Puerarin Suppresses Proliferation of ESCs Figure 1. Estrogen receptor competitive binding assay. ESCs were incubated in the presence of E2 or puerarin solutions for 1 h. After removal of the medium, [3H]-E2 binding capacity was measured. (A) Total binding of [3H]-E2 to ERs in ESCs; the concentrations of [3H]-E2 used were 0, 0.1950, 0.3906, 0.7813, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 nmol/L. (B) Non-specific binding of [3H]-E2. (C) Specific binding of [3H]-E2, calculated by subtracting the non-specific binding values from the total binding values. (D) Measurement of [3H]-E2 binding to ERs in the presence of varying concentrations of E2 (0, 0.0061, 0.0183, 0.0549, 0.1646, 0.4938, 1.4815, 4.4444, 13.3333 and 40 mmol/L) or puerarin (0, 0.0061, 0.0183, 0.0549, 0.1646, 0.4938, 1.4815, 4.4444, 13.3333 and 40 mmol/L). The figures represent data from three experiments; each experiment was performed in triplicate, and data are represented as the mean 6 SD. doi:10.1371/journal.pone.0045529.g001 enzyme in the biosynthesis of prostaglandin E2 (PGE2), which is believed to be implicated in the development of endometriosis and its symptoms [17]. The aromatase P450 (P450arom), which is encoded by the cyp19 gene, is a key enzyme for biosynthesis of estrogen, and is stimulated by PGE2. This results in local production of estrogen, which induces PGE2 formation and establishes a positive feedback cycle, becoming one of the important factors in the pathogenesis of endometriosis [18]. To assess the effects of E2-BSA on expression of these genes, the levels of the mRNA and protein products of the above genes were detected in ESCs subjected to the treatments indicated in Fig. 5. Our results showed that E2-BSA could induce cyclin D1 mRNA expression, and that this effect was suppressed by puerarin. Furthermore, when ESCs were pretreated with U0126, the effect of E2-BSA on cyclin D1 mRNA levels was considerably reduced (Fig. 5A) and puerarin couldn’t suppress the mRNA level any more. In addition, cyclin D1 protein levels, detected by western blotting, were found to correlate with mRNA levels (Fig. 5D). Similar results were obtained with cyp19 (Fig. 5B) and cox-2 (Fig. 5C), showing that the effects of E2-BSA and puerarin on cyclin D1, cyp19 and cox-2 mRNA expression and protein levels are ERK dependent. Discussion The current standard medical treatments for endometriosis include GnRH agonists, contraceptive steroids, progestogens, and androgens [4,19–20], all of which aim to lower circulating E2 concentrations. Of these agents, GnRH agonists appear to be the most effective, but they are expensive, and long-term treatment is not possible because of the loss of bone mineral density. However, puerarin is cheap, and it can be used for long periods without PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45529