The Effect Of Puerarin On Cell Proliferation Of Endometriotic Stromal Cells In Part Via Regulating The Recruitment Of Estrogen Receptor-α

Background and objective:Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epitheliumand stroma outside the uterine cavity, which grows and regresses in an estrogen-dependent manner. It affects up to5-10%of women of reproductive age. The available treatment options of endometriosis are limited.The effects of estrogen are mediated via estrogen receptor-a (ER-a) and ER-B, and subsequently altering transcription. Furthermore, ER-a activity can be regulated through interaction with a multitude of coregulatory factors, termed co-activators and co-repressors. It is now thought that inhibition of steroid receptor function with pharmacological antagonists is not simply a passive process of receptor competition but also involves the active recruitment of corepressor and coactivator proteins.Pueraria is a frequently used traditional Chinese drug. Puerarin, a phytoestrogen derived from puerariae, has the dual properties of anti-estrogen and estrogen-like. In our early clinical practice to treat endometriosis, better therapeutic effect will be achieved if the formula of traditional Chinese medicine (Yu’s Neiyi recipe) included radix puerariae. Thereafter, the effects of puerarin to treat rat models of endometriosis and to suppress invasion and vascularization of endometriosis tissue stimulated by17B-estradiol were elucidated in our Lab. it has potential to treat endometriosis. This study was to investigate whether puerarin could suppress proliferation of the endometriotic stromal cells (ESCs) partially via differential recruitment of nuclear receptor coregulator proteins to the estrogen receptor-a(ER-a) in endometriosis in vitro.Methods:First, the ectopic endometrial tissues were digested using collagenase type Ⅳ, and the endometriotic stromal cells (ESCs) were obtained by screen cloth separation, and the purity was identificated by fluoroimmunoassay of vimentin. Then we investigated the effects of puerarin on the cell cycle and proliferation of ESCs by flow cytometry and Cell Counting Kit-8(CCK-8) assay. Subsequently, we examined puerarin could regulate the transcription of ER-a controlling genes, including Cyclin D1and cdc25A partially via differential recruitment of nuclear receptor coregulator proteins to the estrogen receptor-a (ER-a) through coimmunoprecipitation(CO-IP).Results:The purity of the isolated ESCs was>95%, as determined by the fluoroimmunoassay of vimentin. The down-regulation on both mRNA and protein level of the Cyclin D1and cdc25A expression with the simultaneous block of the cell cycle G1 phase and cell proliferation was observed in ESCs after puerarin treatment, which suggested that puerarin suppresses the nuclear accumulation of Cyclin D1and cdc25A and prevents ESCs cell proliferation stimulated by E2. We also found that E2caused a increased recruitment of the co-activators SRC-1and SRC-3but an decreased recruitment of the co-inhibitors N-CoR and SMRT to ER-a. Whereas, in the presence of puerarin, the recruitment was reversed.Conclusion:This study demonstrated that a range of puerarin concentration(10-7M,10-9M,10-11M) showed an anti-estrogenic effect on ESCs. Futhermore, puerarin showed the strongest antagonistic effect at the concentration of10"9M on the proliferative effect induced by10-8M estradiol, which indicated that puerarin could be used as a novel therapeutic agent for the medical treatment of endometriosis.