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

et al. (2011) Puerarin Suppresses Invasion and Vascularization of Endometriosis Tissue Stimulated by 17b-Estradiol. PLoS ONE 6(9): e25011. doi:10.1371/journal.pone.0025011 Puerarin Suppresses Invasion and Vascularization of Endometriosis Tissue Stimulated by 17b-Estradiol Dan Wang 0 Yuhuan Liu 0 Jie Han 0 Dongxia Zai 0 Mei Ji 0 Wen Cheng 0 Ling Xu 0 Luxi Yang 0 Miaoxia He 0 Jian Ni 0 Zailong Cai 0 Chaoqin Yu 0 Jean-Marc A. Lobaccaro, Clermont Universite, France 0 1 Department of Chinese Traditional Medicine, Changhai Hospital, Second Military Medical University , Shanghai , China , 2 Clinical Research Center, Changhai Hospital, Second Military Medical University , Shanghai , China , 3 Department of Obstetrics and Gynecology, Changhai Hospital, Second Military Medical University , Shanghai , China , 4 Department of Pathology, Changhai Hospital, Second Military Medical University , Shanghai , China , 5 Institute of Micro/Nano Science and Technology, Shanghai Jiaotong University , Shanghai , China Background: Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17b-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 (1028 mol/L) promoted the invasiveness of ESCs by increasing MMP-9 accumulation and decreasing TIMP-1 accumulation. Interestingly, puerarin (1029 mol/L) significantly reversed these effects (P,0.01). The CAM assay indicated that puerarin (1029 mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (1028 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. - . These authors contributed equally to this work. Endometriosis, a common, benign, estrogen-dependent disease affecting 3%10% of women of reproductive age, is defined as the presence of endometrial glands and stroma outside of the uterine cavity [1]. It is not a malignant disorder but it has similarities to a developing tumor. For instance, endometriotic cells can be both locally and distantly metastatic, that is they can attach to, invade and damage other tissues [2,3]. The initial phase of endometriosis is an invasive event that requires extracellular matrix (ECM) breakdown. The key enzymes that regulate the ECM are the matrix metalloproteinases (MMPs), and peritoneal invasion by endometrial tissue is thought to be dependent on MMPs and their corresponding tissue inhibitors of metalloproteinases (TIMPs). MMPs play a pivotal role in the cyclic changes of growth and tissue breakdown that occur in the endometrium. MMPs are usually synthesized during the proliferative phase and are stimulated by estrogen [4]. Several studies have shown an increase in the accumulation of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 in endometriotic tissues. Alteration of MMP-9 and MMP-2 is an important factor in the development of endometriosis [2,3]. Vascularization of endometriotic implants is probably one of the most important factors in the invasion of other tissues by endometrial cells. Angiogenesis is controlled by a number of inducers; of particular importance is the vascular endothelial growth factor (VEGF) family, which is significant in processes characterized by both physiological and pathological angiogenesis. Accumulation of the VEGF gene in normal human endometrial cells is acutely upregulated by E2 in vitro [5]. VEGF immunostaining has been observed in the epithelium of endometriotic implants, particularly in hemorrhagic red implants [6]. Most of the current medical treatments for endometriosis aim to downregulate estrogen activity. Historically, medical therapies have included contraceptive steroids, progestogens, and agonists of gonadotropin-releasing hormone (GnRH), as well as androgens and non-steroidal anti-inflammatory agents [7]. These treatments can only be used for a limited time because of unacceptable side effects. In addition, high recurrence rates after medical treatments are the most significant problem [8]. Therefore, exploring novel therapeutic strategies is necessary for improving the clinical management of patients with endometriosis. Phytoestrogens exhibit estrogenic and anti-estrogenic activities both in vitro and in vivo [9]. Puerarin, a phytoestrogen derived from the Chinese medicinal herb Radix puerariae, has been proven practical in the management of various cardiovascular disorders, alcoholism, and neurological disease [10]. However, whether puerarin could suppress invasion and vascularization of endometriotic tissue is unknown. Establishment of the primary ectopic ESCs Endometriotic stromal cells (ESCs) should be stained positive with anti-vimentin-PE monoclonal antibody by fluoroimmunoassay. The negative cells could fibroblast cells, gland cells and epithelial cells. The purity of ESCs in isolated cells was .95% (Fig. 1). Puerarin suppresses invasion of E2-stimulated ESCs For the following experiments, 1028 mol/L E2 and 1029 mol/ L puerarin were selected because of their efficacy on ESCs in our preliminary experiments. In Fig. 2A, 1028 mol/L E2 showed a stimulatory effect on ESCs invasion compared with the untreated (vehicle) control cells, but the combination of E2 with puerarin reduced this effect (P,0.01). To characterize fully the anti-estrogenic effects of puerarin, we analyzed the accumulation of MMP-9 and TIMP-1 under same treatment. As shown in Fig. 2B, the accumulation of MMP-9 was increased whereas the accumulation of TIMP-1 was decreased after the E2 treatment compared with the untreated control. With the combination of E2 and puerarin treatment, the effect was significantly reversed. Puerarin inhibits angiogenesis in CAM in vivo To explore whether puerarin could inhibit vasculariz (...truncated)