从“心与小肠相表里”理论探讨养心通脉方对冠心病血瘀证机体肠道微生态平衡的影响

Coronary heart disease(CHD) is a disease of serious threat to human health and high mobidity. Recent studies found that gut microbiota play an important role in the prevention and treatment of CHD by " heart-gut-microecologic” axis，which has associated with alleviating inflammation response, improving metabolic characteristics, but the research on the relationship of its specific pathogenesis and traditional Chinese medicine(TCM) syndrome is still unclear.At the same time, the effect of treatment of CHD by clinical application of Yangxin Tongmai decoction is good. However the research of action mechanism is also thorough insufficiently. Based on “the Heart And the Small Intestine Share a Paired Relationship” of the visceral outward manifestation theory and our findings , Yangxin Tongmai decoction is conducive to chronic coronary heart disease blood stasis rats intestinal flora such as lactic acid bacteria, the number of bifidobacteria recovery. Accordingly, our hypothesis is that the therapeutic effect of Yangxin Tongmai decoction on CHD is closely related to gut microbiota by improving metabolism characteristics and inhibiting inflammatory response induced or amplified by intestinal endotoxin. In order to confirmed the hypothesis,we will systemically investigate the relationships between changes in gut microbiota and changes in metabolic profiling and biomolecule by integrating metagenomics and metabonomics analysis,,including LC-TOF-MS,gene sequencing and luminex . This project is expected to lay a theoretical foundation for the clinical application of TCM in the treatment of CHD and provides a new ways to develop drugs for treating CHD.

冠心病是一种严重威胁人类健康且发病率较高的疾病。近来报道肠道微生物通过“心-肠”轴缓解炎症反应、改善体内代谢功能，在防治冠心病中扮演重要角色，但其与血瘀证相关性研究较少；临床应用养心通脉方治疗冠心病效果良好，其作用机制尚未完全阐明。本项目将在前期发现养心通脉方调节大鼠肠道4种菌群作用研究基础上，从“心与小肠相表里”理论出发，探讨肠道及舌苔微生物菌群与冠心病血瘀证机体内代谢特征及各分子的相关性，开展养心通脉方干预大鼠冠心病血瘀证和粪便移植实验，从代谢组学和宏基因组学角度，运用LC-TOF-MS、基因测序、液相芯片等分子生物学技术方法，研究养心通脉方如何调节肠道微生物菌群，抑制炎症反应，改善宿主体内代谢功能。据此阐明肠道及舌苔微生物菌群与冠心病血瘀证的相关性和养心通脉方通过调节肠道微生态治疗冠心病血瘀证的作用机制，为临床中药通过调控机体微生态平衡治疗冠心病和临床药物研发提供理论指导和新思路。

实验基于“心与小肠相表里”理论建立冠心病血瘀证大鼠动物模型，经养心通脉方和粪菌移植干预，探讨冠心病血瘀证与肠道菌群关系，中药干预对冠心病血瘀证大鼠模型肠道菌群、炎症和代谢等的影响及机制。首先通过饮食和药物诱导冠心病血瘀证大鼠模型，造模成功后，随机分组药物干预两周，收集大鼠血清、心脏、小肠等进行生化和病理及透射电镜分析，取粪菌移植肠内容物和回肠黏膜菌群进行16sRNA测序；Western Blot检测Occludin；ELISA检测血清TNF-α、IL-6、IL-10、内毒素；LC-MS/MS 技术分析大鼠血浆非靶向代谢组学并与回肠粘膜菌群相关性分析。结果提示冠心病血瘀证大鼠体态行为、心电图、心肌和小肠组织、血脂血流变指标异常；益生菌群如苏黎世杆菌、双歧杆菌等菌丰度水平降低，致病菌如斯氏菌、韦荣氏球菌等丰度水平上升，模型大鼠小肠黏膜菌群与TNF-α、IL-10、内毒素、血脂、血流变具有相关性；1-甲基组氨酸、N-乙酰组胺、黄体酮和脱氧皮质酮可成为冠心病血瘀证的差异性代谢物。与冠心病血瘀证高度相关的变形杆菌门Hydrogenophaga、Limnohabitans菌属等与5-羟基吲哚、N-乙酰组胺等血浆代谢产物呈正相关，与L-精氨酸、高精氨酸等血浆代谢产物呈负相关；表明大鼠肠道菌群失衡导致的炎症和代谢紊乱与冠心病血瘀证的发生密切相关。养心通脉方干预能够有效改善冠心病血瘀证大鼠血脂指数；恢复肠道微生态平衡，保护乳杆菌、双歧杆菌、阿克曼氏菌等益生菌的恢复，对致病菌如斯氏菌、韦荣式球菌和链球菌等具有抑制作用；能够降低血清TNF-α、IL-6及内毒素水平，升高IL-10水平；能够改善冠心病血瘀证的回肠紧密连接；能调控动物模型血浆代谢；经养心通脉方干预后与其高度相关的拟杆菌门（乳杆菌目）Lactobacillus菌属和泉古菌门Corynebacterium菌属与Boc-β-氰基-L-丙氨酸、水苏碱等血浆代谢产物呈正相关（P<0.05），与5-羟基吲哚、N-乙酰组胺等呈负相关（P<0.05）。以上结果阐明了养心通脉方能够通过调节肠道微生态平衡，小肠黏膜菌群和黏膜机械屏障功能，减少炎性反应和氧化应激反应，改善血浆代谢，来达到治疗冠心病血瘀证之目的。