transduction mechanisms. 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 mitogen- activated protein kinase (MAPK) signaling pathways [8–10]. Puerarin is a major isoflavonoid compound extracted from the Chinese medicinal herb, Radix puerariae. This compound has been suggested to be useful in the management of various disorders, such as coronary artery disease, liver fibrosis, neurotoxicity and bone injury [11–14]. Puerarin, a phytoestrogen with a weak estrogenic action, binds to estrogen receptors, thereby competing with E2 and producing an anti-estrogenic effect. In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. Subsequently, the effects of puerarin to treat rat models of endometriosis, and to suppress invasion and vascularization of endometriotic tissue stimulated by E2 were elucidated in our laboratory [5,15]. However, the underlying molecular mechan- isms mediating such actions of puerarin are still to be further characterized, including the ability of puerarin to bind to ERs and non-genomic effects of puerarin. Therefore, the main aims of the present study were to determine the ability of puerarin to bind to ERs, and to investigate the non-genomic effects of puerarin and its downstream signaling pathway. Results Estrogen Receptor Competitive Binding Assay To test the ability of puerarin and E2 to bind competitively to ERs, ESCs were isolated and cultured, so that the purity of ESCs amongst the isolated cells was around 95% [5]. The radioactivity count was measured in the absence (for the total binding assay) (Fig. 1A) or in the presence (for the non-specific binding assay) of a 100-fold molar excess of unlabeled E2 (Fig. 1B). The Kd value determined from the above two assays was 96 nmol/L (Fig. 1C). In the subsequent experiments, specific binding of [3H]-E2 to ERs was competed by increasing concentrations of unlabeled E2, and the radioactivity value decreased in a concentration-dependent manner. The IC50 value for E2 was found to be 0.28 mmol/L, which was calculated by using Graphpad Prism 5.0 software. Over the same range of concentrations as unlabeled E2, puerarin also competed against the specific binding of [3H]-E2 to ERs, and the IC50 of puerarin was determined to be 0.87 mmol/L (Fig. 1D). The RBA calculated from the assays was 32.2%, suggesting that a direct interaction exists between ERs and puerarin, and that the binding ability of puerarin is one third that of E2. ERK1/2 was Highly Activated by E2-BSA, which was Reversed by Puerarin To discover whether a non-genomic pathway could be activated by E2-BSA and whether it could be reversed by puerarin, ESCs were treated with DMSO vehicle or E2-BSA or E2-BSA plus puerarin or puerarin alone, and human phospho-kinase array assays were performed. As shown in Fig. 2A, ERK1/2, AKT and Chk2 were activated in the E2-BSA treatment group, as compared with the DMSO treatment group, and high levels of phosphor- ylation of all three were further confirmed by western blot (Fig. 2B). Interestingly, the phosphorylations were sharply reduced by treatment with E2-BSA combining puerarin (Fig. 2A), as Puerarin Suppresses Proliferation of ESCs compared with the E2-BSA treatment group. Since the activation of ERK1/2 was a novel finding and had a significant change in this setting, we focused on ERK1/2 in the following experiments. The ESCs were treated with either vehicle or a range of concentrations of E2-BSA (10210 to 1026 mol/L) for 15 min to determine the most effective concentration to cause ERK phosphorylation. Western blotting showed that E2-BSA increased the levels of p-ERK1/2 at all concentrations tested. However, the most effective concentration of E2-BSA was 1028 mol/L, which induced the highest increase in p-ERK1/2 levels, to a level that was about fivefold greater than that seen in control cells (Fig. 3A). To determine the time course of this E2-BSA action, ESCs were treated with E2-BSA (1028 mol/L) for 0, 15, 30, 45 and 60 min. E2-BSA was able to increase ERK1/2 phosphorylation, reaching a peak at 15 min before declining sharply to baseline levels within 1 h (Fig. 3B). To assess the effect of puerarin on the induction of ERK1/2 phosphorylation by E2-BSA, ESCs were treated with puerarin (1029 mol/L), with or without E2-BSA, for 15 min. Fig. 3C shows that puerarin decreased the activating effect of E2-BSA on ERK1/ 2 phosphorylation. To further identify any potential non-classical ER involvement in the induction of ERK1/2 phosphorylation by E2-BSA, the above experiments were repeated in ESCs that had been pretreated with the ER antagonist, ICI182,780. The results showed that pretreatment with ICI182,780 inhibited the effect of E2-BSA on ERK1/2 phosphorylation (Fig. 3C), suggesting that this phosphorylation effect is mediated by the plasma membrane ERs (mER). To investigate the effect of a MAPK/ERK kinases (MEK) inhibitor on the induction of ERK1/2 phosphorylation by E2- BSA, cells were pretreated with U0126 (20 mmol/L) for 30 min, and then E2-BSA alone, E2-BSA plus puerarin, or puerarin alone, were added to the cultures for 15 min. Pretreatment with U0126 abolished ERK phosphorylation elicited by E2-BSA (Fig. 3C), suggesting that ERK phosphorylation in the presence of E2-BSA is MEK-dependent. E2-BSA Induces the Proliferation of ESCs, which was Reversed by Puerarin To find out whether E2-BSA could induce proliferation of ESCs, and whether this induction could be reversed by puerarin, we carried out cell proliferation experiments. As shown in Fig. 4A, when compared with vehicle control, ESCs treated with the indicated concentrations of E2-BSA for 4 d demonstrated a signif- icant increase in cell proliferation, with the maximal effect seen at a concentration of 1028 mol/L E2-BSA (P,0.01). We then investigated the potential effect of puerarin on cell proliferation promoted by 1028 mol/L E2-BSA. Compared with the E2-BSA group, cell proliferation decreased when E2-BSA treatment was combined with puerarin. In addition, puerarin alone was found to suppress cell growth slightly but not statistically significant (P.0.05), as compared with the DMSO group (Fig. 4B). This effect of E2-BSA to stimulate cell proliferation was significantly reduced by pretreatment with U0126 for 60 min, suggesting that the ERK pathway is involved in this process (Fig. 4C). Puerarin Suppresses Related Gene Expression Induced by E2-BSA in ESCs Cyclin D1 serves as a key sensor and integrator of extracellular signals in cells in the early-to-mid G1 phase of cell proliferation and differentiation [16]. Cyclooxygenase-2 (COX-2) is a key PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45529