DataSheet_1_Licorice-Yuanhua Herbal Pair Induces Ileum Injuries Through Weakening Epithelial and Mucous Barrier Functions: Saponins, Flavonoids, and Di-Terpenes All Involved.pdf

In traditional Chinese Medicine (TCM), the licorice-yuanhua herbal pair is one of the most representative incompatible herbal pairs recorded in the “eighteen incompatible herbal pairs” theory. Previous studies of our research group have demonstrated several gut-related side-effects induced by the licorice-yuanhua herbal pair. In this study, we investigated whether and why this incompatible herbal pair could induce gut tissue damage. After licorice-yuanhua treatment, the duodenum, ileum, and colon and serum biomarkers of mice were examined by pathological staining, Western blot, and ELISA assays. The IEC-6 cells and LS174T cells were treated with licorice saponins, yuanhua flavonoids, and di-terpenes; iTRAQ-labeled proteomic technology was then used to explore their synergistic effects on mucosa cells, followed by verification of ZO-1 and MUC-2 protein expressions. The results showed that the licorice-yuanhua herbal pair induced ileum tissue injuries, including epithelial integrity loss, inflammation, and edema. These injuries were verified to be related to epithelial and mucous barrier weakening, such as downregulated ileum ZO-1 and MUC-2 protein expressions. Proteomic analysis also suggested that glycyrrhizic acid and genkwanin synergistically influence tight junction pathways in LS174T cells. Furthermore, licorice saponins, yuanhua flavonoids, and di-terpenes dose/structure-dependently downregulate ZO-1 and MUC-2 protein expressions in mucosa cells. Our study provides different insights into the incompatibility mechanisms and material basis of the licorice-yuanhua herbal pair, especially that besides toxic di-terpenes, licorice saponins and yuanhua flavonoids, which are commonly known to be non-toxic compounds, can also take part in the gut damage induced by the licorice-yuanhua herbal pair.

在中医（Traditional Chinese Medicine，TCM）领域，甘草-芫花药对是“十八反”配伍禁忌理论中记载的最具代表性的相反药对之一。本课题组既往研究已证实，甘草-芫花药对可诱发多种肠道相关不良反应。本研究旨在探究该相反药对是否可诱导肠道组织损伤，及其潜在致病机制。本研究对小鼠给予甘草-芫花干预后，通过病理染色、蛋白质免疫印迹（Western blot）及酶联免疫吸附测定（Enzyme-Linked Immunosorbent Assay，ELISA）实验，检测了小鼠十二指肠、回肠、结肠组织及血清中的生物标志物。分别采用甘草皂苷、芫花黄酮类成分及二萜类化合物处理IEC-6细胞与LS174T细胞；随后借助同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation，iTRAQ）标记蛋白质组学技术，探究其对黏膜细胞的协同作用，并验证紧密连接蛋白ZO-1（Zonula Occludens-1，ZO-1）与黏蛋白2（Mucin-2，MUC-2）的蛋白表达水平。研究结果显示，甘草-芫花药对可诱导小鼠回肠组织损伤，具体表现为上皮完整性丧失、炎症反应及水肿。进一步验证表明，该损伤与上皮及黏液屏障功能减弱密切相关，具体体现为回肠组织中ZO-1与MUC-2的蛋白表达水平下调。蛋白质组学分析还提示，甘草酸与芫花素可协同调控LS174T细胞内的紧密连接通路。此外，甘草皂苷、芫花黄酮类成分及二萜类化合物可分别通过剂量/结构依赖性方式，下调黏膜细胞中ZO-1与MUC-2的蛋白表达。本研究为甘草-芫花药对的配伍禁忌机制与物质基础提供了全新视角，研究发现除已知具有毒性的二萜类化合物外，通常被认为无毒的甘草皂苷与芫花黄酮类成分，同样参与了甘草-芫花药对诱导的肠道损伤过程。