Network pharmacology and experimental validation reveal clotrimazole alleviates dextran sulfate sodium-induced ulcerative colitis by inhibiting intestinal senescence.

Ulcerative colitis (UC) is one of the spontaneous inflammatory bowel disease, which is characterized by chronic and relapsing intestinal mucosal inflammation that principally affects the colon. Chronic inflammation is a hallmark of senescent tissues, and intestinal tissue gradually appears injured with the occurrence of senescence. Therefore, senescence may aggravate the severity of UC. However, little is known about the relationship between UC and senescence. Clotrimazole (CTZ) is a synthetic azole broad-spectrum antifungal drug that has been also reported to have anti-inflammatory and anticarcinogenic effects. However, little research is available about CTZ therapeutic role and underlying mechanisms on UC and senescence. In this study, we identified that dextran sulfate sodium (DSS), a widely recognized inducer of UC, promotes senescence in normal human colon epithelial normal colon mucosa cell line 460 cells and murine colonic tissues. Furthermore, CTZ, identified from a compound library, exhibited potent antisenescence activity in DSS-treated normal colon mucosa cell line 460 cells. Our study revealed that CTZ, as an antisenescence agent, demonstrated significant efficacy in alleviating DSS-induced UC and mitigating colonic senescence. Through network pharmacology analysis, Mendelian randomization, and experimental validation, we identified the potential signaling pathways (AMP-activated protein kinase [AMPK]/ mitogen-activated protein kinase [MAPK]) underlying CTZ's therapeutic effects in UC. We discovered that DSS disrupts the AMPK signaling pathway while excessively activating the MAPK pathway, leading to cellular senescence. However, CTZ treatment significantly restored AMPK activity and inhibited MAPK activation, ultimately reducing cellular senescence. In conclusion, our findings suggest that DSS-induced UC can stimulate senescence in colonic tissues, while CTZ effectively alleviates DSS-induced colonic damage. Our results highlight the potential therapeutic value of CTZ in targeting intestinal senescence for UC treatment, laying a foundation for future antisenescence strategies in colitis therapy. SIGNIFICANCE STATEMENT: This study integrates network pharmacology and Mendelian randomization to identify clotrimazole as a potent inhibitor of intestinal senescence, revealing its therapeutic potential for colitis. Experimental validation confirms that clotrimazole alleviates dextran sulfate sodium-induced colitis by targeting cellular senescence, highlighting antisenescence as a promising strategy for colitis treatment.