基于肠道菌群探讨吴茱萸碱对肾性高血压大鼠VECs功能的影响及机制

Renovascular hypertension-related vascular endothelial cell (VECs) injury was found to be associated with several diseases, which seriously threaten people’s health. Recent work suggests a key role for evodiamine in regulation of VECs function, but the molecular mechanisms involved remain poorly understood. Our results showed that there was a significant increase in the counts of gut microbiota produced short chain fatty acids (SCFAs) in the fecal samples from 2-kidney, 1-clip (2K1C) rats treated with evodiamine. However, the characterization of gut bacterial community mediated by evodiamine in 2K1C rats is currently unknown. Recently, emerging data confirmed the role of SCFAs as the regulators of AMP-activated protein kinase (AMPK) activation. Results also indicated that AMPK was an important regulator of VECs function. In a preliminary experiment we found that AMPK phosphorylation level in VECs of 2K1C rats was decreased. We further observed that treatment of 2K1C rats with evodiamine resulted in an increase of the phosphorylation of AMPK. Therefore, we suppose that evodiamine modulates intestinal microbiota composition of 2K1C rats, which contribute to the regulation of VECs function via AMPK signaling mediated by SCFAs. The aim of this study is to investigate the effect of evodiamine on VECs function of 2K1C rats at whole body, organ and molecular levels. Also, the characterization of gut microbial community mediated by evodiamine in 2K1C rats will be determined. Moreover, we will explore the potential molecular mechanism for the regulation of VECs function by evodiamine based on gut microbiota at cellular and molecular levels. This work has the potential to bring new ideas for the successful prevention and treatment of VECs injury in the patients with renovascular hypertension.

肾性高血压血管内皮细胞（VECs）损伤与多种疾病相关，严重危害人类健康。吴茱萸碱具有VECs功能调节作用，但机制不详。我们研究显示，吴茱萸碱显著提高两肾一夹(2K1C)大鼠肠道菌群中短链脂肪酸(SCFAs)产生菌的含量，但其介导的肠道菌群变化特征尚不清楚。资料表明，SCFAs参与活化AMP激活的蛋白激酶(AMPK)，而后者是VECs功能重要的调控因子。在预实验中，我们发现：2K1C大鼠VECs中AMPK磷酸化水平降低；吴茱萸碱干预后，AMPK磷酸化水平上调。因此，我们推测：吴茱萸碱改变2K1C大鼠肠道菌群结构，并经SCFAs调节AMPK活性，进而影响其VECs功能。本项目拟从整体-血管环-分子层次观察吴茱萸碱对2K1C大鼠VECs功能的影响，分析其对肠道菌群结构的调节作用，并从细胞和分子水平探讨吴茱萸碱经肠道菌群调节VECs功能的分子机制。此研究将为肾性高血压VECs损伤的防治提供新思路。

背景：肾性高血压血管内皮细胞(Vascular endothelial cells, VECs)损伤是动脉粥样硬化的始动因素。对其研究，对于防治高血压血管并发症具有重要意义。肠道菌群在调节VECs功能中发挥作用，但机制不详。.研究内容：本项目采用离体血管环实验、流式细胞仪技术、ELISA、real-time PCR、Western blot等方法观察吴茱萸碱对肾性高血压大鼠VECs功能的影响；采用基因测序等技术分析吴茱萸碱对肠道微生物群落结构的调节作用，进一步在分子、细胞、动物等层面探讨吴茱萸碱影响VECs的机制。.主要结果：（1）吴茱萸碱对肾性高血压大鼠VECs功能的影响：HE结果显示，实验组与对照组主动脉组织形态学未见明显差异；Ach、L-NAME、MB及SNP、L-Arg对血管功能的影响无统计学意义；与Sham组相比，2K1C组主动脉SOD活性下降、MDA水平升高，血清NO浓度降低。分别给予高、中、低剂量吴茱萸碱后，低剂量和高剂量吴茱萸碱能显著降低IL-6、SICAM水平，上调TGF-β1水平，提示吴茱萸碱对肾性高血压大鼠VECs功能具有调节作用。（2）吴茱萸碱对肾性高血压大鼠肠道菌群结构的调节作用：2K1C模型组和吴茱萸碱组大鼠肠道细菌主要隶属于厚壁菌门和拟杆菌门。与2K1C组相比，吴茱萸碱高、中、低剂量组厚壁菌门相对丰度显著增加，而拟杆菌门相对丰度下降。变形菌门、放线菌门无显著差异。在属水平上相比，吴茱萸碱组高、中、低剂量组拟杆菌属、普氏菌属的相对丰度较2K1C组降低。（3）吴茱萸碱经由肠道菌群影响VECs功能的分子机制：与2K1C组相比，吴茱萸碱显著上调主动脉AMPK和ACC的磷酸化水平，下调NADPH氧化酶活性，并促进HAECs细胞释放NO。同时，吴茱萸碱诱导Acetate和Butyrate含量增加，而Hexanoate含量没有显著变化。进一步研究显示，Acetate通过AMPK途径下调NADPH氧化酶活性，并促进HAECs中NO的水平。 .关键数据及科学意义：吴茱萸碱通过调节肠道菌群结构促进Acetate和Butyrate的产生，进而经AMPK信号通路调控VECs功能。此研究为VECs的研究及其防治提供新思路。