Xi Huang Pill exerts anti-triple-negative breast cancer effects by modulating gut microbiota, regulating glycerophospholipid metabolism, and promoting autophagy homeostasis.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype of breast cancer, characterized by high malignancy and poor prognosis. Xihuang Pill (XHP), a traditional Chinese medicine (TCM) formulation, has demonstrated therapeutic efficacy against TNBC. This study investigates the antitumor mechanisms and therapeutic potential of XHP. The chemical composition of XHP was analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). The murine TNBC model was established via orthotopic implantation of 4T1 cells, followed by XHP treatment. Multi-omics analyses including gut microbiota profiling, metabolomics, and molecular biology approaches were conducted, along with the use of a pseudo-germ-free mouse model.XHP inhibited TNBC cell proliferation, induced apoptosis, and suppressed tumor growth, while also enhancing immune function, as evidenced by an increased CD4 + /CD8 + T-cell ratio. Integrated analysis identified four key bacterial genera (Deferribacterota, Mucispirillum, Eisenbergiella, and Monoglobus) and 13 metabolites involved in glycerophospholipid metabolism. XHP downregulated PCYT1A and PCYT2, key enzymes in this pathway. However, antibiotic-induced depletion of gut microbiota abolished XHP's antitumor and metabolic regulatory effects. Furthermore, XHP enhanced autophagy, as indicated by an increased LC3-II/I ratio and decreased p62 expression.In conclusion, XHP exerts antitumor effects against TNBC by modulating gut microbiota-dependent glycerophospholipid metabolism and restoring autophagy homeostasis. These findings provide novel insights into the anticancer mechanisms of TCM by elucidating microecological-metabolic interactions.