牛蒡子苷基于miR-21调控PTEN-c-FLIP通路抑制内皮细胞凋亡防治早期糖尿病视网膜病变

It is identified that retinal endothelial cells (RECs) apoptosis, which induced blood–retinal barrier (BRB) damage, is critical in the early stage of diabetic retinopathy (DR), therefore, new treatments for this mechanism are needed. In our previous study, arctiin improved DR and reduced RECs apoptosis obviously, and we would like to explore the mechanism in this project. As reported, arctiin unregulated the expression miR-21, which associate with cell apoptosis via the PTEN pathway. PTEN negatively regulate the PI3K/Akt pathway, which activity c-FLIP and induce resistance to apoptosis. As our preliminary study showed, the anti-apoptosis effect of arctiin could be suppressed by PI3K/Akt inhibitor, which reduced c-FLIP expression in endothelial cells, therefore, it is well-founded to speculate arctiin regulate the PTEN-PI3K/Akt-c-FLIP pathway and reduce RECs apoptosis through unregulated miR-21. RECs and SD rats interfered with the expression of miR-21 and its downstream gene would be used as models to confirm the hypothesis. It is the first time to explore the pathogenetic mechanism of DR involves the “miRNA-RECs apoptosis” pathway, and it would provide a theoretical basis for the clinical application of arctiin.

内皮细胞凋亡导致的血视网膜屏障破坏是糖尿病视网膜病变（DR）早期的重要病理基础，但目前尚无针对该机制的DR有效治疗药物。申请者前期研究表明中药提取物牛蒡子苷对DR有明显改善作用，并减少视网膜内皮细胞凋亡，本项目旨在阐明其具体分子作用机制。最新研究报道牛蒡子苷可以上调miR-21，而miR-21可以通过抑制PTEN调控PI3K/Akt-c-FLIP凋亡通路。我们的预实验结果显示PI3K/Akt抑制剂可以阻断牛蒡子苷对内皮细胞的保护作用，并减少内皮细胞c-FLIP表达。由此，我们拟构建干预miR-21及其下游基因表达的细胞及动物模型，验证“牛蒡子苷通过上调miR-21进而调控PTEN-PI3K/Akt-c-FLIP通路，抑制视网膜内皮细胞凋亡，治疗早期DR”这一科学假设。本项目将首次从“miRNA-视网膜内皮细胞凋亡”这一全新角度，探索牛蒡子苷治疗DR的分子作用机制，为其临床应用提供理论依据。

糖尿病视网膜病变（DR）是不同类型糖尿病的微血管并发症之一，为工作年龄人群的主要致盲原因。目前临床上DR治疗效果十分有限，且常常伴有严重的副反应。因此，DR早期防治对控制病情发展、降低致盲率尤为重要。课题组前期发现，中药提取物牛蒡子苷具有很好的视网膜保护作用，可能成为早期DR临床治疗药物。然而，阐明牛蒡子苷保护视网膜的分子作用机制将成为其应用于临床前必须解决的问题。本项目旨在阐明其具体分子作用机制。在本项目中，我们研究了牛蒡子素对人视网膜毛细血管的影响内皮细胞(HRCEC)以及牛蒡子素如何抑制高糖诱导内皮细胞的增殖。结果表明，牛蒡子素可降低高糖诱导的HRCECs通过诱导细胞周期阻滞在G0/G1，以剂量依赖的方式增殖。血管形成试验和免疫荧光染色表明牛蒡子苷高糖诱导的HRCECs通过下调VEGF的表达，以剂量依赖的方式一直血管形成。机制研究表明ROCK1/PTEN/PI3K/Akt信号通路在细胞凋亡中起重要作用。此外，对HRCECs进行预孵育Y-27632可减弱牛蒡子苷对细胞周期阻滞、细胞增殖和管状细胞形成抑制。Y-27632也逆转了PTEN的激活，即失活/PI3K/Akt去磷酸化与VEGF下调。综上所述,牛蒡子素可抑制高糖诱导的HRCECs的增殖，通过激活ROCK1和PTEN，失活PI3K和Akt，导致VEGF下调，抑制内皮细胞增殖。