基于microRNA-21和/或microRNA-29探讨黄芪-丹参药对改善高血压肾损害的机制研究

The deficiency of Qi and blood stasis is one of the main pathogenesis of hypertensive nephropathy. The double-drugs compatibility of Salvia miltiorrhiza and Astragalus membranaceus, usually gain desirable efficacy in the treatment of hypertensive nephropathy. The preliminary studies have confirmed its pharmacological mechanism involving multiple signal transduction pathways, in which microRNAs play an important regulatory role. This study is planned to take spontaneously hypertensive rats (SHR) and renal artery endothelial cells as models, screening the target microRNAs of Calycosin - TanshinoneⅡA (C-T, effective monomers of Salvia miltiorrhiza and Astragalus membranaceus) by microRNA chip; the renal artery endothelial cells are transfected by liposome and SHRs are transfected by recombinant lentiviral to overexpress or knockdown of miR-21 (or miR-29b), both in vivo and in vitro, to observe the efficacy of C-T on RhoA/ROCK, FAK/PI3K/AKT, SMAD3/TGF-β signaling pathways by regulating miR-21 and / or miR-29 and to confirm the hypothesis that "miR-21 (or miR-29b) is the target of C-T. This study demonstrates important research value and exploration significance on clarifying the pharmacodynamic mechanism of classic Chinese herbal formula of Buqihuoxue of Salvia miltiorrhiza - Astragalus membranaceus and interpreting the "multi-target effects" of traditional Chinese medicine.

气虚血瘀是高血压肾损害的主要病机之一，补气活血中药黄芪-丹参可明显延缓高血压肾损害的进程，前期研究证实其药理机制涉及多个信号转导通路，microRNA在其中发挥重要的调节作用。本研究拟以自发性高血压大鼠（SHR）和大鼠肾动脉内皮细胞为模型，通过microRNA芯片筛选毛蕊异黄酮-丹参酮ⅡA（黄芪、丹参有效单体）药对的靶microRNA；通过脂质体转染肾动脉内皮细胞、重组慢病毒转染SHR，过表达及敲减miR-21（或miR-29b），体内、体外分别观察毛蕊异黄酮-丹参酮ⅡA药对通过调节miR-21和/或miR-29发挥调节RhoA/ROCK、FAK/PI3K/AKT、SMAD3/TGF-β信号通路的功效，探索验证假说“miR-21（或miR-29b）是毛蕊异黄酮-丹参酮ⅡA药对的直接作用靶点”，对阐明补气活血类中药黄芪-丹参的作用机制，揭示中药的“多靶点效应”具有重要的研究价值和探索意义。

气虚血瘀是高血压肾损害的主要病机之一，补气活血药对黄芪-丹参可明显延缓高血压肾损害的进程。本研究从改善血管紧张素Ⅱ（AngⅡ）诱导的大鼠肾动脉内皮细胞（RRAECs）损伤角度，毛蕊异黄酮-丹参酮ⅡA（黄芪、丹参有效单体药对）配比（最佳浓度为3mg/L+3mg/L）可显著降低细胞凋亡率、促进细胞迁移、减少活性氧（ROS）生成、抑制线粒体自噬、升高线粒体膜电位，且二者配伍的功效优于两单体药物各自应用，其药效与缬沙坦相当，转录组测序联合小RNA测序共筛选出424个差异mRNA和77个差异miRNA，逆转了86个mRNA及23个miRNA的改变，其中差异最显著的是miRNA-200c-3p。ZEB2是miRNA-200c-3p的直接靶基因，AngⅡ通过上调miRNA-200c-3p靶向负调控ZEB2，从而抑制RRAEC增殖和迁移；药对的内皮保护机制，与下调miRNA-200c-3p上调ZEB2表达相关。体内研究中，黄芪-丹参药对灌胃后，共596个mRNA和54个miRNA差异表达，逆转了自发性高血压大鼠（SHR）肾脏211个mRNA和21个miRNA的变化，差异基因富集于PPAR信号通路、轴突导向、炎症介质调节TRP通道、IL-17信号通路等。通过小鼠尾静脉注射AAV-miR-466b-5P，在小鼠肾脏过表达miRNA-466后，尿中β2-MG、NAG、mALB及血清中Cys-C、AngII、CRP水平显著升高，肾脏出现形态学改变和胶原沉积等肾脏损伤的表现，基本复制了高血压肾损害的肾脏表现，且miR-466b-5P过表达可显著降低LEFTY，上调TGF-β、TGFB1、HAS、CD44的表达；黄芪-丹参药对的体内肾脏保护机制，与下调miR-466b-5P、上调LEFTY表达，抑制TGF-β通路激活密切相关。本项目资助下，共发表论文14篇，其中SCI论文4篇，中文核心期刊论文9篇，全部完成项目既定目标。