基于SIRT1/PGC-1α/线粒体轴探讨活血潜阳祛痰方干预肥胖高血压心肌肥厚的机制

Obesity Hypertension (OBH) myocardial hypertrophy significantly increased the incidence of malignant cardiovascular events,which needed to be resolved.The abnormal energy metabolism mediated by SIRT1/PGC-1α/mitochondrial axis was one of the key mechanisms. Our research team found that "Blood stasis-Yang kang-Phlegm" were the main pathological factors of OBH myocardial hypertrophy. Huoxue Qianyang Qutan formula could reduce the degree of OBH myocardial hypertrophy, the Ang II-endoplasmic reticulum stress signaling pathway was one of the main mechanisms. The activation of RAAS、endoplasmic reticulum stress were involved in the regulation of mitochondrial function. On the basis of the previous results, OBH rats and neonatal rat cardiomyocytes are selected as the experimental objects, using cell culture, RT-PCR, Western Blot:(1)To observe the mitochondrial function mediated by SIRT1/PGC-1α signaling pathway in the myocardial hypertrophy of OBH rats; (2)To investigate the effects of Ang II on the mitochondrial function mediated by SIRT1/PGC-1α signaling pathway in OBH rats; (3) To explore the effects of Huoxue Qianyang Qutan formula on the mentioned systems above. The results of this study are helpful to clarify the targets of Huoxue Qianyang Qutan formula on OBH myocardial hypertrophy,and provide a microscopic basis for the application of Chinese medicine.

肥胖高血压（OBH）心肌肥厚显著增加恶性心血管事件的发生，是亟待解决的治疗难点。 SIRT1/PGC-1α线粒体轴介导的能量代谢异常为其关键机制之一。课题组前期研究发现“血瘀、阳亢、痰浊”是OBH心肌肥厚的重要病理因素，活血潜阳祛痰组方可减轻左室肥厚，并与改善AngII-内质网应激有关。而RAAS激活、内质网应激均参与线粒体功能调节。本项目旨在以OBH大鼠及乳鼠心肌细胞为实验对象，采用原代细胞培养、RT-PCR、Western Blot等技术：①观察OBH大鼠心肌肥厚时SIRT1/PGC-1α信号通路调控的线粒体功能变化；②研究AngII刺激对心肌细胞SIRT1/PGC-1α信号通路调控的线粒体功能的影响；③重点探索活血潜阳祛痰组方对上述体系的干预作用。研究结果有助于阐明该方干预OBH心肌肥厚的作用靶点，为中医药的应用提供微观依据。

肥胖高血压（OBH）心肌肥厚显著增加恶性心血管事件的发生，是亟待解决的治疗难点，SIRT1/PGC-1α介导的线粒体功能障碍为其关键机制之一。本项目在课题前期研究基础上，①以高脂饲料喂养自发性高血压大鼠（SHR）构建OBH大鼠模型，造模干预10周后观察大鼠一般状况、易激惹程度，测定理化指标，大鼠宏观行为表现及理化指标可佐证OBH大鼠模型成功建立；②OBH大鼠模型随机分为3组：OBH-HF、OBH-HF/V、OBH-HF/H，分别以饮用水、缬沙坦、活血潜阳祛痰方灌胃，普通饲料喂养的正常血压京都种大鼠（WKY）、SHR灌胃饮用水，用药干预10周后观察大鼠一般状况、易激惹程度，测定理化指标、心脏超声、心脏质量指数、左室质量指数，检测血清及心肌组织AngⅡ含量，HE及WGA染色观察心肌病理改变，qRT-PCR检测ANP、β-MHC、BNP基因表达水平，发现活血潜阳祛痰方可抑制OBH大鼠心肌肥厚，与降压、改善肥胖状态、降低循环及心肌组织中AngⅡ含量及抑制交感神经系统过度激活有关；③进而以透射电镜观察心肌组织超微结构，检测心肌组织线粒体DNA拷贝数、线粒体膜电位、血清及心肌组织线粒体氧化应激指标水平，检测心肌组织线粒体电子传递链复合体Ⅰ-Ⅴ活性，qRT-PCR、免疫共沉淀、Western Blot检测心肌组织线粒体电子传递链亚基及SIRT1/PGC-1α信号通路分子的基因及蛋白表达水平，发现活血潜阳祛痰方可通过上调SIRT1/PGC-1α去乙酰化水平改善线粒体功能，抑制OBH大鼠心肌肥厚；④从新生大鼠的心脏组织中分离心肌细胞，通过细胞生长实验将培养后的心肌细胞分为5组：对照组、模型组、中药低浓度组、中药高浓度组、西药组。荧光免疫实验检测抗α-肌动蛋白；qRT-PCR检测心肌细胞肥大基因标志物基因表达；检测心肌细胞内ROS、线粒体膜电位、心肌细胞ATP含量及心肌线粒体氧化应激损伤相关指标；检测心肌细胞线粒体电子传递链复合体Ⅰ-Ⅴ活性及线粒体DNA拷贝数；检测SIRT1/PGC-1α/线粒体生物轴相关分子基因及蛋白表达，发现活血潜阳祛痰方可减轻Ang II诱导的心肌细胞肥大，可能与抑制心肌细胞内氧化应激水平增高、改善心肌供能及改善心肌细胞线粒体功能有关。