Zailong Cai1* Abstract Puerarin Suppresses Proliferation of Endometriotic Stromal Cells Partly via the MAPK Signaling Pathway Induced by 17ß-estradiol-BSA Wen Cheng1., Lizao Chen1., Shengsheng Yang1., Jie Han 1 , Dongxia Zhai1, Jian Ni2, Chaoqin Yu1*, 1 Department of Traditional Chinese Medicine, Changhai Hospital and Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai, China, 2 Institute of Micro/Nano Science and Technology, Shanghai Jiaotong University, Shanghai, China Background: Puerarin 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). Methodology: ESCs 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 Findings: Our 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/Significance: Puerarin 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. Citation: Cheng W, Chen L, Yang S, Han J, Zhai D, et al. (2012) Puerarin Suppresses Proliferation of Endometriotic Stromal Cells Partly via the MAPK Signaling Pathway Induced by 17ß-estradiol-BSA. PLoS ONE 7(9): e45529. doi:10.1371/journal.pone.0045529 Editor: Luis Eduardo M. Quintas, Universidade Federal do Rio de Janeiro, Brazil Received April 17, 2012; Accepted August 20, 2012; Published September 19, 2012 Copyright: 2012 Cheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the National Natural Science Foundation of China (Project No. 30672742, 30873353, 30930113) and 973 project (2010CB933902). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chqyu81@gmail.com (CY); czl8003@hotmail.com (ZC) . These authors contributed equally to this work. Introduction Endometriosis (EM) is an estrogen-dependent benign disease characterized by extra-uterine implantation and ectopic growth of endometrium. It affects 10–20% of women in their reproductive years, causing chronic pelvic pain and infertility [1]. Current treatments, based on hormonal therapy or surgery, are often insufficient [2–4]. Therefore, exploring novel therapeutic strategies is necessary for improving the clinical management of patients with endometriosis. 17ß-estradiol (E2), which can be locally and distantly secreted by endometriotic cells and ovary, plays a critical role in the development and progression of endometriosis, in which it acts as an important regulator of cell proliferation, survival and differentiation. Clinical and experimental data have clearly established that E2 is the key factor leading to adhesion, aggression and angiogenesis in the pathological process of endometriosis [5]. The predominant physiological effects of E2 traditionally have been considered to be based on interaction of E2 with estrogen receptors (ERa and ERß). The effects are brought about via the regulation of transcriptional processes, involving nuclear trans- location, binding to specific estrogen response elements (EREs) and ultimately regulation of gene expression [6]. However, a number of studies have shown that steroid hormones, particularly E2, are able to elicit cellular responses that are too rapid to be mediated by the activation of transcription and PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45529