Study on the mechanism of action of HuaZhuoKeLi in modulating LCN2-mediated cellular pyroptosis to ameliorate ulcerative colitis.

Ulcerative colitis (UC) is characterized by chronic intestinal inflammation and epithelial barrier dysfunction. Although HuaZhuoKeLi (HZKL) has been clinically used for UC management, its underlying mechanism remains incompletely understood. Lipocalin-2 (LCN2) has been implicated in inflammation-associated cell death, including the NLRP3 inflammasome-mediated pyroptosis. This study aimed to investigate whether HZKL ameliorated UC by modulating LCN2-associated pyroptosis. Integrated bioinformatics analyses were performed using GEO datasets (GSE48959 and GSE75214 as the discovery set; GSE48958 as the validation set). Pyroptosis-related differentially expressed genes (DEGs) were identified using limma and sva normalization, followed by WGCNA to identify UC-associated gene modules. Hub genes were screened via LASSO regression and evaluated by ROC analysis. A dextran sulfate sodium (DSS)-induced UC mouse model was established to assess the effects of HZKL at different doses, using mesalazine as a reference treatment. Drug-containing serum was prepared for in vitro experiments using lipopolysaccharide (LPS)-stimulated Caco-2 cells with LCN2 overexpression or inhibition. Inflammation, pyroptosis, and barrier function were evaluated by histological staining, transmission electron microscopy (TEM), Western blotting, ELISA, qPCR, immunofluorescence, TUNEL assay, CCK-8 assay, and flow cytometry. HZKL notably alleviated colonic inflammation, improved intestinal barrier integrity, and reduced epithelial ultrastructural damage in DSS-induced UC mice. Bioinformatics analyses identified several UC-associated pyroptosis-related genes (LCN2, ZBP1, CD55, and S100A8), all of which exhibited diagnostic potential. Functional experiments demonstrated that HZKL attenuated pyroptosis-related signaling, predominantly accompanied by downregulation of LCN2, reduced inflammatory responses, and enhanced proliferation and migration of intestinal epithelial cells. This study identified LCN2 as a key drug-responsive regulator associated with pyroptosis in UC. HZKL attenuated intestinal inflammation and promoted barrier repair by modulating, at least in part, the LCN2-associated pyroptosis pathway. These findings may provide a mechanistic basis for the therapeutic effects of HZKL, while also highlighting LCN2 as a novel target for further investigation in UC treatment.