Gancao Xiexin decoction alleviates 5-fluorouracil-induced intestinal mucositis by inhibiting ACSL4 and activating Keap1-Nrf2 pathway.

5-Fluorouracil (5-Fu) is a commonly used chemotherapy drug, but it frequently induces toxic side effects such as intestinal mucositis and is closely associated with ferroptosis‌. Gancao Xiexin (GCXX) decoction, a renowned formula in traditional Chinese medicine, is widely employed for treating gastrointestinal inflammatory diseases. However, the therapeutic effects and underlying mechanisms of GCXX on 5-Fu-induced intestinal mucositis remain unclear‌. This study aims to investigate whether GCXX alleviates 5-Fu-induced intestinal mucositis in mice by suppressing ferroptosis. Using a 5-Fu-induced intestinal mucositis mouse model, we found that oral administration of GCXX not only effectively alleviated 5-Fu-induced intestinal mucositis symptoms including weight loss, diarrhea, and colon shortening, but also reduced pathological changes such as villi atrophy, crypt damage, and goblet cell depletion. Furthermore, GCXX significantly mitigated 5-Fu-triggered intestinal barrier dysfunction by restoring proliferating cell nuclear antigen (PCNA) and occludin expression while reducing myeloperoxidase (MPO) expression of colon tissues and serum levels of diamine oxidase (DAO), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Mechanistically, GCXX reduced ferroptosis biomarkers including accumulation of iron and 4-hydroxynonenal (4-HNE) via reducing Kelch-like ECH-associated protein 1 (Keap1) expression and activating nuclear factor erythroid 2-related factor 2 (Nrf2) anti-oxidant pathway, as well as down-regulating long-chain-fatty-acid-CoA ligase 4 (ACSL4) expression and mitogen-activated protein kinases (MAPKs) activation. Molecular docking, dynamics simulation, and drug affinity responsive target stability (DARTS) assays confirmed a stable binding of liquiritin (the main active component of GCXX) with ACSL4 and Keap1, elucidating its anti-ferroptosis mechanism. Our findings demonstrated that GCXX alleviated 5-Fu-induced intestinal mucositis in mice through blocking ferroptosis by activating the Keap1-Nrf2 signaling and inhibiting the ACSL4 and MAPKs pathway, further supporting its clinical utility in mitigating chemotherapy-related toxic side effects.