植物乳杆菌通过调控肠道菌群-胆汁酸轴防治TMAO诱导动脉粥样硬化的作用机制研究

Atherosclerotic cardiovascular disease is the "top killer" of human health. Statins have significant lipid-lowering effects, but are not suitable for all patients with high cholesterol and can cause serious side effects such as severe liver and kidney damage. TMAO, a metabolite of intestinal flora, promotes the development of atherosclerosis by decreasing serum TMAO levels, reducing expression level of cyp7a1, decreasing synthesis and excretion of bile acid and inhibiting reverse cholesterol transport. Therefore, TMAO provides a new target for the prevention and treatment of cardiovascular diseases. Our previous studies showed that Lactobacillus plantarum significantly inhibited TMAO-induced atherosclerosis in ApoE-/- mice by decreasing serum TMAO levels, modulating intestinal flora, increasing fecal bile acid excretion and unconjugated bile acid content, but its specific mechanism was unclear. Therefore, we propose to explore the molecular mechanism of Lactobacillus plantarum on preventing and treating TMAO-induced atherosclerosis in ApoE-/- mouse based on gut microbiota-bile acid axis. In this paper, metatranscriptomics sequencing, fecal microbiota transplantation, uHPLC-MS/MS and molecular biology methods will be used to elucidate the mechanism of Lactobacillus plantarum on TMAO-induced atherosclerosis at the animal, tissue and molecular level, which provides basic data for the clinical application of probiotics in the clinical treatment of atherosclerosis, and provides data support for recognizing TMAO as new target for the prevention and treatment of cardiovascular disease.

动脉粥样硬化性心血管疾病是人类健康的“头号杀手”。他汀类药物虽降脂效果显著，但不适合所有高胆固醇患者，且会造成患者严重肝肾损害。肠道菌群代谢产物TMAO降低肝脏胆汁酸合成酶CYP7A1表达水平，减少肝胆汁酸合成和分泌，影响胆固醇代谢和逆向转运，促进动脉粥样硬化发生。因此，TMAO被认为是防治心血管疾病新的药物作用靶点。我们前期实验表明：植物乳杆菌能减少血清TMAO水平、重塑肠道菌群、增加粪胆汁酸排泄量和非结合胆汁酸含量和抑制TMAO诱导的动脉粥样硬化，但具体机制不清。因此，我们拟以ApoE-/-小鼠为研究对象，采用宏基因组测序技术、粪菌移植技术、液质联用技术和分子生物学技术，从动物、组织和分子水平上阐明植物乳杆菌经肠道菌群-胆汁酸轴防治TMAO诱导动脉粥样硬化的作用机制，为益生菌在动脉粥样硬化临床治疗上的应用提供基础数据，并为TMAO作为防治心血管疾病新药物作用靶点的思路提供数据支撑。

动脉粥样硬化已成为心血管疾病患者死亡的主要原因，也是诱发心血管疾病的主要病理基础，其常规治疗药物疗效有限，且产生各种副作用。肠道菌群代谢产物TMAO与动脉粥样硬化的发生发展密切相关，已成为防治动脉粥样硬化疾病的新靶点。然而，截止申请书提交前，尚无采用益生菌有效抑制TMAO 生成、防治动脉粥样硬化的文献报道。因此，本项目主要研究内容有：①确证植物乳杆菌和产气肠杆菌ZDY01具有干预TMAO 诱导的ApoE-/-小鼠动脉粥样硬化功能；②考察植物乳杆菌和产气肠杆菌ZDY01对肠道菌群多样性、种群结构及功能基因的影响；③采用粪菌移植技术确证植物乳杆菌抑制TMAO 诱导的ApoE-/-小鼠动脉粥样硬化依赖于肠道菌群；④分析粪胆汁酸排泄量和非结合胆汁酸含量增加的分子机制。研究结果表明：植物乳杆菌ZDY04通过增加Alloprevotella, unidentified_Ruminococcaceae, Ruminiclostridium和lactococcus属的丰度，显著降低CDCA、TCA、TCDCA和DCA含量，促进胆固醇的逆向转运相关基因Sr-b1、Abcg5和Cyp7a1表达显著升高，进而抑制TMAO诱导ApoE-/-小鼠体内的动脉粥样硬化。此外，E.aerogenes ZDY01增加了Turicibacter属和unidentified_ Ruminococcaceae属的丰度，上调Asbt和Ostα/β的表达加速门静脉血中胆汁酸的再吸收，减弱胆汁酸依赖性FXR-FGF15激活。FXR- FGF15轴的失活增加了肝脏CYP7A1的表达，加速胆固醇转化成胆汁酸并进一步促进胆固醇的逆向转运从而降低TMAO诱导的动脉粥样硬化作用。该项目为益生菌在动脉粥样硬化临床治疗上的应用提供理论基础，也为TMAO作为临床防治心血管疾病新的药物作用靶点提供依据，具有重要的科学意义。