肾性高血压大鼠肠道菌群结构的变化及其对AMPK介导的血管内皮功能的影响

Renovascular hypertension-related vascular endothelial dysfunction (VED) was found to be associated with several cardiovascular diseases, which seriously threaten people's health. Recent work suggests a crucial role for the gut microbiota in VED, but the molecular mechanisms remain poorly understood. Our results showed that there was a significant reduction in Bifidobacterium and Bacteroides counts in the fecal samples from rats with 2-kidney, 1-clip (2K1C) hypertension. However, the characterization of gut microbial community in rats with renovascular hypertension is currently unknown. In a preliminary experiment we found that AMP-activated protein kinase (AMPK) phosphorylation level in vascular endothelial cells of 2K1C rats was decreased. We further observed that probiotics had a beneficial effect on gut microbiota composition of 2K1C rats, accompanied with elevation of the phosphorylated AMPK level in vascular endothelial cells. Recently, emerging data have indicated that AMPK was an important regulator of vascular endothelial function. Therefore, we suppose that renovascular hypertension leads to changes in the gut microbial community, which contribute to VED in an AMPK mediated manner. The aim of this study is to characterize the gut microbial community structure of 2K1C rats using denaturing gradient gel electrophoresis (DGGE), real-time PCR and pyrosequencing. Also, the effect of gut microbiota on vascular endothelial function and its possible mechanism will be investigated at whole body, organ and cellular levels. This work has the potential to bring new ideas and targets for the successful prevention and treatment of VED in the patients with renovascular hypertension.

肾性高血压血管内皮功能障碍（VED）与多种心血管疾病相关，严重危害人类健康。肠道菌群在VED发生中作用关键，但机制不详。我们研究显示，两肾一夹(2K1C)肾性高血压大鼠肠道中双歧杆菌属和拟杆菌属数量明显减少，但其具体的肠道菌群结构特征尚不清楚。在预实验中，我们发现：2K1C大鼠血管内皮细胞的AMP活化蛋白激酶(AMPK)磷酸化水平明显降低；益生菌改善肠道菌群后，AMPK磷酸化水平上调。资料表明，AMPK是血管内皮功能的重要调控因子。因此，我们推测：2K1C大鼠肠道菌群结构改变，并经AMPK影响其血管内皮功能。本项目拟应用变性梯度凝胶电泳(DGGE)、real-time PCR和焦磷酸测序技术研究2K1C大鼠肠道菌群结构变化特征，并采用siRNA、免疫共沉淀等技术，从整体-血管环-细胞层次观察肠道菌群对血管内皮功能的影响，探讨其分子机制。此研究将为肾性高血压VED的防治提供新思路和新靶点。

肾性高血压血管内皮功能障碍（VED）与多种心血管疾病相关，严重危害人类健康。肠道菌群在VED发生中作用关键，但机制不详。本项目首先建立两肾一夹（2K1C）高血压大鼠模型，提取2K1C模型组大鼠和假手术组大鼠肠道粪便标本总DNA, 使用Illumina Hiseq3000高通量测序平台，采用宏基因组测序技术对2K1C模型组大鼠和假手术组大鼠肠道菌群进行结构组成和主成分分析，使用实时荧光定量聚合酶链反应技术对肠道中优势菌属大肠埃希菌属、双歧杆菌属、乳酸杆菌属及拟杆菌属定量检测，以此阐明肾性高血压大鼠肠道菌群结构特征；进而采用血管环实验、ELISA等方法分析血管内皮功能的变化及其与肠道菌群结构变化的关系；最后采用real-time PCR和Western blot等方法分析肠道菌群结构影响血管内皮功能的分子机制。主要获得如下结果：（1）与假手术组相比，2K1C模型组厚壁菌门相对丰度显著下降（P<0.05），而拟杆菌门相对丰度显著升高（P<0.05）。条件致病菌大肠埃希菌属在2K1C模型组大鼠肠道中相对丰度显著高于假手术组（P<0.05）。粪球菌属、罗氏菌属等短链脂肪酸（SCFAs）产生菌在2K1C模型组大鼠肠道中相对丰度显著低于假手术组（P<0.05）。2K1C模型组拟杆菌门中普氏菌属相对丰度显著高于假手术组（P<0.05）；厚壁菌门中瘤胃球菌属显著高于假手术组（P<0.05）。主坐标分析表明2K1C模型组与假手术组样本在肠道菌群构成上存在明显差异；（2）两组动物主动脉组织形态学观察结果未见明显差异(P>0.05)，乙酰胆碱、L-NAME、MB、SNP、L-Arg等对血管功能的影响无明显差异(P>0.05)；与对照组相比，模型组SOD等降低(P<0.05)，MDA升高(P<0.05)。而且，检测血清中SCFAs含量发现，与对照组相比，2K1C组SCFAs含量发生显著改变(P<0.05)。（3）SCFAs经AMP活化蛋白激酶（AMPK）通路调节 NADPH氧化酶(NADPH oxidase)活性及一氧化氮（NO）水平，进而参与VED的调控。此研究将为肾性高血压VED的防治提供新思路和新靶点。