Abstract
BackgroundPuerarin is a major isoflavonoid compound extracted from Radix puerariae. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E2-BSA).MethodologyESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway.Principal FindingsOur data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E2). E2-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E2-BSA.Conclusions/SignificancePuerarin suppresses proliferation of ESCs induced by E2-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.
Highlights
Endometriosis (EM) is an estrogen-dependent benign disease characterized by extra-uterine implantation and ectopic growth of endometrium
The relative binding affinity (RBA) calculated from the assays was 32.2%, suggesting that a direct interaction exists between estrogen receptors (ERs) and puerarin, and that the binding ability of puerarin is one third that of E2
Puerarin is cheap, and it can be used for long periods without severe side effects; medical treatment with this agent is a good option for avoiding disease relapse after the initial surgical and/or medical therapy
Summary
Endometriosis (EM) is an estrogen-dependent benign disease characterized by extra-uterine implantation and ectopic growth of endometrium. Many rapid effects of estrogen, with an onset in seconds, have been documented in numerous tissues, and the time courses of these events, which are similar to those elicited by growth factors and peptide hormones, suggest that they do not require precedent gene transcription or protein synthesis [7]. Rather, these rapid non-genomic effects of estrogen may be due to activation of membrane-associated signaling pathways, including protein kinase A, phosphotidylinositol-3 kinase and mitogenactivated protein kinase (MAPK) signaling pathways [8,9,10]. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the nongenomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E2-BSA)
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