细胞及阿霉素大鼠为研究对象，采用QRT-PCR、WB、细胞免疫荧光、共聚焦等技术检测足细胞EMT的证据与TGF-β通路中关键信号分子的动态表达规律，在此基础上进一步观察足细胞间充质分化与TGF-β信号通路之间的关系。并应用以稳心草及其黄酮进行干预，通过相关性分析，拟从TGF-β通路介导足细胞EMT角度揭示肾小球硬化发生机制以及"活血消痰、化浊解毒"法对其干预的科学内涵。并为该治法的临床应用和开发防治慢性肾脏病的优势中药提供理论支撑。

慢性肾脏病的发病率在我国和全球呈逐年增高的趋势，成为全人类面临的重大公共卫生问题，慢性肾脏病的防治任重道远。基于中医药干预可有效延缓终末期肾病发生的临床实践和肾小球硬化病及变化规律，以“毒损肾络”理论为基础结合前期工作，建立了“TGF-β信号通路在足细胞间充质转分化（EMT）的肾小球硬化中的响应及稳心草及其黄酮干预的可能作用”的工作假说。以体外培养足细胞及阿霉素大鼠为研究对象，采用实时荧光定量PCR，免疫荧光，免疫印迹，等方法检测阿霉素致肾小球硬化模型大鼠肾脏组织和TGF-β1诱导正常足细胞EMT及稳心草黄酮类化合物槲皮素分别对肾小球硬化模型大鼠肾脏和TGF-β1诱导的足细胞中Nephrin、P-cadherin、WT-1等相关分子的表达特性的变化以及足细胞转化过程中TGF-β通路关键信号分子TGF-βⅡ型受体、Smad2、smad3、Smad4、Smad7、TGF-βⅠ型受体、糖原含酶激酶3β(GSK-β)、β-连环蛋白(β-catenin)的表达变化。结果表明:特定浓度的TGF-β1诱导足细胞EMT和阿霉素致肾小球硬化模型大鼠肾脏组织运用上述实验方法检测后发现Nephrin、ZO-1、P-cadherin、WT-1 、smad-7、β-cadherin表达下调，α-SMA、FSP-1、Vimentin、TGFβ1、Smad2/3、Smad4、GSK3β、、TGFBR1、TGFBR2表达上调；特定浓度的稳心草黄酮类化合物槲皮素干预TGF-β1诱导的足细胞和阿霉素致肾小球硬化模型大鼠肾脏组织用上述实验方法检测后发现Nephrin、ZO-1、P-cadherin、WT-1 、smad-7、β-cadherin表达上调，α-SMA、FSP-1、Vimentin、Smad2/3、Smad4、GSK3β、、TGFBR1、TGFBR2表达下调。结论：1.1TGF-β1能够诱导足细胞形态学改变，促进足细胞EMT的发生发展。1.2Qu能够抑制TGF-β1诱导的足细胞EMT，其作用机制可能与抑制TGF-β信号转导通路中的相关信号分子、维持足细胞生理结构的完整性从而保护肾小球滤过屏障有关。1.3阿霉素能够引起大鼠肾功能受损，肾组织超微结构受损，肾小球足细胞受损，加速肾小球硬化发生发展的进程。1.4槲皮素可以改善大鼠的改善肾功，减少肾小球硬化程度。