肠道菌群代谢物诱导单核细胞亚群变化在AS中的意义与化痰祛瘀解毒通脉方的干预机制研究

Atherosclerosis (AS) is the main pathological basis of cardiovascular and cerebrovascular disease, involving abnormal lipid metabolism, inflammatory reaction, and plaque formation. Traditional Chinese medicine (TCM) believes that AS is the process of combination with ‘phlegm, blood stasis, toxicity’. The abnormal lipid metabolism or "phlegm and blood stasis" which was closely related to diet and gastrointestinal function. It is having been proved that gut microbiota was a new target for many diseases. Therefore, we hypothesize that the imbalance of gut microbiota and abnormal metabolites of bacteria can lead to the change of monocyte subsets in circulatory system, and that was the underlying mechanisms of AS pathogenesis. HTQYJDTMF was proved of the effect of regulating blood lipid, inhibiting inflammatory reaction and inhibiting the formation of plaque. In this study, the purpose is to prove that it is one of the mechanisms of anti-AS by regulating the intestinal flora, improving the metabolites and inhibiting the proinflammatory response of monocyte. The stool and blood samples of patients with atherosclerosis of intermin-gled phlegm and blood stasis syndrome were collected, and the relationship between intestinal flora imbalance, metabolites and phenotypic changes of monocytes was studied. Then, we use ApoE-/- mouse AS model and cell model to investigate the mechanism of drug intervention to clarify the regulation role on interaction chain of “gut microbiota- bacteria metabolites- monocyte subsets-AS “.

动脉粥样硬化（AS）是心脑血管病主要的病理学基础，涉及脂质代谢异常、炎性反应、斑块形成多个环节。中医学认为AS是痰、毒、瘀、痰瘀互结相互促生和发展的过程。无论是脂质代谢异常还是“痰、瘀”与饮食不节、胃肠功能等密切相关。研究证明，肠道菌群是多种疾病的新靶点。故提出肠道菌群失调、菌群代谢产物异常导致循环系统单核细胞亚群的变化，诱发血管内皮炎性反应、斑块形成的AS发生机制假说。“化痰祛瘀解毒通脉方”具有调节血脂、抑制炎性反应、抑制斑块形成等作用。本研究欲证明该方通过调节肠道菌群、改善代谢产物，抑制单核细胞的促炎反应是抗AS的作用机制之一。临床采集动脉粥样硬化痰瘀互结证患者的粪便和血液样本，研究肠道菌群失衡、代谢物与单核细胞表型变化之间的关系。然后，应用ApoE-/-小鼠AS模型与细胞模型考察药物的干预机制，阐明本方对“肠道菌群-菌群代谢物-单核细胞-AS”作用链的调控作用。

血脂异常的发病率越来越高，有效防治血脂异常对于预防心脑血管事件的发生有重要意义。目前针对血脂异常的研究缺乏基于系统生物学层面的综合表征和系统认知。双参芎连颗粒（SSXL）作为取得新药临床批件的中药复方药物，具有调节血脂、改善血液流变性、抗血小板聚集、抗血栓形成、抗炎、改善痰瘀互结证的证候表征等确切的药理作用。进一步研究双参芎连颗粒从肠道途径干预血脂异常发挥有效作用的机制可以为中医药从肠道微生物途径预防和治疗血脂异常提供新的思路与方法。课题选择研究健康受试者（NOR组）、血脂异常者（DYS组）与动脉粥样硬化合并血脂异常者（AS组）的肠道微生态特征、临床血清学指标与痰瘀互结证候特征在疾病演变过程中的特点差异，从动物实验整体水平分析挖掘SSXL从肠道途径干预血脂异常发挥有效作用的机制，探寻中药从肠道菌群治疗血脂异常的新靶点。 课题利用16S rDNA高通量测序和代谢组学液相色谱质谱（LC-MS）联用技术，分析研究临床中血脂异常者、动脉粥样硬化合并血脂异常者与健康人群肠道菌群的差异特征，应用流式细胞术检测单核细胞亚群的变化，明确肠道菌群-粪便代谢物-炎性指标-单核细胞亚群-血脂异常-AS之间的关系。进一步选取人源临床典型动脉粥样硬化合并血脂异常者粪便样本，通过灌胃移植到germ-free C57BL/6J小鼠体内，联合高脂饲料饲喂3个月制备血脂异常痰瘀互结证模型。观察SSXL干预后对血脂异常痰瘀互结证相关指标及肠道微生态的改善作用。我们观察到如下结果：①血脂异常、动脉粥样硬化合并血脂异常的临床表现为：收缩压、痰瘀互结证候评分明显高于正常人群；血脂TC、LDL-C、ApoB水平升高和HDL-C水平降低，并伴随凝血功能障碍，MCP-1、M-CSF水平等炎症因子的升高；肠道菌群F（Firmicutes）/B（Bacteroidetes）值逐渐增大，肠道中厚壁菌门增多是导致动脉粥样硬化合并血脂异常相关生物标志物升高的原因。②SSXL可通过促进肠道菌群平衡状态、调控胆汁酸代谢途径发挥调节血脂异常痰瘀互结证及相关指标发挥改善作用。