基于TGF-β1/Smads对腹膜间皮细胞EMT影响的黄芪抗腹膜纤维化机制研究

New results show that PMCs EMT can adjust peritoneal fibrosis. An important signaling pathway, TGF beta 1 / Smads, mediated the PMCs EMT and reduced the peritoneal fibrosis. Long-term clinical treatment shows that Astragalus could inhibit the antagonists of tissue fibrosis induced by TGF beta. We found astragalus can reduce the damage to PMCs by peritoneal dialysate with high concentration sugar. This application will focus and research the mechanism of Astragalus inhibiting tissue fibrosis by the TGF beta 1 / Smads pathway, including the peritoneal fibrosis rat model building and cultured HPMCs EMT, siRNA HPMCs of Smad2/3, Ⅰ and Ⅱ type TGF-β R. We will measure tissue cell morphology, EMT markers and the change of Smads protein after the Astragalus injection pretreatment. The most important, we will express Ⅰ and Ⅱ type TGF-β R in vitro and research the interaction of proteins and the ingredients in the astragalus root extract using NMR technology. This project will reveal the molecular mechanisms of the Astragalus treating peritoneal fibrosis and provide further theoretical and experimental basis for the clinical application of Astragalus.

最新研究发现PMCs EMT是腹膜纤维化的起始和可逆环节。而TGF-β1/Smads是介导PMCs EMT的重要信号通路，有证据表明阻断TGF-β1∕Smads可逆转PMCs EMT，已知黄芪能抑制TGF-β拮抗器官组织纤维化，减轻高糖腹透液对PMCs的损伤，减轻腹膜纤维化。本课题采用高糖腹透液构建腹膜纤维化大鼠、以Smad2基因转染大鼠，观察黄芪预处理后腹膜形态学、EMT标记物、TGF-β1、Smads信号蛋白的变化；在体外培养HPMCs、以Smad2/3、Ⅰ和Ⅱ型TGF-βR siRNA转染HPMCs，观察黄芪对TGF-β1诱导的HPMCs EMT标记物、Smads信号蛋白的影响，明确黄芪对腹膜纤维化及PMCsEMT的作用，通过上调与沉默TGF-β1/Smads信号蛋白及受体蛋白探讨黄芪的调控机制，并运用NMR技术体外鉴定可能对受体蛋白起作用的黄芪提取物成分，为该药临床新用提供依据。

摘要：研究发现PMCs EMT是腹膜纤维化的起始和可逆环节。而TGF-β1/Smads是介导PMCs EMT的重要信号通路，有证据表明阻断TGF-β1∕Smads可逆转PMCs EMT，已知黄芪有抑制TGF-β拮抗器官组织纤维化的作用，我们的前期研究发现黄芪能减轻高糖腹透液对腹膜的损伤，对PMCs具有保护作用。本课题选用腹膜纤维化大鼠和原代培养HPMCs为研究对象，并通过建立Smad7基因过表达及敲除PMCs细胞株，以明确黄芪对抗腹膜纤维化及PMCs EMT的作用，探讨黄芪对TGF-β1/Smads信号通路的调控机制。结果：① 黄芪能通过抑制单核细胞趋化蛋白（MCP-1）的表达，减少腹膜透析大鼠腹膜内巨噬细胞的募集，减轻腹膜TGF-β1的表达及TGF-β1/Smads信号通路的活化，对抗腹膜纤维化。② 黄芪能通过增强抑制性Smad7的表达，对TGF-β1/Smads信号通路起抑制作用，进而抑制TGF-β1诱导的PMCs EMT。