Integrating multi-omics approaches to elucidate the mechanism of XiaoPi Decoction in treating hyperplasia of mammary glands via AMPK signaling pathway activation.

BACKGROUND: Hyperplasia of Mammary Glands (HMG) is a non-inflammatory, non-neoplastic condition characterized by structural abnormalities in breast tissue, resulting from varying degrees of mammary parenchyma and stroma hyperplasia and incomplete regeneration. XiaoPi Decoction (XPD), an empirical formula for HMG in our hospital, has shown clinical efficacy by reducing breast nodule volume, alleviating mammary pain, and regulating sex hormone levels and T lymphocyte subset activity. It also exhibits high safety, but its precise therapeutic mechanisms remain unclear. The primary aim of this study is to systematically elucidate the therapeutic mechanisms of XPD in treating HMG.

METHODS: First, UPLC-Q-TOF-MS was used to analyze XPD's chemical components. Subsequently, an estrogen-progesterone induced HMG rat model was established to verify XPD's efficacy and explore its molecular mechanisms. Network pharmacology (with multi-database cross-validation) and transcriptomic analysis (on mammary tissue of control, model, and medium-dose XPD groups) were applied to identify potential molecular targets and pathways. Additionally, non-targeted metabolomics (on the same three groups as transcriptomics) was used to assess metabolic changes in HMG rats after XPD intervention, with a three-tier quality control protocol to mitigate batch effects.

RESULTS: Fifty-two chemical components were identified in XPD, mainly including thioglucosides, phenolic acids, and alkaloids. Animal studies showed XPD normalized mammary tissue structure and hormone levels: it decreased estradiol (E2), prolactin (PRL), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) concentrations, increased progesterone (P) levels, and downregulated mRNA expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and prolactin receptor (PRLR) in mammary tissue. Network pharmacology and transcriptomics indicated XPD targeted the AMPK/mTOR/HIF1/VEGF signaling pathways. Metabolomics revealed XPD affected fatty acid biosynthesis. Western blot confirmed XPD reduced protein expression of AMPK, mTOR, ACC, p62, and VEGF, while upregulating ULK1 and TSC2 expression and their phosphorylation.

CONCLUSION: This study suggests XPD exerts therapeutic effects on HMG by modulating hormone levels. Through AMPK activation, XPD inhibits fatty acid synthesis and energy metabolism, which reduces intracellular lipid accumulation and suppresses excessive cell proliferation. Additionally, it improves cellular energy status, thereby alleviating abnormal cell proliferation caused by energy metabolic disorders. XPD also promotes autophagy by inhibiting the downstream mTOR pathway, further suppressing cell proliferation. Moreover, it inhibits VEGF expression to exert anti-angiogenic effects, reducing blood supply to hyperplastic breast tissue and restricting its growth-thus contributing to HMG treatment.