基于肠道菌群调节的湿热内蕴实质及葛根芩连汤苦寒燥湿机制研究

The study on the pathogenesis of hyperlipidemia which is of initial factors related diseases and complications, belong to Dampness-heat and lipid turbidity of traditional Chinese medicine helps us to improve Zheng diagnosed and clinical effects. We think that sweetness and fatness can cause Dampness-heat interior intestinal, which induces intestinal conduction loss and separating the clear from the turbid dysfunction, mainly displays in the intestinal flora imbalance. To simulate Dampness-heat accumulating interior intestinal of the body, we use high fat diet induced hyperlipidemia. We adopt the technology of sequence on 16SrRNA and bioinformatics analysis to get the targets of gut microbiota that influence on the triglycerides on absorption of Damp-heat interior gut, using kinetic analytical methods to solve the problem of the false on the static analysis. Next, we take ultra performance convergence chromatograph to detect the short chain fatty acids of gut bacteria metabolites to discuss damp and hot condensing mechanism of intestinal conduction disorders. This method of discussion on Dampness-heat condensation and mechanism of disturbance on interior intestinal provides technical methods for the objective study on the etiology and pathogenesis of TCM. The efficiently formula Gegenqinlian decoction(GQD) for treating Dampness-heat interior accumulation with clinical and experimental basis is used as a tool drug, using pathway analysis for reverse proof on Dampness-heat agglomerate gut. Then, we employ germ-free mice experiment to verify the mechanism of biological effect, clarifying the scientific connotation on drying dampness of GQD regulation the gut microbiota by sweetness and fatness causing Dampness-heat interior intestinal elevated triglycerides, to provide a scientific basis for prevention and treatment Dampness-heat on clinical TCM.

高脂血症是相关疾病的始发因素和并发症，属中医“湿热”、“脂浊”范畴，其病因病机的研究，有助于临床辨证、提高临床疗效。本项目认为：肥甘厚腻可导致湿热内蕴肠道，肠道传导失司，泌别清浊功能紊乱，其主要因素为肠道菌群失调。本课题以高脂饲料所致湿热内蕴肠道发病过程为研究对象，以高脂饲料诱导高脂血症，模拟湿热蕴结肠道，采用16SrRNA测序方法，引入生物信息学分析技术，获得湿热内蕴肠道影响甘油三酯入血吸收的肠道菌群靶点，进行动力学分析解决静态分析假阳性问题，采用超高效合相色谱检测菌群产物短链脂肪酸构成比，探讨湿热凝聚肠道传导失常机制，为中医病因学客观化研究提供技术方法；以临床和实验治疗湿热内蕴证有效的方药葛根芩连汤（GQD）为工具药，进行途径分析，以对湿热内蕴肠道反向求证，以目标菌群在无菌小鼠中进行验证，阐明GQD调节肥甘厚腻所致湿热内蕴甘油三酯升高的燥湿科学内涵，为中医临床防治湿热内蕴证提供科学依据

高脂血症是相关疾病的始发因素和并发症，属中医“湿热”、“脂浊”范畴，其病因病机的研究，有助于临床辨证、提高临床疗效。通过本项目研究阐明了GQD（葛根芩连汤）调节肥甘厚腻所致湿热内蕴甘油三酯升高的燥湿科学内涵。1.60%高脂饲料易诱发湿热内蕴甘油三酯水平升高的高脂血症大鼠模型，该模型存在肥胖型和胰岛素抵抗型。同时，构建了大鼠中医临床症状评分标准。2.筛选了影响肥甘厚腻诱发湿热内蕴肠道的高脂血症大鼠模型甘油三酯入血吸收的肠道菌群靶点。研究结果发现：与空白组相比，模型组肠道菌群门水平上菌群种类相同，但比例不同。属水平上Akkermansia菌在明显存在差异，且与既往文献报道有差别。同时，也发现了一些与既往研究相一致的菌群。如Bacteroides在正常组中丰度更高，Blautia和Anaerotruncus在模型组中更高。不同剂量GQD对肠道菌群的影响有差异，低剂量使用时，能够改变肠道菌群，但是与正常组菌群结构区别较大，使用中等和高剂量GQD后，肠道菌群更相似，并与正常组菌群结构相近。阳性药物组使用后改变菌群结构，但是并未向正常组恢复。3. GQD降低湿热内蕴甘油三酯水平升高的高脂血症大鼠甘油三酯水平，且使得肠道紧密连接蛋白Claudin1、ZO1、肠碱性磷酸酶IAP和蛋白Occluding的表达量上升(P＜0.01)，GQD中剂量组乙酸/丙酸显著上调（P＜0.01），丙酸/丁酸显著下调（P＜0.01）。综上数据显示：GQD可以通过调节SCFAs构成比例来降低湿热内蕴甘油三酯水平升高的高脂血症大鼠患糖尿病风险。