基于“肠道菌群—5-HT信号系统”探讨自发性高血压大鼠中枢-胃肠响应机制及镇肝熄风汤的影响

There is still no complete and unified understanding of the pathogenesis about essential hypertension. Based on the relations that gut microbiota with the clinical risk factors (cholesterol, type 2 diabetes mellitus, obesity, atherosclerosis) of the hypertension, with the chronobiology disorders, with the regulation of blood pressure by the central nervous system, as well as the research that gut microbiota participate in controling hypertension, combined with the characteristics of 5-HT signaling system, about the pathogenesis of hypertension ,this study puts forward a hypothesis that the changes of the community characteristics of gut microbiota might affect blood pressure and thus cause cascade reactions characterized by changes in 5-hydroxytryptamine(5-HT) signaling systems, so as to establish response mechanism between the center and the gastrointestine.Spontaneously hypertensive rats(SHR)will be selected to study. Firstly, the changes of blood pressure in male SHR and homologous male Wistar-Kyoto rats(WKY) before and after fecal microbiota transplantation will be analyzed,the differences of the community characteristics will be detected in gut microbiota after transplantation，and the accompanying changes of intestinal mucosal barrier and 5-HT signaling system will be investigated.Secondly, the effect of Zhenganxifeng decoction and benazepril respectively on ‘Gut microbiota-5-HT signaling system’ in SHR can be described.The community characteristics of gut microbiota will be determined by high-throughput sequencing techniques,and the levels of the other indexes will be tested by Real-time RT-PCR,Western blot, HPLC-ECD and immunohistochemical method. On this basis, the relationship between gut microbiota and hypertension will be provided direct evidence,and some new ideas and study prospects about hypertension pathogenesis may be explored and discovered.Also,the experimental basis that Zhenganxifeng decoction embodying spleen-stomach theory and the possible mechanism that influence blood pressure may be illustrated.

高血压发病机制至今没有完整统一的认识。本项目基于肠道菌群与高血压危险因素、生物钟紊乱、中枢血压调控等关系以及肠道菌群参与高血压控制的研究探索，结合5-HT信号系统的特性，提出高血压发病机制中，肠道菌群结构的改变可能导致血压异常，进而引发以5-HT信号系统变化为特征的级联反应并形成中枢-胃肠响应的假说，选用自发性高血压大鼠（SHR）模型，首先对比雄性SHR和同源雄性WKY大鼠粪菌移植前后血压变化，分析移植后肠道菌群的结构和多样性以及伴随发生的肠粘膜屏障、5-HT信号系统变化；其次观测镇肝熄风汤、贝那普利分别对SHR“肠道菌群—5-HT信号系统”的影响。采用高通量测序、Real-time RT-PCR、Western blot、HPLC-ECD及IHC等法测定相关指标。从而为肠道菌群和高血压关系提供直接证据，为研究高血压提供新的思路；阐释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。

背景和方向：高血压发病机制至今没有完整统一的认识。本项目基于肠道菌群与高血压危险因素、生物钟紊乱、中枢血压调控等关系以及肠道菌群参与高血压控制的研究探索，结合5-HT信号系统的特性，提出高血压发病机制中，肠道菌群结构的改变可能导致血压异常，进而引发以5-HT信号系统变化为特征的级联反应并形成中枢-胃肠响应的假说，分析了粪菌移植对自发性高血压大鼠（SHR）肠道菌群的结构和多样性及其5-HT信号系统变化的影响，同时观测了镇肝熄风对SHR“肠道菌群—5-HT信号系统”的影响，并从肠道菌群角度阐明了镇肝熄风汤调节血压的可能机制。.研究内容：本研究通过16SrDNA测序和分子生物学等方法分析了镇肝熄风汤对SHR血压、心率、“肠道菌群—5-HT信号系统”的影响；同时观察了血浆神经递质的变化，检测了海马、额叶组织中5-HT1A受体、5-HT1B受体、5-HT7受体mRNA的表达，下丘脑、海马、额叶及结肠组织中5-HT1A受体、5-HT1B受体、5-HT7受体的蛋白表达。.结果和数据：.（1）粪菌移植降低了SHR的收缩压、舒张压、平均动脉压。.（2）粪菌移植增加了SHR粪便OTUs数目、软壁菌门、变形菌门相对丰度，降低了疣微菌门相对丰度，提高了SHR菌群α多样性。.（3）粪菌移植可影响SHR血浆神经递质5-HT、甘氨酸的水平。.（4）粪菌移植降低了SHR额叶组织中5-HT1AR、5-HT7R mRNA的表达；降低了SHR海马组织5-HT7R、额叶组织5-HT1AR、下丘脑组织5-HT1BR的蛋白表达。.（5）镇肝熄风汤降低了SHR的收缩压、舒张压、平均动脉压。.（6）镇肝熄风汤减少了SHR粪便OTUs总数，增加了拟杆菌门、疣微菌门相对丰度，降低了SHR菌群丰富度及多样性。.（7）镇肝熄风汤可影响SHR血浆中5-HT、肾上腺素、天冬氨酸水平。.（8）镇肝熄风汤降低了SHR海马组织中5-HT1AR、5-HT1BR的基因和蛋白表达，升高了额叶组织中5-HT1AR、5-HT1BR、5-HT7R的基因及蛋白表达；镇肝熄风汤还降低了下丘脑组织中5-HT1BR、5-HT7R的蛋白表达和结肠组织中5-HT1AR、5-HT1BR、5-HT7R的蛋白表达，.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。