从TLR4偶联肠道微生态-粘膜免疫探讨化浊祛湿通心方治疗冠心病机制

Huazhuo Qushi Tongxin decoction is a clinical effective decoction in treating coronary heart disease which from Chinese medicine master Lu Zhi-zheng based on theory of regulating spleen and stomach to cure CHD. In previous study, we found that it can improve the symptoms of angina pectoris and regulate blood lipid. It’s effective component salvianolic acid B can protect hypoxic injured cardiomyocytes by regulating the TLR4-NFκB pathway. Recent studies have found that intestinal microbiota is involved in the regulation of intestinal mucosal immunity through TLR4-NFκB pathway. Therefore, we propose Huazhuo Qushi Tongxin Decoction in the treatment of coronary heart disease may be related to regulating the TLR4 coupling intestinal microecology and mucosal immune. This research adopts the heart and spleen deficiency in rats with coronary heart disease as the research object with the treatment of Huazhuo Qushi Tongxin decoction. 16S rRNA sequencing and HPLC-MS technology were used to analyze the distribution of intestinal flora and secondary metabolites in intestinal tracts, and to detect the changes of TLR4-NFκB pathway in mesenteric and small intestinal epithelial cells. According to the experimental results of different selected metabolites, blocking or agonisting the TLR4 pathway, using Huazhuo Qushi Tongxin effective components intervention in rat intestinal epithelial cells and rat myocardial cells were cultured alone and two cells co-culture under deoxidized condition, detecting the change of TLR4-NFκB pathway under three conditions. From the cell and the overall animal level to discuss the mechanism of Huazhuo Qushi Tongxin decoction regulating TLR4 coupling intestinal microecology and mucosal immune to cure coronary heart disease. Instructing clinical medicine use and explaining the specification of traditional Chinese medicne in treatment of coronary heart disease.

化浊祛湿通心方是名老中医临床调理脾胃治疗冠心病的有效经验方，课题组前期研究发现该方有改善心绞痛症状和调节血脂作用，其有效组份丹参酚酸B能调节TLR4及其通路保护缺氧损伤心肌细胞。最新报导肠道微生物通过TLR4通路参与肠粘膜免疫调控。因此，我们提出该方治疗冠心病可能与调节TLR4偶联的肠道微生态-粘膜免疫相关。本研究构建心脾气虚型冠心病大鼠，予化浊祛湿通心方动态干预；用16s rRNA测序和HPLC-MS技术联合分析肠道微生态：大鼠肠道菌群和次级代谢产物变化；检测肠系膜淋巴和小肠上皮细胞TLR4通路改变和心脏综合功能；体外采用大鼠小肠上皮细胞、乳鼠心肌细胞在常氧和缺氧状态下单独及共培养，予中药有效组份联合差异次级代谢产物、TLR4阻断剂或激动剂干预，检测TLR4通路变化。多层面解析化浊祛湿通心方调节TLR4偶联的肠道微生态-粘膜免疫治疗冠心病的靶点和机制，为传承名老中医特色治疗提供科学依据。

冠心病是目前世界上致死率和致残率最高的重大疑难疾病之一。化浊祛湿通心方是国医大师路老调理脾胃治疗冠心病的经验方，临床疗效确切，但其作用机理尚未研究。本研究通过动物和细胞两大部分的实验研究，探讨了化浊祛湿通心方对冠心病大鼠的肠道菌群和代谢产物的调节作用；探讨了化浊祛湿通心方对TLR4及其炎症通路相关分子的调节作用；探讨了化浊祛湿通心方对冠心病大鼠心脏结构和功能学指标的保护作用。结果表明化浊祛湿通心方能明显增加肠道有益菌群杆菌门、乳杆菌科、乳杆菌的种类和数量，明显减少有害菌群变形菌纲、毛螺菌科、消化链球菌科、蔷薇属细菌的种类和数量。明显增加有益代谢产物苯甲酸、α-亚麻酸、吲哚乙酸、花生四烯酸、富马酸、草酸的浓度，明显减少有害代谢产物氢肉桂酸、十二烷酸和α-羟基异丁酸的浓度。化浊祛湿通心方有效组份能显著抑制缺氧损伤乳鼠心肌组织TLR4、NFκB和TNFα的分子表达。然而，化浊祛湿通心方有效组份不能明显抑制缺氧损伤小肠上皮细胞TLR4及其炎症通路的分子表达水平。本研究客观真实地探讨了冠心病肠道微生态紊乱及化浊祛湿通心方的调节作用。化浊祛湿通心方通过调节心肌梗死大鼠的肠道菌群和物质代谢，抑制TLR4及其通路的炎症损伤保护了心脏的结构和功能，减少了心肌梗死面积，改善了心脏超微结构和线粒体能量代谢，提高了心脏射血分数。化浊祛湿通心方对缺氧损伤小肠上皮细胞TLR4及其炎症通路的分子调节作用可能依赖于其对肠道菌群和代谢产物的调节，二者之间可能存在一定的分子机制，值得我们进一步去深入研究和探讨。上述研究用现代科学技术方法深刻阐明了化浊祛湿通心方调理脾胃治疗冠心病的深刻内涵。为传承和发展名老中医经验方和临床治疗冠心病新药开发提供了科学数据。