基于调控Wnt/β-catenin阻抑腹膜间皮细胞EMT的黄芪抗腹膜纤维化机制研究

It is believed that epithelial to mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) plays a key role in the early stage of peritoneal dialysis (PD) associated peritoneal fibrosis (PF). It has been proved by some studies that astragalus is able to stop EMT of PMCs by regulating TGF-β1/Smads signal cascade. Our previous studies have found that the classical pathway TGF-β1/Smads is partly involved in the process of astragalus stopping EMT of PMCs, and astragalus could not only significantly increase the expression of Smad7 which is the inhibitory protein of β-Catenin , but also decrease the expression of Vimentin which is the main marker protein of β-Catenin able to induce EMT. The result reminds us that Wnt/β-Catenin is also involved in the process of astragalus stopping EMT of PMCs. It is planned in the study to observe the functions of β- Catenin in EMT of PMCs and the intervention result by astragalus in virtue of PF rats induced by high glucose peritoneal dialysis solution and HPMCs EMT model induced by TGF-β1,meanwhile, observe the EMT marker protein，β-Catenin activity and the change of its downstream pathway after intervention by astragalus with the help of Smad7 knock-out and over-expressed animal and cell model in order to clarify the exact signaling pathways activated in the intervention by astragalus on PMCs EMT. The study may supply a new therapy and research thinking for prevention and treatment of PD associated peritoneal fibrosis.

PMCs EMT是形成PD相关PF早期病变的关键环节。有研究显示黄芪能调控TGF-β/smads阻抑PMCs发生EMT。我们前期研究发现TGF-β/smads这一经典途径只部分参与了黄芪阻抑PMCs EMT的过程，并发现黄芪能显著上调β-Catenin的抑制蛋白（Smad7），下调β-catenin诱导EMT的主要标记蛋白（Vimentin），提示Wnt/β-Catenin也参与黄芪阻抑PMCs EMT的过程。本课题借助高糖腹透液诱导的PF大鼠模型与TGF-β1诱导的HPMCs EMT模型，观察β-Catenin在PMCs EMT中的作用及黄芪的干预效果；利用敲除和过表达Smad7的动物与细胞模型，观察黄芪处理后EMT标记蛋白、β-Catenin活性及其下游蛋白的改变，阐明黄芪干预PMCs EMT的确切信号通路，为PD相关PF的防治提供新的方法和研究思路。

PMCs发生EMT是腹膜纤维化的起始与可逆环节，多条信号传导途径参与调控这一过程。我们前期研究证实了黄芪作用于Smad7阻断TGF-β1/Smads信号传导从而拮抗腹膜纤维化的机制，此外有证据表明β-catenin与Smads蛋白存在交互作用。本研究的第一部分拟检测PD患者腹透流出液中的TGF-β1、β-catenin表达水平，为深入探讨Wnt/β-catenin信号传导调控EMT机制、探索腹膜纤维化干预手段提供新的临床证据；第二部分分别运用体内/体外模型证明Wnt/β-catenin信号通路参与调控PMCs EMT，并阐明黄芪对通路介导的EMT的调控作用，深入探讨黄芪抗腹膜纤维化机制；第三部分研究β-catenin与Smads蛋白的交互关系，并运用转基因细胞/大鼠模型探讨黄芪作用Smad7抑制β-catenin的活性的分子机制。结果：（1）PD患者腹透流出液中β-catenin、TGF-β1水平升高，其表达与透析龄相关；（2）Wnt/β-catenin信号在高糖腹透液诱导的PF大鼠中被激活，黄芪能抑制其介导的大鼠腹膜EMT及纤维化；（3）Wnt/β-catenin信号通路调控TGF-β1介导的PMCs EMT，黄芪通过调控该信号途径阻抑PMCs EMT过程；（4）黄芪调控β-catenin通路的机制在于抑制TGF-β1介导的GSK-3β磷酸化，恢复胞浆中GSK-3β与β-catenin结合，促进β-catenin磷酸化，降低游离β-catenin蛋白水平；（5）Smad7能与β-catenin结合，而黄芪通过上调Smad7表达，抑制β-catenin的活性，促进其降解，从而阻抑EMT及腹膜纤维化。本研究为防治腹膜透析相关腹膜纤维化提供了实验依据。