基于“肠道菌群-药动学-代谢组学”整体关联性的千金子炮制减毒机理研究

Semen Euphorbiae,one kind of toxic traditional Chinese medicine must be processed in order to reduce the toxicity and remove oil so that it could be taken safely.While the attenuated processing mechanism of traditional Chinese medicine is still not explicit. Our previous studies have found that the amount of major toxicity-active ingredient groups,Diterpenoid esters,decreased after processing and the key attenuated mechanism of Semen Euphorbiae were closely related to intestinal inflammatory factors,P-gp and CYP450 enzymes. Under the guidance of the overall concept of TCM, we introduced the16S rRNA quantitative PCR,UPLC-Q-TOF/MS and other forefront technology to the processing of traditional Chinese medicine field innovatively. Taking the gut micro-ecology,pharmacokinetics and the dynamic changes of the endogenous metabolic componet as starting point, we built and evaluated the overall correlation of metabolic networks in vivo and vitro, interaction network between drug and body by means of the overall identification and hierarchy-relation analysis.On the basis of the cognition of unique properties of Semen Euphorbiae and the practice of manufacturing pulveratum method for resolving and reducing toxicity, this project will study and clarify the attenuated mechanism of Semen Euphorbiae after processing from the overall, tissular and molecular levels, which the scientific connotation of the hypothesis will be expressed by regulating gut micro-ecology, mediating changes in time and space of ADME and endogenous metabolic components as well as changes in specific metabolic pathway in organism.This project will not only offer a scientific basis for the clinical safety of Semen Euphobiae,but also provide new thoughts and techniques for the study of toxicity-attenuated processing mechanism of traditional Chinese medicine.

有毒中药千金子需经去油制霜后入药用，炮制目的是减毒，但其减毒机理尚不明确。前期研究发现制霜后主要毒-效成分群二萜酯类含量降低，减毒机制与肠道炎性因子、P-gp、CYP450酶等因素密切相关，因此在中医药整体观的指导下，本项目创新性地将16S rDNA荧光定量PCR、UPLC-Q-TOF/MS等前沿技术引入炮制减毒机理研究，以肠道菌群微生态环境、药动学、内源性代谢组分动态变化为切入点，通过整体辨识和层次分析，构建“体内外”交叉代谢网络、药物与机体相互作用网络的整体关联性。本项目基于对千金子药性理论的认识和制霜解毒降毒、缓和药性的实践，从整体、组织、分子水平研究，以探明千金子制霜减毒，通过调控肠道菌群微生态环境、介导机体吸收、分布、代谢、排泄，以及机体内源性代谢组分时空变化与特异性代谢通路改变而发挥作用之假说的科学内涵，为其安全用药提供科学依据，也为炮制减毒作用机制研究提供新的思路与技术手段。

本项目以肠道菌群微生态环境、药动学、内源性代谢组分动态变化为切入点，研究千金子制霜减毒的作用机制。采用平板菌落计数法以及16S rDNA荧光定量PCR技术研究了千金子制霜前后对大鼠粪便中4类代表性肠道菌群双歧杆菌、乳酸杆菌、大肠杆菌、肠球菌的数量变化。实验结果表明，千金子制霜前后提取物均会影响肠道菌群的生物多样性，影响肠道菌群平衡，且制霜后对4类肠道菌群的作用减弱，引起肠道菌群紊乱程度减小，这与千金子霜泻下作用缓和的研究结果相一致。基于UPLC-MS/MS技术研究千金子制霜前后主要二萜醇酯类成分千金子素L1、千金子素L2和千金子素L3在体内的药代动力学特征，揭示其制霜前后主要二萜醇酯类成分吸收入血、到达靶器官发挥整体药效或毒性作用及体内排泄特征的差异，结果表明制霜可影响千金子在体内的ADME过程，制霜后3种成分体内的吸收减少、在各组织中的分布降低、在粪便和尿液中的排泄增加，初步的代谢研究结果表明制霜后3种成分的代谢物发生了量的变化，推测千金子制霜后毒性降低、作用缓和可能与其主要活性成分及代谢产物在体内的变化有关。基于UPLC-Q-TOF/MS代谢组学技术，结合主成分分析（PCA）、偏最小二乘法-判别分析（PLS-DA）和正交偏最小二乘法-判别分析（OPLS-DA），探讨高、中、低剂量的千金子生品和霜品提取物的量效、时效关系，对千金子制霜前后大鼠的粪便与尿液代谢谱进行分析，找出其内源性差异代谢产物，结果表明在尿液样品中鉴定出25个内源性差异代谢物，在粪便样品中鉴定出16个内源性差异代谢物，通过对千金子制霜减毒作用的生物标志物进行通路富集，发现千金子制霜后通过调节脂质代谢、氨基酸代谢、能量代谢和脂肪酸代谢，以及改善肠道菌群等代谢途径来发挥减毒作用。本研究从整体动物、组织、分子水平研究千金子制霜减毒的作用机理，为其安全用药提供科学依据，也为炮制减毒作用机制研究提供新的思路与技术手段。