自发性高血压大鼠肠道菌群的结构特征分析与镇肝熄风汤的影响

Based on analysis of the relations between gut microbiota and the clinical risk factors (cholesterol, atherosclerosis, obesity, metabolic syndrome, type 2 diabetes mellitus) of the essential hypertension, as well as antihypertensive effect of lactic acid bacteria, this research puts forward a viewpoint that gut microbiota might be an environmental factor that play an important role in the development of essential hypertension. Spontaneously hypertensive rats(SHR)will be selected to study the community characteristics of gut microbiota，which collected from the five segments (jejunum, ileum, caecum, colon and rectum) of the rats. First, the classification of gut microbiota will be identified and analyzed under the culture and non-cultured conditions, further, the diversity and difference of gut microbiota will be detected by PCR-DGGE and Real time fluorogenetie quantitative PCR(RT-PCR). Meanwhile, the levels of gut microbiota metabolites(shortc hain fatty acid and D-lactic acid) will be tested by gas chromatograph and UV spectrophotometry. The main components of renin-angiotensin-aldosteronesystem (RAAS) will be detected, the levels of plasma renin, AngⅡ,Ang(l-7)tested by enzyme-linked immunoabsorbent assay, the expressions of the mRNA of ACE，AT1R，ACE2 and MasR detected by RT-PCR as usual, the protein of ACE2 detected by Western blot test. Moreover, the correlation between RAAS and gut microbiota will be studied. In the process, the effects of Zhenganxifeng decoction and benazepril hydrochloride tablet can be described. On this basis, the relationship between hypertension and gut microbiota will be explored from angle of molecular biology, and some new ideas and study prospects about hypertension pathogenesis may be given. Also, the experimental basis that Zhenganxifeng decoction embodying spleen-stomach theory and the possible mechanism that influence blood pressure will be illustrated, in addition, adding new contents in the mode of action of benazepril hydrochloride tablet in treating hypertension.

本项目基于肠道菌群与高血压危险因素之间的关系，并结合乳酸菌降压研究，以肠道菌群可能为高血压发病的环境因素为假说，选用自发性高血压大鼠（SHR）模型，取空肠、回肠、盲肠、结肠和直肠五个肠段肠道菌群，首先采用培养和非培养条件鉴定分析菌群的分类特点；然后进一步应用PCR-DGGE法、Real-Time PCR法分析肠道菌群的多样性和差异性；并检测肠道菌群代谢产物（采用GC、UV法测定）及肾素-血管紧张素-醛固酮系统（RAAS）主要元件变化（采用ELISA、Real-Time PCR、Western blot法测定），分析RAAS与肠道菌群的相关性。同时，观测镇肝熄风汤、贝那普利对其影响。从而探索肠道菌群与高血压的关系，为深入发现和证明高血压尚不明了的发病机制提供新的思路和研究方向；并从肠道菌群角度阐明镇肝熄风汤体现脾胃学说的实验基础及影响血压的可能机制，为贝那普利的作用方式研究增添新的内容。

背景和方向：高血压发病机制至今没有完整统一的认识。本项目以肠道菌群可能为高血压发病的环境因素为假说，分析自发性高血压大鼠（SHR）肠道菌群的结构特征以及肠道菌群代谢产物、肾素-血管紧张素-醛固酮系统（RAAS）主要元件变化，观测镇肝熄风汤对其影响。.研究内容：本研究通过16S rDNA测序技术分析了镇肝熄风汤对SHR肠道菌群的影响；同时检测了血清中内毒素、D-乳酸、二胺氧化酶、Renin、AngⅡ、Ang1-7等的变化及肾皮质和结肠组织ACE、AT1R、ACE2和MasR mRNA的表达，肾皮质及结肠组织ACE2的蛋白表达。.结果和数据：.（1）镇肝熄风汤改善SHR的饮食和饮水，并降低SHR收缩压、舒张压、平均动脉压及心率。.（2）镇肝熄风汤上调SHR肠道菌群肠杆菌、双歧杆菌和乳杆菌数量，降低肠球菌、产气荚膜杆菌数量、肠道菌群球/杆比及厚壁菌门/拟杆菌门比。.（3）SHR空肠菌群厚壁菌门和拟杆菌门数量减少，变形菌门和放线菌门数量增加；回肠菌群厚壁菌门和放线菌门数量减少，变形菌门和拟杆菌门数量增加；盲肠菌群厚壁菌门和放线菌门数量减少，拟杆菌门和变形菌门数量增加；结肠菌群厚壁菌门和变形菌门数量增加，拟杆菌门数量减少，放线菌门数量相同；直肠菌群厚壁菌门数量减少，拟杆菌门和变形菌门数量增加，放线菌门数量相同。经α多样性分析，SHR 5个不同肠段肠道菌群丰度和多样性高于WKY；经OTU 聚类分析，SHR与WKY物种构成存在差异。镇肝熄风汤减少SHR空肠、回肠、盲肠、结肠、直肠等肠段变形菌门数量，减少回肠和盲肠的拟杆菌门数量、空肠放线菌门数量和结肠厚壁菌门数量；增加空肠、回肠、盲肠、直肠等肠段厚壁菌门数量，增加空肠、结肠、直肠等肠段拟杆菌门数量和回肠的放线菌门数量；镇肝熄风汤也改变上述各肠段的肠道菌群多样性，调整菌群丰度，改变其肠道菌群物种构成，升高结肠产乙酸菌、产丙酸菌和产丁酸菌所占比例。.（4）镇肝熄风汤可以降低SHR吲哚、内毒素、D-乳酸、二胺氧化酶和AngⅡ含量，升高其Ang（1-7）含量。.（5）镇肝熄风汤可以明显降低SHR大鼠肾皮质、结肠中ACE和AT1R基因表达，明显升高肾皮质中ACE2、MasR基因表达和结肠中ACE2蛋白表达。.科学意义：本项目探索了肠道菌群与高血压的关系，并阐明镇肝熄风汤体现脾胃学说的实验基础及影响血压的可能机制。