黄葵素干预循环外泌体miRNAs调控糖尿病肾病肾小管上皮细胞EMT的机制研究

Snail1-mediated renal tubular epithelial cell mesenchymal transition (EMT) is a key component of renal interstitial fibrosis in diabetic kidney disease (DKD). Snail1 is targeting regulated by circulating exosomal miRNAs. It has been found that flavonoids compounds can intervene the expression of circulating exosomal miRNAs. Therefore, we propose the research hypothesis that total flavone of Abelmoschus Manihot(TFA) may play a protective role in kidney via circulating exosomal miRNAs/Snail1 pathway. DKD rat model and NRK-52E cell EMT model will be established, observing the effect of TFA on the expression of circulating exosomal miRNAs, Snail1 and renal tubular EMT markers through the techniques such as gene chip, cell transfection, qPCR and Western blot, confirming the molecular mechanism that TFA can down-regulate the level of Snail1 and delay renal tubular EMT in DKD through intervening the expression of circulating exosomal miRNAs. The implementation of this project provides a new thinking of the treatment in renal interstitial fibrosis of DKD.

Snail1介导的肾小管上皮细胞-间充质转化（EMT）是糖尿病肾病（DKD）肾间质纤维化的关键环节，Snail1受到循环外泌体miRNAs的靶向调控，而黄酮类化合物能够干预循环外泌体miRNAs的表达。申请人前期研究发现，黄葵素（黄蜀葵花总黄酮）可以有效减少细胞外基质的沉积，延缓DKD肾间质纤维化，因此，我们提出黄葵素可能通过干预循环外泌体miRNAs的表达，改善Snail1介导的肾小管上皮细胞EMT过程，发挥肾保护作用。本项目建立DKD大鼠模型及NRK-52E细胞EMT模型，通过基因芯片、细胞转染、qPCR、Western blot等技术，观察黄葵素对血清外泌体miRNA、Snail1及EMT标志蛋白的影响，证实黄葵素通过改善循环外泌体miRNAs的表达，下调Snail1水平，延缓DKD肾小管上皮细胞EMT的分子机制。该项目的实施，为黄葵素治疗DKD肾间质纤维化提供新思路。

背景：Snail1受血浆外泌体miRNA的靶向调控，其介导的肾小管上皮细胞EMT是DKD肾间质纤维化的关键环节。黄葵素（TFA）可改善肾间质纤维化，我们推断TFA可能通过干预血浆外泌体miRNA表达，延缓Snail1介导的肾小管上皮细胞EMT进程。.研究内容：建立DKD小鼠模型及TCMK-1细胞EMT模型，通过miRNA测序、细胞转染、RT-qPCR、Western blot等技术，观察TFA干预对血浆外泌体miRNA、Snail1、E-cadherin和α-SMA表达的影响。.关键数据：db/db小鼠血浆外泌体miR-296-5p低表达、miR-34a高表达（P＜0.05; P＜0.05），双荧光素酶实验及细胞转染技术证实miR-296-5p负调控Snail1，miR-34a正调控Snail1。TFA单体金丝桃苷（HYP）干预后，db/db小鼠肾组织miR-296-5p表达上调，miR-34a表达下调（P＜0.05; P＜0.05），Snail1 mRNA和蛋白表达下降（P＜0.01; P＜0.05），E-cadherin mRNA和蛋白表达上升（P＜0.01; P＜0.01），α-SMA mRNA及蛋白表达下降（P＜0.05; P＜0.01）。db/db小鼠血浆外泌体干预后，TGF-β1诱导的TCMK-1细胞miR-34a表达上调（P＜0.05），Snail1 mRNA和蛋白表达上升（P＜0.05），E-cadherin mRNA表达下降（P＜0.05）。不同剂量TFA、HYP干预后，TGF-β1诱导的TCMK-1细胞miR-296-5p表达上调，miR-34a表达下调（P＜0.05; P＜0.05），Snail1 mRNA和蛋白表达下降（P＜0.05），E-cadherin mRNA表达上升（P＜0.05），α-SMA mRNA及蛋白表达下降（P＜0.05）。.重要结果：TFA通过上调血浆外泌体miR-296-5p表达或下调血浆外泌体miR-34a表达，延缓Snail1介导的肾小管上皮细胞EMT进程，改善DKD肾间质纤维化。.科学意义：阐明TFA干预血浆外泌体miRNA改善DKD肾间质纤维化的分子机制，为TFA的中药现代化进程提供新证据。血浆外泌体miRNA是不同细胞间基因调控的新途径，为探索DKD发病机制及治疗手段提供新思路。