从miR-483-3p/高血压心脏重构相关靶基因研究清眩降压汤的药效作用机制

From the molecular marker of miR-483-3p of hypertension of hyperactivity of liver Yang, and it’s target genes AGT, ACE-1, ACE-2, AT2R from Renin angiotensin system(RAAS), IGF-1, MAPK, TGF-β, MMPs from IGF-1/MAPK and TGF-β/smad signaling pathway, construct lentiviral expression vector, infecting the cardiac myocytes and fibroblasts stimulated by AngII. At the same time, miR-483-3p lentiviral vectors are injected into spontaneous hypertensive rat (SHR) by the tail vein and intragastric administration with Qingxuanjiangya Decoction, using rat tail blood pressure meter, small animal ultrasound, laser confocal microscopy, HE staining, phalloidin staining, IHC, Q-PCR, western blot ,etc to analysis the miR-483-3p and it’s target genes expression, blood pressure level, systolic and diastolic function of heart, cardiac pathology, cell morphology, and the expression of related proteins, then to analysis mechanism of Qingxuanjiangya Decoction to control blood pressure and inhibit cardiac remodeling systematically, lay a foundation of theory and experiment for the prevention and treatment of hypertension of hyperactivity of liver Yang.

拟以高血压肝阳上亢证分子标志miR-483-3p为支点，其调控的肾素血管紧张素系统（RAAS）的关键基因AGT、ACE-1、ACE-2、AT2R及其下游IGF-1、MAPK、TGF-β、MMPs等IGF-1/MAPK、TGF-β/smad信号通路的靶基因为切入点，构建慢病毒表达载体，感染AngII刺激心肌细胞和成纤维细胞模拟的心脏重构细胞模型。同时鼠尾静脉注射miR-483-3p慢病毒表达载体，感染自发性高血压大鼠，以平肝潜阳中药——清眩降压汤干预，鼠尾血压计、小动物超声、激光共聚焦显微镜、HE染色、鬼笔环肽染色、IHC、Q-PCR、western blot等方法，分析miR-483-3p的表达及其对靶基因的调控作用和由此产生的血压、心脏舒缩功能、心脏病理、细胞骨架形态、及相关蛋白的表达变化，系统分析清眩降压汤控制血压、抑制心脏重构的作用机制，为高血压肝阳上亢证的防治奠定理论和实验基础。

课题组前期研究发现，高血压患者血清miR-483-3p表达显著升高，拟构建miR-483-3p敲除自发性高血压大鼠模型，探讨miR-483-3p或清达颗粒对高血压心脏重构的影响及其相关作用机制，研究清达颗粒是否通过调控miRNA的表达发挥其药效作用。实验证明，miR-483-3p或清达颗粒可以降低血压、改善自发性高血压大鼠心脏功能、腹主动脉脉冲波传导速度、心脏重量指数等，明显减轻SHR的心肌组织结构紊乱程度及炎症细胞的浸润，明显抑制SHR中ACE2的表达水平以及MAPK通路的活化，清达颗粒可以降低自发性高血压大鼠血清miR-483-3p表达水平。miR-483-3p敲除可缓解高血压心脏重构；清达颗粒可能通过调控MAPK通路减轻高血压大鼠心脏重构。