Quercetin Modulates the Senescence-Fibrotic Microenvironment in Uterine Fibroid Cells: A Potential Non-Hormonal Therapeutic Approach

Introduction: Uterine fibroids are common benign tumors of the reproductive tract, affecting millions of women during their reproductive years. Fibroids are characterized by excessive deposition of extracellular matrix (ECM) proteins such as collagens, fibronectin, and proteoglycans. The most common symptoms include heavy and prolonged menstrual bleeding, pelvic pain, and infertility. Treatment options are limited, particularly non-hormonal medical therapies. Recently, we and others have reported that fibroids exhibit a senescence-fibrotic microenvironment. Cellular senescence is characterized by expression of cell cycle inhibitors such as p16Ink4a. Senescent cells, arrested in division, secrete inflammatory cytokines and growth factors, collectively known as the senescence-associated secretory phenotype (SASP), which may promote fibrosis. Quercetin, a natural compound found in fruits, vegetables, and beverages like red wine and black tea, possesses anti-inflammatory and anti-fibrotic properties. We hypothesized that quercetin would modulate the senescence-fibrotic microenvironment in fibroid cells.

Materials and

Methods: Fresh fibroid samples were collected in HBSS immediately after surgery under approved IRB protocols and established primary cultures following mechanical (cutting into 1mm pieces) and enzymatic (Collagenase/DNase) approaches. Early passages (1-5) of these primary fibroid cell cultures were exposed to different concentrations of quercetin (1, 10, 50, and 100 µM) or to a vehicle (DMSO) for 48 hours. Protein expression was quantified using western blotting. Gene expression was quantified using real-time PCR after treating the cells with 50 µM quercetin for 48 hours. Data were analyzed using the Mann-Whitney U test in GraphPad Prism 10 (version 10.2.0, build 392), with significance determined at a p-value of ≤ 0.05.

Results, Conclusions, and Discussions: Treatment of fibroid cells with quercetin led to a dose-dependent significant reduction in p16 protein levels, by 19.9% at 50 µM (p=0.0087) and by 39.4% at 100 µM (p=0.0043) compared to control fibroid cells (Fig 1A). The mRNA levels of p16 (CDKN2A) also significantly decreased with 50 µM quercetin (p=0.0022) (Fig 1B). Quercetin treatment significantly reduced TNFA levels (p=0.0022) and increased IL1A, IL1B, and IL8 levels (p=0.0022) (Fig 1C). Several fibrosis markers, including fibronectin, collagen, plasminogen activator inhibitor-1 (PAI1), and α-smooth muscle actin (α-SMA), were evaluated. Quercetin treatment led to a dose-dependent reduction in protein levels of fibronectin (39.3% at 50 µM, p=0.02 and 63.3% at 100 µM, p=0.0002), COL1A1 (32.6% at 50 µM, p=0.24 and 33.98% at 100 µM, p=0.02), PAI1 (46.5% at 50 µM, p=0.002 and 75.3% at 100 µM, p=0.002), and α-SMA (32.3% at 50 µM, p=0.049 and 44.7% at 100 µM, p=0.003) (Fig 1D). Correspondingly, mRNA levels of FN1, COL1A1, COL3A1, PAI1, and ACTA2 were significantly reduced with quercetin (p=0.002) (Fig 1E). Gene expression of Prolyl 4-hydroxylase, alpha polypeptide I (P4HA1), an enzyme involved in collagen synthesis, was also significantly reduced by quercetin (p=0.0022) (Fig 1E). Mechanistically, quercetin (100 µM) significantly reduced YAP signaling (p=0.04) but not Smad (p=0.87) or β-catenin (p=0.38) (Fig 1F). This YAP-specific action was further confirmed by a reduction in the protein levels of the YAP-responsive gene CTGF (55.5% at 100 µM, p=0.0003) (Fig 1G). Additionally, the YAP target gene ANKRD1 mRNA levels significantly decreased after quercetin treatment (p=0.0022) (Fig 1H).
Taken together, these results indicate that quercetin acted as a senolytic agent, altering the senescence-fibrotic microenvironment of uterine fibroids. Quercetin reduced p16 protein and mRNA levels, indicating it may attenuate cellular senescence in fibroid cells. Quercetin treatment selectively affected SASP markers, reducing TNFα while increasing IL1α, IL1β, and IL8, suggesting a complex regulation of the inflammatory microenvironment. Quercetin also significantly reduced key fibrosis markers such as fibronectin, collagen, PAI1, and α-SMA, suggesting its potential as an anti-fibrotic therapeutic agent for uterine fibroids.

Acknowledgements (Optional): NIH grant R01HD111243 to JS, MAB, JHE.