雷公藤甲素靶向调控TET介导的DNA去甲基化逆转足细胞EMT的机制研究

Epithelial mesenchymal transition (EMT) is usually considered as the main mechanism of podocyte injury, which is a key factor in the formation of glomerular proteinuria. In our previous study, we successfully induced the EMT model of podocyte by TGF-β1 in vitro, and found triptolide (TP) could significantly increase the expression of the key molecules of podocyte phenotype to reverse the EMT process. Further experiments show that TP can also increase the expression of TET double oxygenase (TET). Considering the TET protein is mainly involved in the demethylation process of DNA cytosine, we put forward the novel hypothesis of this topic: “TP may reverse the podocyte EMT process through targeting TET mediated DNA demethylation”. In order to verify this hypothesis, we attempt to study on the epigenetic molecular mechanism of TP intervention in podocyte injury by both in vivo and in vitro experiments. To elucidate the effect of TP on podocyte EMT, both the regulation of TP on TET molecules and the effect of TET on methylation status of medium CpG density region in the promoter of key podocyte phenotype molecules are sought to be analyzed. Through these investigations, we hope to uncover the molecule mechanism of TP in alleviating podocyte associated glomerular diseases. In addition, we wish it will provide novel intervention targets and effective measures of podocyte for the prevention and treatment of chronic kidney disease.

足细胞发生上皮-间充质转分化（EMT）是足细胞损伤的主要机制，而足细胞损伤是肾小球性蛋白尿形成的关键因素。在前期研究中，我们采用TGF-β1成功诱导了体外足细胞EMT模型，并发现雷公藤甲素（TP）可显著上调足细胞标志分子的表达从而逆转足细胞EMT过程，进一步的实验显示TP可上调TET双加氧酶（TET）的表达。鉴于TET蛋白主要参与DNA胞嘧啶的去甲基化过程，本项目提出“TP通过靶向调控TET介导的DNA去甲基化从而逆转足细胞的EMT过程”的假说，并拟通过体内外实验开展TP干预足细胞EMT的分子机制研究。通过揭示TP对TET蛋白的调控以及TET进而对足细胞表型关键分子启动子中等CpG密度区域甲基化状态的调节，阐明TET在TP逆转足细胞EMT过程中的作用。通过项目的实施，最终揭示TP缓解足细胞相关肾小球疾病的分子机理，为慢性肾脏疾病的防治提供新型干预靶点及有效措施。

足细胞上皮-间质转分化(EMT)是多种慢性肾脏疾病蛋白尿产生及疾病进展的重要病理机制。抑制足细胞EMT已成为慢性肾脏疾病防治研究的热点。临床及体外证据均表明雷公藤甲素（TP）可改善足细胞病变，但其具体疗效机制尚不明确。既往文献显示TET双加氧酶不仅在自然分化过程中具有重要作用，还参与一些诱导分化过程，但其介导的DNA去甲基化是否在足细胞EMT过程中发挥作用尚不清楚。在本项目中，我们采用体外TGF-β1诱导的足细胞EMT模型结合体内单侧肾切除联合双次阿霉素尾静脉注射法构建的大鼠局灶节段性肾小球硬化模型揭示TP保护足细胞的潜在分子机制。我们发现TET1-3在成熟足细胞中均有表达，TET1和TET3的表达较低，TET2的表达最高。在TGF-β1诱导的足细胞EMT模型中，TET2表达下调，TP干预可上调足细胞中内源性TET2的表达。亚硫酸盐扩增子高通量测序（BSAS）结果显示TP可改变足细胞裂孔隔膜主要标记分子NEPH1和Nephrin启动子中等密度CpG区域特定位点的甲基化状态。借助shRNA慢病毒技术敲低TET2会导致NEPH1和Nephrin启动子具有较高的甲基化状态从而在TP干预的情况下也不能恢复正常的表达水平。体内，我们发现TP可保护FSGS大鼠足细胞损伤和增加TET2的表达。以上结果表明TET2介导的DNA去甲基化可能在一定程度上参与了足细胞损伤，为进一步揭示TP缓解足细胞相关肾小球疾病的新的分子机制有一定的帮助，为慢性肾脏疾病的防治提供新型足细胞干预靶点及有效措施。