基于肠道菌群-宿主代谢的甘遂醋炙减毒存效机制研究

Kansui, a kind of toxic traditional Chinese medicine for Junxia, was stir-baked with vinegar to reduce the toxicity in Chinese medicine. The literature and previous research of our group confirmed that stir-baked with vinegar could reduce the significant gastrointestinal toxicity of kansui, while retaining the purgative effect. The toxicity and effect sites of kansui have close relation with intestinal tract, but existing research results could not answer the scientific problems, for example, the biological mechanism of kansui stir-baked with vinegar for detoxification and retaining the effect. Hence, under the guidance of the theory of traditional Chinese medicine, this project is proposed to study the material basis and mechanism of action of kansui Stir-baked with vinegar for detoxification and retaining the effect based on the gut microbiota-host co-metabolism. The research work of this project were listed as follows: Firstly, based on gene sequencing and real time PCR Technology, the construction fingerprintsof animal gut microbiota were built for normal and model groups both treated by kansui; Secondly, through metabonomic methods and multivariate statistical techniques, explain the gut microbiota-host co-metabolism pathways related to gastrointestinal tract irritation and purgation of kansui, and the inherent relationship between "host metabolism-gut microbiota-biological effects-active components; Thirdly, through the multi-angle verification of the interaction of the gut microbiota and chemical components, and micro ecological intervention, this project could confirm the relationship between the toxicity and effect of kansui and gut microbiota. In brief, this project was carried out to reveal the material foundation and biological mechanisms of kansui stir-baked with vinegar for detoxification and retaining the effect, provide a scientific basis of safe and effective application for clinical, and supply the exploration of ideas and methods for scientific research of toxic traditional Chinese medicine.

中医临床对峻下有毒中药甘遂采用醋炙以减毒，文献与本课题组前期研究均证实醋炙在降低甘遂强烈胃肠刺激性毒性同时保留其泻下作用。甘遂毒效作用部位均与肠道密切联系，但现有研究结果尚未能解答甘遂醋炙减毒存效生物学机制等科学问题。据此，本项目在中医药理论指导下，提出基于肠道菌群-宿主代谢开展甘遂醋炙减毒存效物质基础与生物学机制研究。内容包括：1.应用基因测序和实时PCR技术，构建给予甘遂各样品后正常/腹水模型动物肠道菌群结构指纹谱；2.应用代谢组学与多变量统计技术，解析与甘遂肠道刺激和泻下作用相关的肠道菌群-宿主代谢通路并挖掘“宿主代谢-肠道菌群-生物效应-活性成分群”间内在联系；3.通过微生态制剂干预、化学干预及肠道菌群与化学成分群相互作用等多维验证，确证甘遂醋炙减毒存效与肠道菌群关系。以揭示甘遂醋炙减毒存效物质基础与生物学机制，为甘遂临床安全有效应用提供科学依据，为有毒中药科学应用基础研究提供探索。

本项目基于肠道菌群-宿主代谢，对给予生/醋甘遂各样品的正常及癌性腹水模型动物，通过毒/效指标测定、内源性代谢物分析及采用16S rDNA高通量测序技术、GC-MS及LC-MS等测定其中肠道菌群种类/丰度、粪便短链脂肪酸和胆汁酸的含量，对比了生/醋甘遂各样品对正常及腹水模型大鼠毒/效作用、粪便代谢组学与肠道菌群的差异，探讨了甘遂醋炙减毒存效生物学机制与物质基础。研究结果表明甘遂毒性与肠道有益菌Lactobacillus，潜在致病菌Blautia、Ruminococcus（瘤胃球菌属）、Alloprevotella（拟普雷沃氏菌属）等菌属及肠道菌调节的短链脂肪酸、胆汁酸含量相关，表现出一定的醋炙减毒作用。给予生/醋甘遂粉末后，大鼠肠道中与腹水模型高度相关的有益菌（Lactobacillus）比例提高，与腹水模型高度相关的潜在致病菌（Anaeroplasma、Lachnospiraceae）比例降低，肠道菌调节的短链脂肪酸和保护性胆汁酸含量升高、细胞毒性胆汁酸含量降低，表现出一定的模型缓解作用。生/醋甘遂均可使模型大鼠体内异常代谢有所恢复，相比生甘遂，醋甘遂对正常大鼠内源性成分影响程度低。生/醋甘遂对正常大鼠的干预作用可能与视黄醇代谢、初级胆汁酸生物合成、赖氨酸降解、嘌呤代谢等4条代谢通路的调节有关；对模型大鼠的干预作用可能与苯丙氨酸代谢、苯丙氨酸，酪氨酸和色氨酸生物合成、色氨酸代谢、甘油磷脂代谢、糖基磷脂酰肌醇生物合成等5条代谢通路的调节有关。肠道菌中Lactobacillus属、Blautia属等通过调节嘌呤代谢、胆汁酸代谢、视黄醇代谢、色氨酸代谢发挥醋炙减毒作用，Lachnospiraceae属、Anaeroplasma属等通过调节甘油磷脂代谢、不饱和脂肪酸的生物合成、苯丙氨酸代谢、色氨酸代谢发挥甘遂醋炙存效作用。伪无菌鼠和给予筛选出的益生菌（Lactobacillus）、短链脂肪酸和熊脱氧胆酸(UDCA)等的确证实验表明，虽然肠道菌群不是甘遂发挥毒/效作用的必要条件，但肠道菌群稳态，特别是其中益生菌的合理丰度及其调节的短链脂肪酸（乙酸、丙酸、丁酸）的合理含量与胆汁酸的合理比例，不仅对肠道保护有重要意义，还可与甘遂泻水逐饮产生一定协同作用。同时，作为甘遂醋炙减毒存效主要成分群和单体类成分的醋甘遂成分群B、C和代表性二萜类成分，其毒/效作用与肠道菌群有一定的相关性。