To investigate the potential mechanism of Xiaoyao Pill in intervening breast precancerous lesions using network pharmacology, molecular docking, and molecular dynamics simulations.

Traditional Chinese medicine databases and platforms were used to retrieve the active components and potential targets of Xiaoyao Pill. Overlapping targets between Xiaoyao Pill and breast precancerous lesions were identified. A protein-protein interaction network was constructed, and core targets were screened from the overlapping targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were then performed. Key active components and core targets were subjected to molecular docking, and molecular dynamics simulations were conducted using GROMACS software to validate the network pharmacology results.

A total of 99 active components of Xiaoyao Pill, 1093 drug targets, 893 disease-related targets, and 265 overlapping targets were obtained. Gene Ontology enrichment analysis identified 2524 biological process terms, 53 cellular component terms, and 175 molecular function terms. Kyoto Encyclopedia of Genes and Genomes enrichment analysis identified 187 significantly enriched signaling pathways, primarily involving the AMPK signaling pathway, estrogen signaling pathway, and central carbon metabolism in cancer. Molecular docking showed that key targets, including AKT1, PIK3CA, ESR1, and CCND1, exhibited strong binding affinities with several core active components of Xiaoyao Pill, such as β-sitosterol, paeoniflorin, and quercetin. Molecular dynamics simulations further confirmed stable binding between the active components of Xiaoyao Pill and these targets.

Xiaoyao Pill may act on multiple signaling pathways, including the AMPK/mTOR and estrogen signaling pathways, by regulating core targets such as AKT1. It may thereby reduce abnormal energy metabolism in precancerous cells, modulate hormone-related signaling, regulate the cell cycle and immune microenvironment, and ultimately exert an interventional effect on breast precancerous lesions. This study provides new research evidence supporting the potential clinical application of this classical Chinese formula in the prevention and treatment of breast cancer.