方证相应指导下的三草降压汤对自发性高血压大鼠（SHR）降压机制及其与调控IL-1β关联通路TLR4/NF-κB和NLRP3炎症小体的相关性研究

Sancao Jiangya Decoction (SCD), initially proposed by Professor Liu Du-zhou, has been proved as an effective compound prescription in the treatment of essential hypertensive (EH) with liver fire hyperactivity syndrome. Previous studies have demonstrated that SCD showed a remarkable antihypertensive efficacy that is equivalent to the administration of nifedipine, and furthermore had some more advantages such as short onset time, persistence and less target organs damage. But, recent studies found that a significant difference in the antihypertensive efficacy has been found when using SCD to treat the liver fire hyperactivity syndrome and non-liver fire hyperactivity syndrome, which indicated that the SCD had high selectivity aiming at the TCM syndrome. Therefore, the purpose of this study is to clarify the underlying mechanism of SCD highly selecting the syndrome. In this current study, the SCD is used to administrate the Spontaneously Hypertensive Rats that are screened out in accordance with the syndrome criterion. The antihypertensive efficacy is then determined and compared when the prescription (SCD) is conforming to the syndrome or not, in order to identify the characteristic of correspondence of the prescription and the syndrome. To further elucidate whether the mechanism of SCD highly selecting the syndromeare associated with the inflammatory reaction, the inflammation is then brought into this study as a key breakthrough point, owing to its crucial roles in the liver fire hyperactivity syndromeas well as the EH. Particularly, it is need to deeply explore that the TLR4/NF-κB pathway and NLRP3 inflammasome closely related to the production and secretion of primary inflammatory factor IL-1β will play important roles in the antihypertensive effect of SCD. This research will be great importance in deepened understanding the mechanism of SCD exerts antihypertensive, and will provide new ideas for clarifying the scientific connotation of the correspondence of the prescription and the syndrome and building a bridge between the Chinese medicine and orthodox medicine.

三草降压汤是刘渡舟教授治疗高血压肝火亢盛证的经验方。前期研究证明，该方降压效应与硝苯地平相当，且起效时间短，持续时间长，并可减轻靶器官损害。另发现该方具有很强的证候选择性，对肝火亢盛证与非肝火亢盛证降压效果存在较大差异。为了揭示其证候选择性强的内在机制，本课题拟对SHR进行证候筛选，并通过三草降压汤对不同组别SHR的干预，明确在相同疾病条件下，方证相应与否的疗效差异度，以确定其方证相应特点。继之以与中医肝火亢盛证及高血压均有紧密联系的炎症反应为切入点，进行该方抗炎及其他降压机制相关性的研究，阐释其证侯选择性是否与其抗炎作用相关。并以与原发性促炎因子IL-1β的生成与分泌相关的信号通路TLR4/NF-κB和NLRP3炎症小体为核心，深入探讨抗炎与降压相关的内在机制。本研究不仅对研究中药降压的效应机制有重要意义，而且对揭示方证相应的科学内涵，搭建高血压中西医沟通的桥梁具有重要价值。

背景：目前，全世界近三分之一成年人患有高血压，我国高血压患者已经达到三亿之多，且具有年轻化趋向，对于高血压的起病原因、预防及治疗的研究已经成为一项重要的课题。.目的：通过网络药理学研究，挖掘SCD潜在核心靶点，预测其降压作用通路；探索SCD的药效、靶器官保护作用以及发挥降压作用的具体机制。.方法：.①通过网络药理学，筛选SCD降压的具体作用机制.②通观察血压、旷场实验、旋转耐受时间、易激惹程度、宏观表征等变化，评估SCD对SHR血压以及肝火亢盛程度的影响.③通过检测心脏重量指数（HMI）与左心室重量指数（LVMI）测定，并进行HE染色以及MASSON染色，观察SCD对SHR心脏病理损伤的影响.④通过检测尿微量白蛋白（mA1b）、尿肌酐（UCRE）和尿蛋白（UP）等指标，HE染色以及MASSON染色法，观察SCD对SHR肾损伤的影响.⑤采用免疫组化法、ELISA法、WB法以及QPCR法检测NF-κB/NLRP3/IL-1β相关指标，观察SCD对SHR心脏、肾脏TLR4/NF-κB/IL-1β通路的影响.⑥通过cck8检测SCD对HK-2细胞的毒性，之后WB法检测TLR4、NF-κB蛋白表达量，探索SCD与AngⅡ适宜加药浓度.⑦检测AngⅡ诱导损伤的HK-2细胞中ROS、MDA、SOD含量，观察SCD对AngⅡ诱导损伤的HK-2细胞氧化应激损伤的影响.⑧WB法检测HK-2细胞TLR4/NF-κB/IL-1β通路相关指标，观察SCD对于肾小管上皮细胞TLR4/NF-κB/IL-1β通路的影响.结果：.①网络药理学研究预测得到NF-K B/NLRP3/IL-1β信号通路在SCD治疗高血压时起到重要作用.②SCD在降低SHR血压的同时，能够改善肝火亢盛症状.③SCD能改善SHR左心室肥厚的程度，改善高血压下心脏的病理损伤以及炎性浸润，保护靶器官.⑤SCD能通过降低血压，改善尿液生化异常，降低血肌酐、尿素氮和血尿酸水平，改善肾脏组织病理形态等方面改善高血压肾损伤.⑥SCD能下调NF-K B/NLRP3/IL-1β信号通路相关蛋白的表达，抑制炎症反应，这可能是其发挥降压、靶器官保护作用的分子学机制.⑦SCD可以通过抑制氧化应激损伤、调控tlr4/NF-κB/NLRP3信号通路，保护AngⅡ刺激下的肾小管上皮细胞，从而保护肾小管功能，改善高血压下的肾脏组织损伤