Target prediction and preliminary validation of quercetin in treatment of endometriosis

To investigate the multi-target mechanisms of quercetin in treating endometriosis (EMT) through integrative network pharmacology analysis.

Active targets of quercetin were collected from the TCMSP database, while EMT-related differentially expressed genes (DEGs) were identified through the Gene Expression Omnibus (GEO) dataset. A comparative analysis was conducted to pinpoint potential therapeutic targets of quercetin for EMT treatment. Functional enrichment analyses were employed to investigate the biological functions associated with these targets, and a protein-protein interaction (PPI) network was conducted to identify core targets. Molecular docking and dynamics simulations were performed to validate the binding characteristics between quercetin and the core targets. The top 2 target protein pairs, HSP90AB1 and AR, exhibiting the lowest binding energy, were selected for subsequent cellular experimental validation. Human EMT-immortalized ectopic endometrial epithelial cell line 12Z (n=6, independent replicates) was subjected, and CCK-8 assay was used to determine ehe effects of quercetin on cell viability and proliferation, and the half-maximal inhibitory concentration (IC50) was calculated at 48 h after treatment. Then the 12Z cells were treated with quercetin at a concentration gradient of 0, 30, 60 and 90 μmol/L, the migration and invasion abilities were assessed with cell scratch and cell invasion assays. Western blotting was conducted to detect the changes in the expression of HSP90AB1 and AR proteins after different doses of treatment.

There were 49 potential EMT-related therapeutic targets and 10 core targets identified. Functional enrichment analyses revealed that these targets were significant enriched in inflammation-related signaling pathways, including AGE-RAGE, ErbB and TNF; immune-related pathways, such as Th17 cell differentiation, T/B cell receptor signaling; angiogenesis-related pathways like VEGF; and hormonal regulatory pathways involving estrogen and GnRH. Molecular docking demonstrated that quercetin exhibited favorable binding activity (binding energy <-5 kcal/mol) with all core target proteins, with particularly strong binding energies (<-7 kcal/mol) observed for AR, EGFR, FOS, ERBB2, and HSP90AB1. Molecular dynamics simulations revealed that quercetin forms sustained hydrogen bond interactions with AR and HSP90AB1, facilitating the formation of stable complexes. CCK-8 assay, cell scratch assay, and transwell invasion assay indicated that quercetin inhibited the proliferative activity, and migrative and invasive abilities of 12Z cells in a concentration-dependent manner, with more pronounced inhibitory effects observed at 60 and 90 μmol/L quercetin (P<0.001); Western blotting revealed that treatment of 12Z cells with varying quercetin concentrations for 48 h up-regulated the expression of HSP90AB1 and AR, with the most significant increase observed at 90 μmol/L quercetin (HSP90AB1, P<0.05; AR, P<0.001). The restored expression levels of HSP90AB1 and AR showed positive correlations with the proliferative activity, migrative and invasive abilities of ectopic endometrial cells.

Quercetin effectively addresses endometriosis through multiple molecular targets and signaling pathways, and stabilization of the HSP90AB1/AR complex and subsequent protein upregulation represents a key therapeutic mechanism.