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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.

frontiers in pharmacology

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