基于microRNA-155探讨半夏白术天麻汤改善肥胖性高血压选择性瘦素抵抗的机制研究

MicroRNA (miR) transcription and post-transcriptional regulation play important roles in the pathological process of obesity-related hypertension, in which miR-155 is one of the most important regulatory molecules in selective leptin resistance in obesity-related hypertension. Clinical application and preliminary experimental studies have shown that Banxia Baizhu Tianma decoction (BBT) demonstrates beneficial weight loss and antihypertensive efficacy with vague pharmacological mechanism. This study is planed to take rat aortic endothelial cells and high-fat diet-induced obesity-related hypertensive rats as models, observing the effect of BBT on the microRNA expression profile of ox-LDL -AngII induced aortic endothelial cells and screening the target microRNAs of BBT by microRNA chip; the rat aortic endothelial cells are transfected by liposome and high-fat diet-induced obesity-related hypertensive rats are transfected by recombinant lentiviral to overexpress or knockdown miR-155, both in vivo and in vitro, to observe the efficacy of BBT on MAPK/ERK, JAK2/STAT3 and NF-κB signaling pathways by regulating miR-155 and confirm the hypothesis that "miR-155 is the target of BBT". This study demonstrates important research value and exploration significance on clarifying the pharmacodynamic mechanism of classic Chinese herbal formula of BBT and interpreting the "multi-target effects" of traditional Chinese medicine.

microRNA(miR)的转录和转录后调节在肥胖性高血压发生发展中具有重要的作用，其中miR-155是肥胖性高血压选择性瘦素抵抗的重要调节分子之一。临床应用和前期研究显示半夏白术天麻汤具有明确的降压减重药效，但药理机制不明。本研究拟以大鼠主动脉内皮细胞和高脂饲料诱导的肥胖性高血压大鼠为模型，通过miR芯片检测，观察半夏白术天麻汤对ox-LDL-AngII联合诱导的内皮细胞miR表达谱的调节和影响，筛选有效靶miR；通过脂质体转染主动脉内皮细胞、重组慢病毒转染肥胖性高血压大鼠，过表达及敲减miR-155，体内、体外分别观察半夏白术天麻汤通过调节miR-155发挥调节MAPK/ERK、JAK2/STAT3、NF-κB信号通路的功效，探索验证假说“miR-155是半夏白术天麻汤的直接作用靶点”，对阐明半夏白术天麻汤的药理机制，揭示中药的“多靶点效应”具有重要的研究价值和探索意义。

本研究中，体外培养人主动脉内皮细胞(HAEC)，50µg/mL氧化低密度脂蛋白(ox-LDL)造模，半夏白术天麻汤可剂量依赖性(0.1-50mg/mL)地降低内皮细胞凋亡率、提高细胞迁移、改善细胞骨架、降低VE-钙粘蛋白分布，且无细胞毒性，2mg/mL为最佳浓度。经转录组联合microRNA测序，ox-LDL诱导了719个mRNA和90个miRNA差异表达(DE)；半夏白术天麻汤干预逆转了51.8%由ox-LDL所诱导的mRNAs (854 DE mRNA)和66.67%的miRNA (157 DE miRNA) 改变，其中差异最显著的是miR-217。双荧光素酶报告基因实验和siRNA敲减/过表达miR-217，证实ox-LDL通过下调miR-217靶向上调EGR1，导致内皮细胞的增殖抑制和IL-6、IL-1β、ICAM-1、TNF-α升高，造成炎性损伤；半夏白术天麻汤的药效在miRNA-217敲减后下降，而miRNA-217过表达则可增强其内皮保护作用。体内研究中，半夏白术天麻汤(17.8g/kg)灌胃给药8周后，显著降低了肥胖性高血压小鼠的体重、收缩压、舒张压、血清总胆固醇，减轻主动脉增生肥厚和主动脉根部脂质沉积；血浆非靶向代谢组学共鉴定出94种差异代谢物，显示半夏白术天麻汤下调了24种甘油磷脂和10种脂肪酰水平，主要调节鞘磷脂信号通路、逆行性内源性大麻素信号通路、鞘磷脂代谢、液体剪切应力和动脉粥样硬化，还参与调节FcγR介导的吞噬作用、NF-κB通路、Th1、Th2、Th17细胞分化、NK细胞介导的细胞毒性等炎症免疫过程。通过CRISPR/Cas9构建miR-217缺失小鼠，对比观察半夏白术天麻汤灌胃高脂饮食诱导的肥胖性高血压小鼠和miR-217缺失小鼠，血清IL-1β、IL-6、TNF-α、MCP-1下降，主动脉Irak1分布减少、Sirt1增加，主动脉JAK2、STAT3、NF-κB表达降低且SOCS3升高，其作用因miR-217缺失而降低，即半夏白术天麻汤通过激活miR-217抑制JAK2/STAT3、NF-κB信号通路，发挥降压减重药效；但并未因miR-217缺失而完全阻断，体现了中药的“多靶点效应”。本项目资助下，共发表论文12篇，其中SCI论文7篇，中文核心期刊论文3篇，科技核心期刊论文2篇，全部完成项目既定目标。