Enhancing the Efficacy of Ulcerative Colitis Treatment by Inhibiting LCN2-Mediated Pyroptosis Through Traditional Processing Techniques: With the rhizome of Atractylodes macrocephala Koidz. as an Example.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes Macrocephala Rhizome (AMR), the dried rhizome of Atractylodes macrocephala Koidz (family Asteraceae), is a commonly used Chinese herbal medicine for treating ulcerative colitis (UC) and has extensive applications in both Traditional Chinese Medicine (TCM) and modern medicine. The therapeutic effects of its distinctive processed variety, rice-washed water processed Atractylodis Macrocephalae Rhizoma (Migan Shui Piao Baizhu, R-AMR), have yet to be reported, and its potential mechanisms of action require further exploration. AIM OF THE STUDY: This study aims to compare the therapeutic effects of R-AMR and raw AMR on UC mice and further explore their mechanisms of action, thereby providing insights into the processing mechanism of AMR using rice-washed water. METHODS: Firstly, a DSS-induced mouse UC model was established in vivo. The efficacy of treatment with rice-washed water processed AMR in improving UC was evaluated by DAI scores, histopathological changes, serum inflammatory cytokines, tight junction proteins (ZO-1, Claudin-1), and mucin (MUC2) levels. Then, bioinformatics, machine learning, and molecular docking were integrated to identify core disease targets and explore the mechanism of R-AMR in treating UC. Nextly, an LPS-induced UC cell model was established using NCM460 cells, and LCN2 siRNA-transfected cell experiments were conducted. The biological effects and mechanisms of action of key targets were validated in vitro through immunofluorescence, WB, and RT-qPCR techniques. Finally, we validated the roles of the above-identified pathways in the treatment of UC with AMR through in vivo experiments. RESULTS: In in vivo efficacy experiments, both R-AMR and raw AMR effectively reduced DAI scores and histopathological scores in UC mice. They downregulated proinflammatory factors and upregulated anti-inflammatory factors while increasing tight junction protein expression (ZO-1, Claudin-1) to restore damaged intestinal epithelial mucosal barriers. R-AMR demonstrated superior therapeutic effects compared to raw AMR. Bioinformatics and machine learning results indicate that Lipocalin 2 (LCN2) and Tissue inhibitor of metalloproteinase 1 (TIMP1) is a key target in UC, with the NOD-like receptor signaling pathway being one of its representative pathways. Molecular docking results reveal that Atractylenolide I (Atr I) has a strong affinity for LCN2 and is one of the active components enhancing the efficacy of R-AMR. In vitro experiments demonstrate that Atr I restores tight junctions between LPS-induced NCM460 cells. Targeted inhibition of LCN2 reduces NLRP3 inflammasome, Caspase 1 activation, and GSDMD-N expression, decreases proinflammatory factor release (IL-1β, IL-18, TNF-α, IL-6), and alleviates UC inflammatory responses. In vivo experiments verified that the therapeutic effect of AMR on UC is associated with the LCN2/NLRP3/ Caspase 1/GSDMD pyroptosis pathway. CONCLUSIONS: Both raw AMR and R-AMR exhibit protective effects against UC, with R-AMR displaying superior therapeutic outcomes. Its bioactive constituent Atr I attenuates pyroptosis through targeted inhibition of LCN2, leading to preservation of the intestinal epithelial mucosal barrier and subsequent amelioration of UC symptoms.