Shaoyao decoction promotes ISCs proliferation by activating Wnt/β-catenin and YAP1/TAZ signaling to enhance epithelial regeneration in damp-heat colitis.

Ethnopharmacological relevance: Shaoyao decoction (SYD) is a classic traditional Chinese medicine formulation that presented a significant therapeutic effect on damp-heat colitis. However, its potential pharmacological mechanisms remain not fully elucidated.

Aim of the study: This study aimed to investigate the effect of SYD on intestinal epithelial regeneration by analyzing the activity of intestinal stem cells (ISCs) in a mouse model of damp-heat colitis.

Materials and methods: LC-MS/MS was employed to identify the main compounds in SYD extract. A damp-heat colitis mouse model was established by combining three factors: the high-sugar and high-fat diet, high heat (33 ± 1 °C) and high damp (90 ± 2 %) environment, and DSS treatment. SYD was given orally to evaluate its therapeutic efficacy. The therapeutic efficacy of SYD was assessed via clinical indicators and histopathological observation. ELISA and RT-qPCR were performed to measure cytokine levels (IL-18, IL-21, IL-22, IL-33 and EGF) in distal colon tissue. Western blotting and RT-qPCR were applied to detect the expression levels of proteins (Lgr5, EpCAM, Wnt3a, LRP6, β-catenin, TCF4, FZD1, Gα13, YAP1, TAZ, α-catenin and Muc2) and mRNA (Lgr5, Ki67, TCF4, Fzd1, Ereg, Egfr) in colon. Additionally, immunofluorescence and immunohistochemistry were used to observe the localization of key proteins (EpCAM, Wnt3a, β-catenin, YAP1, Muc2, Hes1) in colon.

Results: 91 compounds were identified in SYD extract. SYD significantly alleviated clinical symptoms and reduced histopathological damage of mice with damp-heat colitis. SYD downregulated the levels of pro-inflammatory cytokines (IL-18 and IL-21), and upregulated levels of epithelial regeneration-associated cytokines (IL-22, IL-33 and EGF). Mechanistically, SYD promoted ISC activation and epithelial proliferation by increasing expressions of Lgr5, Ki67 and EpCAM. Concurrently, SYD upregulated protein expressions in canonical Wnt/β-catenin pathway (Wnt3a, LRP6, β-catenin and TCF4) and alternative Wnt-YAP1/TAZ signaling axis (FZD1, Gα13, YAP1 and TAZ). Finally, SYD promoted mucus barrier repair by increasing Muc2 expression.

Conclusions: This study suggested that SYD exerts significantly therapeutic efficacy on damp-heat colitis. Mechanistically, SYD enhances ISC proliferation and differentiation by activating both the canonical Wnt/β-catenin pathway and alternative Wnt-YAP1/TAZ signaling axis, thereby promoting injury-induced intestinal epithelial regeneration.