活血潜阳祛痰方调控NLRP3炎性小体通路改善肥胖高血压心肌纤维化的机制研究

Obesity Hypertension (OBH) myocardial remodelling significantly increases the incidence of malignant cardiovascular events, and fibrosis is one of the main features of remodelling. The activation of RAAS is one of the key mechanisms of myocardial fibrosis. Our research group found that "Blood stasis-Yang kang-Phlegm" were the main pathological factors of OBH myocardial remodelling, and Huoxue Qianyang Qutan formula could improve OBH myocardial remodelling through lowering Ang II level，which was the core factor of RAAS. It was reported recently that the activation of NLRP3 inflammasome pathway (NLRP3/csapase-1/IL-1β) was involved in the myocardial fibrosis induced by Ang II. Therefore, we hypothesize that the NLRP3 inflammasome pathway may be the interference target of Huoxue Qianyang Qutan formula on the OBH myocardial fibrosis. This project intends to select OBH rats and neonatal rat cardiac fibroblasts (CFs) as experimental objects, and use cell culture, RT-PCR, Western Blot and immunofluorescence technologies. The aims of this project are as follows: ① To observe the changes of NLRP3 inflammasome pathway in the myocardial fibrosis of OBH rats; ② To investigate the function of NLRP3 inflammasome pathway in the fibrosis model of CFs induced by Ang II; ③ Mainly to explore the effects of Huoxue Qianyang Qutan formula on the mentioned systems above. The results will provide scientific basis for the reversing of OBH myocardial fibrosis with the application of Traditional Chinese Medicine.

肥胖高血压（OBH）心肌重构显著增加恶性心血管事件发生，纤维化是重构的主要特征之一，RAAS激活是其发生的关键。课题组发现“血瘀、阳亢、痰浊”是心肌重构的重要病理因素，活血潜阳祛痰方可改善OBH心肌重构，机制与降低RAAS中的核心因子AngII有关。晚近报道，NLRP3炎性小体通路（NLRP3/csapase-1/IL-1β）激活参与了AngII的促心肌纤维化效应。因此，我们提出假说，该通路可能是活血潜阳祛痰方干预OBH心肌纤维化的作用靶点。项目拟以OBH大鼠及乳鼠心肌成纤维细胞（CF）为实验对象，采用原代细胞培养、RT-PCR、Western Blot、免疫荧光等技术：①观察OBH大鼠心肌纤维化时NLRP3炎性小体通路的变化；②研究NLRP3炎性小体通路在AngII诱导的CF纤维化模型中的作用；③重点探索活血潜阳祛痰方对上述体系的干预作用，以期为中医药逆转OBH心肌纤维化提供科学依据。

肥胖相关性高血压(OBH)更易导致心肌肥大及间质纤维化，显著增加恶性心血管事件，药物治疗手段有限，心肌成纤维细胞（CFs）是最主要的纤维化反应细胞，NLRP3 炎症小体可能是CFs参与心肌重构的重要分子靶点。课题组前期研究发现，“血瘀、阳亢、痰浊”是OBH心肌重构的重要病理因素，活血潜阳祛痰方可减轻OBH大鼠的左室重构，但具体作用机制尚未完全阐明。本研究从细胞和整体动物水平，基于NLRP3炎性小体探讨活血潜阳祛痰方改善OBH心肌纤维化的机制。本研究通过构建OBH动物模型、体外Ang II诱导的CFs纤维化模型，采用HE、MASSON染色、免疫荧光、免疫组化、细胞转染、荧光定量PCR、Western blot和ELISA等研究方法发现：（1）OBH大鼠呈现明显的心肌纤维化，伴有Col I、Col III等分子表达升高，同时心肌中NLRP3、caspase-1和IL-1β水平显著增加，活血潜阳祛痰方可显著降低OBH大鼠血压、Lee’s指数，缓解大鼠心肌重构，减轻纤维化面积，降低血中IL-1β含量，并抑制心肌组织NLRP3/Caspase 1/IL-1β通路分子表达；（2）体外根据IC50选择不同浓度中药进行体外试验，活血潜阳祛痰方可以抑制Ang II诱导的CFs细胞增殖，降低其向肌成纤维细胞转化，抑制Hyp分泌及胶原分泌。干扰NLRP3可以减轻Ang II诱导的CFs纤维化，过表达NLRP3表达可减弱活血潜阳祛痰方的保护作用。综上可见，中药复方的抗心肌纤维化的作用是通过部分抑制NLRP3炎症小体信号通路实现的。本研究证明了NLRP3炎性小体通路激活是OBH心肌纤维化的重要机制；证实了活血潜阳祛痰方改善OBH心肌纤维化的分子机制是抑制NLRP3炎性小体通路激活，为OBH的治疗提供新思路，丰富了中医药治疗的科学内涵。