Ruhe Sanjie Tablet ameliorates depression-associated mammary gland hyperplasia by modulating estrogen receptor signaling and CDK2.

[ETHNOPHARMACOLOGICAL RELEVANCE] Mammary gland hyperplasia (MGH) is prevalent estrogen-dependent disorder involving hormonal imbalance and psychological comorbidity, which significantly elevates breast cancer risk. Ruhe Sanjie Tablet (RST), a traditional Chinese herbal formula, is clinically effective in alleviating MGH symptoms, yet its underlying mechanisms, particularly concerning associated depressive behaviors, remain insufficiently elucidated.

[AIM OF THE STUDY] This study investigated the therapeutic effects and integrated mechanisms of RST on MGH in a rat model.

[MATERIALS AND METHODS] The rat MGH model was established through sequential injections of estradiol benzoate for 25 days and followed by progesterone for 5 days. The effects of RST on MGH were evaluated via mammary nipple morphology and depression-like behaviors (open field, sucrose preference, and forced swim tests). Hematoxylin and eosin (H&E) staining was utilized for pathological examination on mammary and ovarian tissues. Serum concentrations of cytokines (IL-6, IL-1β, TNF-α) and sex hormones (E, PRL, P, FSH, LH) were measured by ELISA. Protein expressions were confirmed via Western blotting (CDK2, Cyclin E) and immunohistochemistry (ERα, ERβ, Cyclin E1, Ki-67, PR, and PCNA in mammary gland, and GnRH, ERα, and ERβ in hypothalamus). An integrated approach employing network pharmacology, molecular docking, dynamics simulations, and untargeted metabolomics was utilized to identify core targets and metabolites of RST constituents.

[RESULTS] RST significantly reduced mammary nipple diameter/height and alleviated depression-like behaviors (increased exploration, sucrose preference, active coping). It decreased serum pro-inflammatory cytokines and rebalanced sex hormones (lowered E/PRL, elevated P/FSH/LH). Histopathological improvements in mammary and ovarian tissues were observed. Mechanistically, network pharmacology and molecular docking identified CDK2 as a core target, and Epmedin C exhibiting superior binding affinity and stability with a higher relative content (the third) in RST. Experimental validation confirmed that RST downregulated expression of ER, PR, PCNA, CDK2, and Cyclin E in mammary, indicating a disruption of estrogen signaling and G1/S phase cell cycle progression. Metabolomics revealed that RST mainly modulated 10 differential metabolites, upregulating anti-inflammatory lipids and downregulating pro-inflammatory mediators.

[CONCLUSION] RST alleviates MGH and its comorbid depressive behaviors through multi-target actions involving hormonal rebalancing, suppression of estrogen receptor signaling, inhibition of CDK2-mediated cell cycle progression, and modulation of inflammatory metabolites.