Atractylodis Rhizoma-Atractylodis Macrocephala Rhizoma herbal pair restores intestinal mucosal barrier function in ulcerative colitis via activating Epac1/Rap1 pathway and inhibiting PI3K/AKT pathway

Ulcerative colitis (UC) is a persistent, diffuse intestinal inflammation and ranks among the most challenging chronic diseases worldwide. Atractylodes lancea (Thunb.) DC. and Atractylodis macrocephala Koidz. are traditional Chinese medicines (TCMs) with a long history of clinical application, particularly for gastrointestinal disorders. Both Atractylodis Rhizoma (AR) and Atractylodis Macrocephala Rhizoma (AM) have shown significant efficacy in managing UC; however, the underlying mechanism by which the AR-AM herbal pair promotes intestinal mucosal healing remains poorly understood. The therapeutic effects of the ethanolic extract of AR-AM (EEAR-AM) were evaluated in a murine UC model induced by dextran sodium sulfate (DSS). A network pharmacology approach was employed to explore the anti-UC properties of EEAR-AM, including identification of active compounds, prediction of potential targets, and construction of a protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were subsequently performed to preliminarily elucidate the mechanisms of EEAR-AM in UC treatment. Finally, the proposed molecular mechanisms were validated in both DSS-induced UC mice and Caco-2 cells. In vivo results demonstrated that EEAR-AM significantly attenuated DSS-induced weight loss, reduced colon shortening, lowered the disease activity index (DAI) score, and modulated the spleen coefficient. Moreover, EEAR-AM improved colonic tissue architecture, reduced inflammatory infiltration, restored goblet cell density, enhanced mucin MUC2 expression, and elevated levels of tight junction (TJ) proteins. Additionally, EEAR-AM suppressed the expression of matrix metalloproteinase 2 (MMP-2) and MMP-9. Network pharmacology analyses indicated that EEAR-AM may ameliorate intestinal mucosal dysfunction through modulation of the exchange protein directly activated by cAMP 1 (Epac1)/Ras-associated protein 1 (Rap1) pathway and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathways. These actions potentially enhance cellular barrier integrity and reduce the release of inflammatory mediators. Western blotting results confirmed that EEAR-AM activated the Epac1/Rap1 pathway while downregulating the PI3K/AKT pathway in both DSS-induced UC mice and Caco-2 cells, consistent with predictions from network pharmacology. This study represents the first evidence that the EEAR-AM herbal pair improves intestinal mucosal barrier function in UC, with therapeutic effects likely mediated by activation of the Epac1/Rap1 pathway and inhibition of the PI3K/AKT pathway.