The Effects and Possible Mechanisms of Puerarin to Treat Endometriosis Model Rats

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2015, Article ID 269138, 11 pages http://dx.doi.org/10.1155/2015/269138 Research Article The Effects and Possible Mechanisms of Puerarin to Treat Endometriosis Model Rats Jin Yu,1 Li Zhao,1 Danying Zhang,2 Dongxia Zhai,2 Wei Shen,2 Lingling Bai,2 Yiqun Liu,2 Zailong Cai,3 Ji Li,4 and Chaoqin Yu2 1 Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Department of Gynecology of Traditional Chinese Medicine, Changhai Hospital, Second Military Medical University, Shanghai 200433, China 3 Department of Biochemistry and Molecular Biology, Second Military Medical University, Shanghai 200433, China 4 Department of Gynecology of Traditional Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China 2 Correspondence should be addressed to Ji Li; and Chaoqin Yu; Received 16 September 2014; Accepted 14 January 2015 Academic Editor: Tsung-Chieh Jackson Wu Copyright © 2015 Jin Yu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. To explore the effects of puerarin to treat endometriosis (EMT) model rats and the possible regulatory mechanisms. Methods. EMT model rats were surgically induced by autotransplantion of endometrial tissues. The appropriate dosage of puerarin to treat EMT model rats was determined by observing the pathologic morphology of ectopic endometrial tissues and by detecting the levels of estradiol (E2) and prostaglandin E2 (PGE2) of both serum and ectopic endometrial tissues. The related genes and proteins of ectopic endometrial tissues were analyzed by Real-time PCR and immunohistochemistry (IHC) to explore the possible mechanisms. Results. Puerarin could reduce the levels of E2 and PGE2 and prevent the growth of ectopic endometrium tissues by inhibiting the expression of aromatase cytochrome P450 (p450arom) and cyclooxygenase-2 (cox-2); puerarin could adjust the anabolism of E2 by upregulating the expression of 17𝛽-hydroxysteroid-2 (17𝛽-hsd-2) and downregulating the expression of 17𝛽hydroxysteroid-1 (17𝛽-hsd-1) of the ectopic endometrium tissues; puerarin could increase the expression of ER𝛽 and improve the inflammatory microenvironment of EMT model rats. Conclusions. Our data suggest that puerarin has a therapeutic effect on EMT model rats and could be a potential therapeutic agent for the treatment of EMT in clinic. 1. Introduction Endometriosis (EMT) causes chronic pelvic pain and infertility [1], affects 5%–10% of women in their reproductive ages [2, 3], and is an estrogen-dependent benign disease characterized by extrauterine implantation and ectopic growth of endometrium [4]. The pathogenesis of EMT is still unclear and one of the most popular theories is “ectopic endometrium implantation” proposed by Sampson in 1921. Current treatments, including surgery and hormonal therapy, are often insufficient and imperfect for high rate of relapse and various side effects such as hepatic injury and osteoporosis [5– 7]. Therefore, it is necessary to explore novel therapeutic strategies and drugs for reversing the clinical symptoms of EMT patients and improving their quality of life. In our early clinical practice to treat endometriosis (EMT), a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae [8, 9]. Puerarin, extracted from Radix puerariae, is widely known as a natural conditioner of selective estrogen receptors (ERs) [10] and has an antiestrogenic effect for its weak estrogenic action by binding to ERs as we have studied in vitro before. This study was to investigate the effects of puerarin to treat the EMT model rats and the possible regulatory mechanisms in vivo, to provide scientific basis for clinical treatment of EMT patients. 2 Evidence-Based Complementary and Alternative Medicine 2. Materials and Methods Table 1: The primer sequences of target genes. 2.1. Animals. SPF grade female Sprague-Dawley (SD) rats (body weight 160–180 g; 𝑛 = 140) were purchased from Shanghai Sippr-bk Laboratory Animals co., Ltd. (license: SCXK (HU) 2008-0016) and raised in Fudan university animal center, 25∘ C constant temperature (Humidity 50%), 12 h light: 12 h dark cyclical alternates, without feeding soybean and alfalfa products. All procedures described here were reviewed and approved by the Ethical Committee of Fudan Medical University. Target gene 2.2. Drugs. Puerarin (purity ≥ 98%) was purchased from Sigma (batch number: P5555); raloxifene hydrochloride (RLX, purity 98%) was purchased from Sigma (batch number: 1598201); sodium carboxymethyl cellulose (CMC, 800 cps) was purchased from J&K (batch number: 241297). 17𝛽-HSD-2: 2.3. Rat Model. At the first 10 days, the estrous cyclicality of all rats was checked with histological screening of vaginal exfoliated cells [11] and the rats with irregular estrous cyclicality were eliminated, while the normal ones were prepared for modeling. The EMT rat model, as Vernon and Wilson reported [12], was induced by autotransplantation of endometrial tissues. The surgery was taken in aseptic conditions and the right part of the uterus tissue about 1 cm long was cut out with surgical scissors; 4 pieces of the endometrium were stripped carefully and stitched to the following parts, respectively, with nonabsorbable suture (USP8/0-2 number, ZENMA): the root of mesentery (a piece of endometrium (3 mm × 3 mm)), the left side of the ovary (a piece of endometrium (3 mm × 3 mm)), and abdominal wall on both sides (a piece of endometrium (5 mm × 5 mm) on the right side and a piece of endometrium (3 mm × 3 mm) on the left side). After surgery, all rats were treated with cephalosporin (0.1 g/per rat) by intraperitoneal injection for 3 days. 2.4. Drug Intervention. The successful EMT model rats verified by exploratory laparotomy were randomly divided into five groups: control group (CMC), low-dose group (LSI), middle-dose group (M-SI), and high-dose group (H-SI), which intraperitoneally received 0.1% CMC (0.01 mL/body weight (g)/day), 5, 20, and 80 mg of puerarin/body weight (kg)/day, respectively, and positive control group (RLX) which orally received RLX (dose: 10 mg/body weight (kg)/day). Both of puerarin and PLX were dissolved in CMC prorata and all rats of the five groups were respectively administered for 4 weeks between 9:00 and 10:00 a.m. every day. 2.5. RIA Analysis. Biochemical assessments of rat serum were detected by RIA analysis. The abdominal aortic blood (about 4 mL) was obtained from the experimental rats anaesthetized by 1% pentobarbital sodium (6 mL/kg body mass). In addition, ectopic endometrium tissues were stripped from the implant sites, homogenated with the refiner, and stored at −80∘ C refrigerator. At last, the (...truncated)