六味地黄汤调控PHD2/HIF-1α信号途径干预肾间质纤维化的机制研究

Chronic hypoxia is a key factor leading to renal interstitial fibrosis, but the specific mechanism is not clear. Hypoxia play a role mainly by inducible factor 1 alpha (HIF-1 alpha), HIF-1 alpha can regulate a variety of fibrosis factor, proline hydroxylase 2 (PHD2) is a key enzyme in the regulation of HIF-1 alpha. Six-ingredient Rehmannia Decoction is an effective prescription for treatment of chronic kidney disease, its pharmacological effects has antioxidation, hypoxia tolerance, we hypothesized that PHD2/HIF-1 alpha signal pathways involved in renal interstitial fibrosis, Six-ingredient Rehmannia Decoction may interfere with the regulation of PHD2/HIF-1 alpha signaling pathway in renal interstitial fibrosis. This topic used VHL-/- mice (over expression of HIF-1 alpha) to establish the animal model of 5/6 nephrectomy in vitro and to induce hypoxia in human renal tubular epithelial cells by CoCl2 in vivo, in vivo and in vitro experiments combined,using recombinant plasmids and gene interference technique, to observe the effect of fibrosis factor CTGF, PAI-1,TIMP-1 protein and gene expression after the HIF-1 alpha overexpression and deletion, to investigate the PHD2/HIF-1 alpha signaling pathway in the molecular mechanism of renal interstitial fibrosis and Six-ingredient Rehmannia Decoction intervention, offer the experimental basis for clinical prevention and treatment of chronic kidney disease.

慢性缺氧是导致肾间质纤维化的关键因素，然其具体作用机制不清。缺氧主要通过诱导因子1α（HIF-1α）发挥作用，HIF-1α可调控多种促纤维化因子，脯氨酸羟化酶2（PHD2）是调控HIF-1α的关键酶。六味地黄汤是临床治疗慢性肾脏病有效方剂，具有抗氧化、耐缺氧的药理作用，我们推测PHD2/HIF-1α信号途径参与肾间质纤维化，六味地黄汤可能通过调控PHD2/HIF-1α信号途径延缓肾间质纤维化。本课题拟用VHL-/-小鼠 (HIF-1α过表达)建立5/6肾切除动物模型和CoCl2体外诱导人肾小管上皮细胞HK-2缺氧，体内和体外实验相结合，利用重组质粒和基因干扰技术，观察HIF-1α过表达和缺失分别对促纤维化因子CTGF、PAI-1、TIMP-1蛋白和基因表达的影响，探讨PHD2/HIF-1α信号途径在肾间质纤维化中的作用及六味地黄汤干预的分子机制，为临床防治慢性肾脏病提供实验依据。

慢性肾脏病（chronic kidney disease，CKD）是世界范围内的公共健康问题。有效防治 CKD 已成为肾脏疾病研究领域的焦点之一。近年来的研究表明，慢性肾脏病的发病过程与肾小管间质存在的慢性缺氧关系密切。缺氧主要通过诱导因 子1α（HIF-1α）发挥作用，HIF-1α可调控多种促纤维化因子，脯氨酸羟化酶2（PHD2）是调 控HIF-1α的关键酶。中药复方六味地黄汤是临床治疗慢性肾脏病有效方剂，具有抗氧化、耐缺氧的药理作用，我们推测PHD2/HIF-1α信号途径参与肾间质纤维化，六味地黄汤可能通过调控PHD2/H IF-1α信号途径延缓肾间质纤维化。.本课题从动物实验和细胞培养两方面进行了研究。一方面，通过5/6肾切除制备慢性肾衰大鼠模型，检测24h尿蛋白、血尿素氮、血肌酐等生化指标观察了大鼠肾功能的变化，HE染色、Masson染色观察了大鼠肾组织形态结构的变化，免疫组织化学法、Western blot法和RT-PCR法观察了PHD2、HIF-1α、CTGF、PAI-1、TIMP-1等PHD2/HIF-1α信号途径相关因子的蛋白和基因表达变化。结果表明，六味地黄汤可上调PHD2的蛋白和基因表达，进而下调HIF-1α的表达，后者抑制CTGF、PAI-1、TIMP-1等促纤维化因子的表达，从而延缓肾间质纤维化。另一方面，通过CoCl2体外诱导人肾小管上皮细胞HK-2缺氧，观察PHD2/ HIF1α信号途径对肾间质纤维化的影响以及六味地黄汤含药血清干预作用。结果表明，六味地黄汤可能通过上调 PHD2 的表达，促进 HIF1α 的降解，后者下调 CTGF 的表达，从而 抑制肾间质纤维化。.本项目分析了PHD2/HIF-1α信号途径对肾间质纤维化的影响以及六味地黄汤对PHD2/H IF-1α信号途径的作用，从缺氧的角度阐明了六味地黄汤抗肾间质纤维化的作用机制。该研究为进一步认识慢性肾脏病的发病机制，为临床采用六味地黄汤治疗慢性肾脏病提供了理论和实验依据。