基于肠道微生态/屏障系统的芫花致胃肠毒性物质基础及作用机制研究

The disorder of Intestinal microflora and injury of intestinal barrier function can lead to a variety of gastrointestinal adverse reactions. Previous reports including preliminary studies of our research group have confirmed that Genkwa Flos can cause significant gastrointestinal toxic side effects, but its toxic components and mechanism have not been clearly elucidated yet. Accordingly, this study is putting forward a new resolution involving in the intestinal microflora and the intestinal barrier function, to reveal the material basis and mechanism behind the gastrointestinal toxicity of Genkwa Flos, which includes the work as follows: (1) Establish the standard material library of chemical composition and the chemical composition information database of Genkwa Flos using modern chromatographic isolation and analytical methods; (2) Illustrate how and which chemical compounds from Genkwa Flos regulate the intestinal microflora and identify the target microbial populations by the metagenome sequencing of the next generation; (3) Investigate the influence of the effective constituents from Genkwa Flos on the intestinal barrier function based on Ussing Chamber, Western blot and PCR techniques (focusing on the transport function, permeability and tight junction protein expression of intestinal epithelial cells). The aim of this study is to uncover the mechanism of the gastrointestinal toxicity of Genkwa Flos by of all kinds of ingredients through regulating the intestinal microflora and barrier functions, and also to lay a foundation for further exploring the method to reduce the toxicity of Genkwa Flos, and finally to provide support for clinical safe medication of Genkwa Flos.

肠道微生态紊乱/屏障功能损伤可导致多种胃肠道不良反应的发生。前人报道及本课题前期研究均证实芫花致胃肠毒副作用显著，但其致毒的物质基础及其作用机制尚未阐明。据此，本项目提出从肠道微生态/屏障功能的角度，揭示芫花致胃肠毒性的化学物质基础及作用机制研究，拟开展工作如下：（1）采用现代色谱分离、分析方法，构建芫花化学组分标准物质库及各化学组分信息库；（2）采用基因测序方法分析芫花各化学组分对动物肠道微生物调控，鉴定特异微生物种群；(3)基于Ussing Chamber，PCR及Western blot技术探讨芫花各化学组分对动物肠道屏障功能（肠道上皮细胞转运功能、通透性及紧密连接蛋白表达）的影响；旨在揭示芫花各类成分通过调控肠道微生态/屏障系统致胃肠毒性的作用机制，为芫花临床安全用药提供支撑，也为进一步深入探索降低芫花毒性奠定基础。

本项目基于肠道微生态/屏障功能的角度，以芫花为研究对象，揭示其致胃肠毒性的物质基础及作用机制，为芫花临床安全用药提供支撑，也为进一步深入探索降低芫花毒性奠定基础。（1）采用现代色谱分离、分析方法，共分离鉴定了芫花中44个化学成分，包括二萜类化合物、黄酮类化合物、酚类化合物、脂肪类化合物等。采用UPLC-Q-TOF / MS分析技术，对胃肠毒性部位组分进行全面表征，构建芫花化学组分标准物质库及各化学组分信息库。（2）采用高通量测序方法、PCR、Western blot技术揭示了芫花对肠道微生态及屏障功能的调控作用。芫花可通过破坏肠道粘液化学组成、下调肠粘液蛋白、肠道紧密连接蛋白表达、升高炎症因子表达诱发肠道屏障损伤及炎症；并能改变肠道菌群群落结构，差异菌属Rikenella上调，Mucispirillum下调，主要差异基因代谢通路集中在Type II general secretion pathway和Dissimilatory sulfate reduction。（3） 阐释了芫花导致胃肠毒性的物质基础及作用机制。芫花导致胃肠毒性的物质基础主要是其中的二萜部位、芫花酯甲成分、芫花素成分，可通过破坏肠道粘液层、下调肠粘液蛋白MUC-2、肠道紧密连接蛋白ZO-1导致肠道屏障损伤；其中二萜成分能显著引起肠道菌群群落结构的变化，差异菌属 Prevotella、Paraprevotella,、Rikenella与芫花所致肠道毒性呈正相关。通过项目实施在国内外学术刊物发表论文9篇，其中SCI收录7篇；培养博士研究生1名。项目相关成果申请并获得2019年陕西高等学校科学技术奖一等奖。