Puerarin Suppresses Invasion and Vascularization of Endometriosis Tissue Stimulated by 17β-Estradiol

BACKGROUND: Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17β-estradiol (E2) and producing an anti-estrogenic effect. This study was to investigate whether puerarin could suppress the invasion and vascularization of E2-stimulated endometriotic tissue. METHODOLOGY/PRINCIPAL FINDINGS: The endometriotic stromal cells (ESCs) were successfully established and their invasive ability under different treatments was assessed through a Transwell Assay. Simultaneously, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by western blotting. Vascularization of endometriotic tissues was observed by chicken chorioallantoic membrane (CAM) assay. The staining of MMP-9, intercellular adhesion molecule 1 (ICAM-1), TIMP-1, and vascular endothelial growth factor (VEGF) in grafted endometriotic tissues was examined using immunohistochemistry analysis. The purity of ESCs in isolated cells was >95%, as determined by the fluoroimmunoassay of vimentin. E2 (10(-8) mol/L) promoted the invasiveness of ESCs by increasing MMP-9 accumulation and decreasing TIMP-1 accumulation. Interestingly, puerarin (10(-9) mol/L) significantly reversed these effects (P<0.01). The CAM assay indicated that puerarin (10(-9) mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (10(-8) mol/L) treatment (P<0.05). Accordingly, immunohistochemistry showed that the accumulation of MMP-9, ICAM-1, and VEGF was reduced whereas that of TIMP-1 increased in the combination treatment group compared with the E2 treatment group. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that puerarin could suppress the tissue invasion by ESCs and the vascularization of ectopic endometrial tissues stimulated by E2, suggesting that puerarin may be a potential drug for the treatment of endometriosis

Representative photomicrographs of the fluoroimmunoassay for vimentin in the isolated ESCs.

<p>The cells were stained with DAPI (a, d), mouse IgG1 PE (b) and mouse-anti-human vimentin PE (e). The photographs (a and b; d and e) were merged on the right (c; f) respectively.</p

Puerarin inhibits angiogenesis in CAM in vivo.

<p>10⁻⁸ mol/L E2 or combination with 10⁻⁹ mol/L puerarin or normal saline containing 0.1% DMSO (v/v) were loaded on gelatin sponges which were loaded on the chick chorioallantoic membranes of chick embryos respectively. After 72 h incubation, 10% v/v formaldehyde was added onto the surface of CAMs to fix the blood. The disc and surrounding CAMs were incised carefully and photographed; representative photographs show the CAMs of 10⁻⁸ mol/L E2 or combination with 10⁻⁹ mol/L puerarin or normal saline containing 0.1% DMSO (v/v) treatment groups (A). Number of visible blood vessel branch points are statistically shown (B). n = 6, * P<0.05, ** P<0.01.</p

MMP-9, TIMP-1, ICAM-1, and VEGF staining of the endometriotic xenografts in the CAM model treated with vehicle, E2, and E2 + puerarin.

<p>The cells with brown color represent protein positive. E2-treated xenografts show a stronger signal for MMP-9, ICAM-1, and VEGF, and a weaker signal for TIMP-1. Co-treatment with puerarin significantly suppressed this effect. −: very weak staining; +: weak staining; ++: strong staining; +++: very strong staining. n = 6. NC: Negative control, Immunostaining for untreated group slice (omitting primary antibody).</p