Revealing the lactation-promoting mechanism of the phenolic extracts in Hemerocallis citrina Baroni based on network pharmacology, transcriptomics, proteomics, and molecular docking

Abstract Impaired lactation often causes early cessation of exclusive breastfeeding, increasing health risks for infants and mothers. Hemerocallis citrina Baroni, as a food ingredient, is renowned for its potential to improve lactation. However, its active compounds and specific lactation-promoting mechanisms remain unclear. Herein, we employed an integrated multi-omics strategy combining network pharmacology, transcriptomics, proteomics, molecular docking and experimental validation to analyze the lactation-promoting mechanism of H. citrina. Our results showed that ethanol extract of H. citrina (HEE) significantly upregulated serum lactation-related hormone levels and promoted mammary gland development, which resulted in improved lactation. In total, 159 phenolics were identified by ultra-high performance liquid chromatography-Q-exactive orbitrap-mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). Based on the network pharmacology result, 67 intersecting target genes between HEE phenolics and lactation-related targets were identified. The constructed HEE phenolics-target-pathway network indicated that HEE might act on key targets (tumor necrosis factor (TNF), protein kinase 1 (AKT1), epidermal growth factor receptor (EGFR), hypoxia-inducible factor 1 subunit alpha (HIF1A) and cysteinyl aspartate-specific protease 3 (CASP3)) through active phenolics including caffeic acid, 2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one, gnaphaliin, chrysoeriol, and diosmetin. Further combined analysis using network pharmacology and multi-omics (transcriptomics and proteomics) suggested that the regulation of AKT1, CASP3, and the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway could be important mechanisms by which HEE improves impaired lactation. Molecular docking and molecular dynamics (MD) simulation validated that the key active phenolics in HEE bind well and stably with core targets. Cellular experiments further showed that HEE significantly promoted mammary epithelial cell proliferation and enhanced milk components synthesis via the PI3K-AKT signaling pathway, providing new insights into exploring the development and application of H. citrina to alleviate impaired lactation.