基于lncRNA-p21/p53研究钩藤莱菔子组分药对干预高血压内皮祖细胞自噬的作用机制

Endothelial progenitor cells (EPCs) play an important role in the repair of vascular injury, and enhancing autophagy can antagonize the EPCs damage in the pathological process of hypertension. LncRNA-p21 is an important regulator of autophagy. We have been focusing on the study of vascular endothelial protection and vascular remodeling. Previous studies have demonstrated that components of uncaria and semen raphani have a good effect on reducing blood pressure, protecting blood vessels and enhancing autophagy, nevertheless, the specific mechanism is unknown. In this study, according to EPCs damage in hypertension, we will combine the molecular biology and epigenetics and take regulation of autophagy by lncRNA as the breakthrough point, obversing the target lncRNA of components of uncaria and semen raphani by high-throughput sequencing which regulat autophagy to antagonize AngII-induced EPCs damage. LncRNA-p21 was respectively overexpressed and interfered, both in vivo and in vitro, exploring the mechanism of components in targeting intervention lncRNA-p21 to enhance autophagy and antagonize EPCs damage. LncRNA expression profile and peripheral blood EPCs in hypertensive patients were also detected. This study aims to further interpret the mechanism of multi pathways and multi targets of anti-hypertension traditional Chinese medicine, and provide the basis for traditional Chinese medicine in the treatment of hypertension and target organ protection.

内皮祖细胞（EPCs）参与血管损伤的修复，而增强自噬能够拮抗高血压进程中EPCs的损伤，lncRNA-p21是自噬的重要调节分子。申请人专注高血压血管内皮保护与血管重构，前期研究发现钩藤莱菔子组分药对具有较好的降压、保护血管和增强自噬的作用，但更深入的药理机制不明。本研究采用分子生物学和表观遗传学的思路，以高血压EPCs损伤为切入点，从lncRNA调控自噬的视角，基于高通量测序技术构建lncRNA表达谱，通过AngII体外诱导的EPCs损伤模型明确钩藤莱菔子组分药对调控自噬以拮抗EPCs损伤的靶lncRNA；体外、体内分别过表达及敲减lncRNA-p21，探晰该组分药对靶向干预lncRNA-p21以增强自噬，从而拮抗EPCs损伤的作用机制，并通过检测患者lncRNA表达谱及外周血EPCs进行最终验证，以期深入解读中药多通路多靶点抗高血压的机制，为中医药抗高血压、保护血管靶器官提供依据。

本项目从高血压内皮祖细胞损伤的视角，探究抗高血压中药钩藤莱菔子组分药对（钩-莱组分药对）的有效靶lncRNA，揭示组分药对靶向干预lncRNA调控细胞自噬拮抗内皮祖细胞损伤的自我保护机制，揭示中医药多通路、多靶点的内涵本质。研究结果（一）建立原代大鼠骨髓源EPCs的体外培养方法，以AngII为干预因素，发现钩-莱组分药对的主要药效成分钩藤总生物碱和莱菔子水溶性生物碱能够提高细胞自噬水平从而改善细胞功能，拮抗EPCs损伤；同时，采用高通量测序技术和基于碱基配对的lncRNA-mRNA互作分析筛选鉴定lncRNA序列表达谱，筛选出钩-莱组分药对的靶lncRNA——lncRNA-p21，并证实lncRNA-p21是调控细胞自噬的关键靶点。研究结果（二）基于lncRNA-p21/p53通路揭示钩-莱组分药对上调细胞自噬修复EPCs细胞功能的作用靶点，发现钩-莱组分药对通过上调lncRNA-p21，促进p53的转录活性，提高AMPK磷酸化和p-TSC2表达，抑制mTOR磷酸化和激活，从而提高细胞自噬水平而修复AngII诱导的EPCs细胞功能障碍。研究结果（三）以SHR大鼠为研究对象，在体验证钩-莱组分药对作用机制及靶点，发现钩-莱组分药对通过调控lncRNA-p21/p53激活SESN2/AMPK/TSC2通路，从而增强SHR大鼠EPCs自噬，拮抗EPCs损伤，改善EPCs功能，促进SHR大鼠内皮损伤修复。研究结果（四）以藤菔降压片干预高血压病肝阳上亢证或高血压病阴虚阳亢证患者，发现藤菔降压片均能有效降压及改善临床症状，具有一定的有效性和安全性，且能升高高血压患者外周血EPCs水平，提高外周血EPCs增殖、黏附、迁移、成管能力，改善细胞功能，拮抗细胞损伤。