补中益气汤经肠道菌群的调控作用治疗脾虚证的分子机制研究

Buzhong Yiqi decoction is a prescription commonly used on treatment of spleen deficiency syndrome in traditional Chinese medicine (TCM), however the functional mechanism is unclear. With genomics technology and animal model of human fecal transplantation, we attempted to reveal the mechanism of Buzhong Yiqi decoction in the treatment of spleen deficiency syndrome by the regulation of gut microflora, based on the gut characteristic microbiome of spleen deficient patient found in our previous research. Firstly, we would compare the effects of Buzhong Yiqi decoction on spleen deficient mice, pseudo germ-free mice as well as the mice transplanted with the health and spleen deficient patient faeces in vivo, by assessment of the gut immune function, gut characteristic microbiome and gut microflora composition, 5-HT level and associated gut microbiome. Further, incubation technology in vitro would be used to clarify whether the therapeutic effect of Buzhong Yiqi decoction on spleen deficiency was directly acting on gut characteristic microbiome and gut microflora composition. Thus, the Buzhong Yiqi decoction improving gut microflora to exert the treatment of spleen deficiency syndrome would be illustrated. It would provide a theoretical basis for the clinical application of Buzhong Yiqi decoction, and help to build a better understanding of the modern pharmacological mechanism of TCM prescriptions, the theory of diagnosis and treatment of TCM.

补中益气汤是脾虚证治疗常用方剂，但具体作用机制不明。本项目从转化医学角度，基于前期脾虚证患者肠道特征菌组，采用基因组学技术和人源粪便移植动物模型,通过比较补中益气汤给予脾虚证小鼠、伪无菌小鼠、正常/脾虚证患者粪便移植小鼠肠黏膜免疫功能、肠道特征菌组和肠道菌群组成、5-HT及调节5-HT生成菌组变化，探讨不同模型动物体内肠道菌群介导的补中益气汤治疗脾虚证的调节作用机制，为补中益气汤临床应用提供理论依据。在此基础上，采用体外孵育技术，通过比较补中益气汤体内外对肠道特征菌组、肠道菌群组成和调节5-HT生成菌组的作用差异，明确补中益气汤的调节作用是否直接作用于特征菌组/肠道菌群，进一步验证其经肠道菌群调控作用治疗脾虚证的分子机制。深入阐明补中益气汤治疗脾虚证的作用机制，并予以现代医学的解释，对于阐释中药方剂的现代药理作用机理和中医诊治理论，以及中医药现代化的实现具有重要意义。

补中益气汤是脾虚证治疗常用方剂，但具体作用机制不明。本项目从转化医学角度，基于前期脾虚证患者肠道特征菌组，采用基因组学技术对其进行生物学分析并建立了定量分析方法，经对该方法的灵敏度、重复性和特异性等进行考察，结果表明该方法灵敏可靠、稳定性和重复性良好。成功建立了粪菌移植脾虚证小鼠模型，通过塑造番泻叶灌胃及脾虚证患者粪菌移植脾虚证动物模型，比较补中益气汤对两种脾虚证模型小鼠肠道免疫功能、5-HT水平变化，脾虚证特征菌及肠道菌群组成的影响，探讨补中益气汤经肠道菌群的调节作用治疗脾虚证的作用机制。结果表明，补中益气汤对两种脾虚证小鼠肠组织的IgA有升高作用，对IL-1β、TNF-α、5-HT、CD8+/CD3+细胞百分率有降低作用，对IL-2、IFN-γ含量的变化有双向调节作用，细胞因子IFN-γ、IL-2在两种脾虚证模型中变化不一致可能是与不同肠道菌群结构有关；补中益气汤可以升高脾虚证模型小鼠Akkermansia muciniphila ATCC BAA-835 （AKK）菌含量并降低Bacteroides uniformis （BU）菌含量；Acinetobacter属、Parabacteroides属、Ruminococcus属可能为补中益气汤治疗脾虚证的潜在生物标志物。采用模拟人体胃肠道代谢方法，体外考察补中益气汤与健康人群/脾虚证患者粪便共孵育后AKK和BU菌不同时间点的动态变化及肠道菌群组成差异，验证补中益气汤对肠道菌群的调节作用。结果表明，补中益气汤体外对健康人/脾虚证患者粪便样品中肠道菌群具有一定的调节作用；补中益气汤可能不直接作用于AKK菌，可能通过调节菌群结构间接影响AKK菌相对丰度；而补中益气汤可以直接作用于BU菌，降低肠道中BU菌含量。本研究证明，补中益气汤可通过调节肠道菌群及脾虚证特征菌群，同时影响肠组织细胞因子和5-HT水平，发挥抗炎、增强肠黏膜免疫功能的作用，这对于补中益气汤的临床应用，阐释中药方剂的现代药理作用机理和中医诊治理论具有积极意义。