PurposeThe objective of this study was to investigate the effects of agrimonolide (AM) on mice with dextran sulfate sodium (DSS)-induced colitis and elucidate its protective mechanisms. MethodsA 3 % DSS solution was used to induce colitis, and intragastric administration of AM at doses of 25 and 50 mg/kg was performed. A comprehensive assessment was conducted to evaluate inflammatory responses and mucosal integrity in the colon. Inflammatory factors were quantified using enzyme-linked immunosorbent assay (ELISA). The proportions of T helper cell 17 (Th17) and regulatory T cells (Treg) cells in mesenteric lymph nodes (MLNs) was analyzed through RT-qPCR and flow cytometry. Proteins associated with the Notch and JAK2/STAT3 pathways were examined via RT-qPCR, western blotting, and immunofluorescence. Additionally, the impact of AM on Treg and Th17 cell differentiation was investigated in vitro. ResultsPre-treatment with AM significantly alleviated colon inflammation in mice, as evidenced by reduced body weight loss, shorter colon length, lower disease activity index (DAI) score, and decreased myeloperoxidase (MPO) content. Notably, AM pre-treatment attenuated the production of pro-inflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6, in mice with DSS-induced colitis. Additionally, AM pre-treatment significantly enhanced the expression of tight junction proteins (Occludin and ZO-1), thereby preserving gut barrier function. Moreover, we observed that AM administration decreased the ratio of Th17 cells while increasing the frequency of colonic Treg cells, thus modulating the Th17/Treg balance both in vivo and in vitro. Furthermore, in the AM-treated group, the expression of Notch-1, Jagged1, delta like 4 (DLL4), phospho-janus kinases 2 (p-JAK2)/JAK2, and p-signal transducer and activator of transcription 3 (STAT3)/STAT3 in colonic tissue was reduced compared to the DSS group. Remarkably, the therapeutic effects of AM in colitis mice were blocked by a Notch activator. ConclusionThese findings underscore the effectiveness of AM in alleviating symptoms and pathological damage in DSS-induced colitis mice by rebalancing Th17/Treg cell homeostasis through modulation of the Notch and JAK2/STAT3 signaling pathways. These insights into AM's mechanisms of action offer potential avenues for novel therapeutic strategies.