Network pharmacology and experimental validation reveal that betulin alleviates 5-fluorouracil-induced intestinal injury by inhibiting intestinal senescence and enhances antitumor efficacy.

5-Fluorouracil (5-FU) remains the first-line chemotherapeutic agent for colorectal cancer. Although 5-FU significantly improves patient survival, its severe gastrointestinal toxicity-particularly intestinal injury and diarrhea-impairs treatment adherence and patient quality of life, often leading to therapeutic failure. Thus, effective interventions to prevent or mitigate these adverse effects are urgently needed. Betulin (BET), a natural pentacyclic triterpenoid derived primarily from birch bark, exhibits various biological activities, including anti-inflammatory, antioxidant, antiviral, and antitumor effects. Its anti-inflammatory and antioxidant properties suggest betulin (BET) as a promising candidate for alleviating chemotherapy-induced tissue damage. However, its impact on 5-FU-induced intestinal injury remains unclear. The findings of this study revealed that 5-FU led to significant intestinal injury by promoting cellular senescence and exacerbating the inflammatory response. BET mitigates these effects by decreasing senescence-associated β-galactosidase activity and downregulating key senescence markers such as p53, p21, and p16. Moreover, BET modulates senescence-associated secretory phenotype factors, thereby reversing the proinflammatory microenvironment elicited by 5-FU. Integrating network pharmacology, Mendelian randomization, and experimental validation, we identified the mechanistic target of rapamycin/mitogen-activated protein kinase signaling pathway as a pivotal mediator of BET's protective effects against 5-FU-induced intestinal injury. In conclusion, our study reveals that 5-FU-induced intestinal damage is driven by cellular senescence, which BET effectively ameliorates through suppression of senescence and inflammation. These findings provide a novel framework for targeting antisenescence strategies to alleviate chemotherapy-associated intestinal toxicity. SIGNIFICANCE STATEMENT: This study identifies betulin as a novel agent that alleviates 5-fluorouracil-induced intestinal injury by inhibiting cellular senescence and inflammation via the mechanistic target of rapamycin/mitogen-activated protein kinase pathways. These findings highlight antisenescence as a promising therapeutic strategy to mitigate chemotherapy-induced gastrointestinal toxicity.