A total of 32 eight-week-old female Wistar rats (weight, 180–220 g) were obtained from the Laboratory Animal Center of Heilongjiang University of Chinese Medicine (Harbin, China). The experimental protocols were conducted in accordance with the Guidelines for Animal Experiments (US Government Principle for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training) (9), and were approved by the Ethical Committee of Heilongjiang University of Chinese Medicine (no. SCXK Hei2008004).

WSC was purchased from Duoduo Pharmaceutical Co., Ltd. (Jiamusi, China); the enzyme-linked immunosorbent assay (ELISA) kit for estradiol (E₂) was obtained from Beijing Yuande Biomedical Engineering Co., Ltd. (Beijing, China); the radioimmunoassay (RIA) kit for laminin (LM) was purchased from Beijing North Institute of Biotechnology (Beijing, China); the ELISA kit for fibronectin (FN), rabbit polycolonal antibodies against LM (BA1033) and FN (BA1049), and the streptavidin-biotin complex (SABC) and 3,3′-diaminobenzidine (DAB) kits were all purchased from Boster Biological Technology, Ltd. (Wuhan, China); rabbit polycolonal antibodies against ERα (SC542), anti-ERβ (SC8974) and anti-PR (SC538) were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China); the mifepristone and misoprostol tablets were obtained from Beijing Zizhu Pharmaceutical Co., Ltd. (Beijing, China).

Female Wistar rats were mated with male rats of proven fertility in the evening, at a ratio of 2:1, as previously reported (10). If sperm was found in the vaginal smears the following morning, that day was considered as day 1 of gestation. On day 7 of gestation, 32 rats were randomly divided into four groups, which included the normal (group N), pregnant control (group P), model (group M) and WSC-treated (group W) groups, with eight rats in each group. Abortions were induced in each rat from groups M and W using 8.3 mg/kg mifepristone, followed by 100 µg/kg misoprostol 10 hours later, which were applied via intragastic administration. Cotton balls of the same weight, which were hemi-wrapped with plastic films, were subsequently placed into the vagina of the rats to verify the success of the abortion and to prevent blood leakage. From day 8 of gestation, the rats in groups N, P and M were treated with 1 ml per 100 g body weight distilled water each day for seven days, whereas the rats in group W were treated with 1 ml per 100 g body weight WSC suspension at a concentration of 0.25 g/ml per day for seven days.

On day 14 of gestation, all the rats were anesthetized with ether and visceral organs were removed. The unilateral uterine specimens were weighed. The uterine indexes were subsequently calculated by dividing the uterine weight with the body weight of each rat. The specimens were fixed in 4% formaldehyde for 24–48 h, embedded in paraffin, serially sectioned and restained with hematoxylin-eosin. Following incubation at 37°C and sealing with neutral gum, the uterine shape was investigated under a microscope (CX21; Olympus Corporation, Tokyo, Japan).

From day 8 of gestation, a new cotton ball was replaced in the vagina at an interval of 6 h from 6:00, until no blood stain was observed. The duration of uterine bleeding was recorded and the cotton balls were stored at −20°C for the determination of the uterine bleeding volume. On day 14 of gestation, a 0.02-ml blood sample was collected from the tail vein using a hemoglobin pipet, added to 4 ml (V₁) NaOH solution (5%) and mixed. The cotton balls from each rat were placed in a beaker, soaked and washed with the appropriate amount of NaOH solution that was adjusted to the quantity of bleeding. The washed solution was then preserved in an additional beaker. The process was repeated once or twice as required, and the total volume (V₂) of NaOH solution used was recorded. Following complete mixing of the washed solution, a 5-ml sample of the mixture was filtrated using a filtrator (Beijing Bomei Glass Co., Ltd., Beijing, China). The absorbance (A) values of the filtered extract and the 4-ml NaOH solution containing the tail vein blood were recorded at 546 nm using an ultraviolet-visible light detector (Helios Gamma; Thermo Fisher Scientific, Waltham, MA, USA), with a 5% NaOH solution used as the blank control. The volume of uterine bleeding was calculated using the following equation: Volume (ml) = tail vein blood (0.02 ml) × (A of filtered extract × V₂)/(A of tail vein blood × V₁).

Blood samples were collected from the femoral artery of the rats on days 1, 8 and 14 of gestation. Following centrifugation at 600 × g for 10 min, the serum was aspirated and stored at −20°C. The E₂ level was analyzed with an ELISA kit, according to the manufacturer's instructions. For the measurement of FN and LM levels, plasma samples were collected on day 14 of gestation and analyzed by ELISA and RIA, respectively, according to the manufacturer's instructions.

Paraffin-embedded tissues prepared for hematoxylin-eosin staining were also used in the immunohistochemistry assay. Tissue sections were blocked with 5% bovine serum albumin at 37°C for 30 min, and incubated with primary rabbit polyclonal antibodies targeted against ER, PR, FN and LM at 4°C overnight. The samples were subsequently incubated with a biotin-conjugated secondary antibody (BST06K11A; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Staining was developed by incubation of the sections with the SABC and visualized using DAB. All the slides were counterstained with hematoxylin, and images were captured using an inverted microscope (magnification, x100) (IX71-21PH; Olympus Corporation). Immunoreactive spots from five randomly selected fields in each section were further selected for analysis and quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

The data are expressed as the mean ± standard deviation. Statistical analyses were conducted using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The statistical significance of differences was evaluated using the Mann-Whitney U-test, where P<0.05 was considered to indicate a statistically significant difference.

As shown in Table I, the uterine indexes in groups P and M were significantly higher compared with that observed in group N (P<0.01); whereas the uterine index in group W was notably lower when compared with group M (P<0.05).

The pathological results obtained from the paraffin sections of the uterine tissue revealed that there were a number of trophoblastic cells observed in group P when compared with the normal uterine shape in group N. However, in group M, there was a predominance of decidual cells and few trophocytes, in addition to evidence of extravasated blood. In group W, there were few decidual cells observed and no evidence of extravasated blood (Fig. 1).

No evidence of uterine bleeding was observed in groups N and P. The duration of uterine bleeding was prolonged and the volume of uterine bleeding increased significantly in group M when compared with group P (P<0.01); whereas the duration of uterine bleeding was reduced and the volume of uterine bleeding decreased significantly in group W when compared with group M (P<0.01 and P<0.05, respectively; Table II).

Table III shows the E₂ level in the rats from the various groups. No statistically significant changes were observed in the E₂ level on days 1, 8 and 14 of gestation in group N. However, in group P, the E₂ level exhibited an increasing trend following the experimental days. In addition, the E₂ level in group M increased initially at day 8 of gestation and then decreased by day 14 of gestation. The serum E₂ level in group W was significantly elevated when compared with that in group M on day 14 of gestation (P<0.01).

As shown in Table IV, the plasma levels of FN and LM in group M were significantly elevated when compared with group P (P<0.01 and P<0.05, respectively). However, the levels of FN and LM in group W were markedly lower than that observed in group M (P<0.01 and P<0.05, respectively).

Immunohistochemistry staining results showing the expression of the ER, PR and FN in the uterine tissues are shown in Fig. 2. Since no evident expression of LM was detected in the uterine tissue (data not shown), LM expression was not analyzed with immunohistochemistry. The ERα and PR were predominately expressed in the endometrial glandular epithelial cells, simple columnar epithelial cells and were also detected in the smooth muscle cells of the uterine endometrium. No statistically significant differences were observed between the optical density (OD) values for the ERα and PR in groups P and N. However, the OD values of the ERα and PR were significantly decreased in group M when compared with group P (P<0.01 and P<0.05, respectively), whereas the OD values of the ERα and PR were significantly increased in group W when compared with group M (P<0.01 and P<0.05, respectively). FN was primarily expressed in the myometrial and endometrial interstitial cells and cells of the decidual tissues. No statistically significant difference was observed between the OD value of FN in groups P and N. The OD value of FN was significantly increased in group M when compared with group P (P<0.05), whereas the OD value of FN was significantly decreased in group W when compared with group M (P<0.01; Table V).

As shown in Table VI, no statistically significant differences were observed in the hemodynamic indexes when comparing groups P and N. The plasma viscosity, the whole blood viscosity at shear rates of 1 mPa•s and 5 mPa•s, the whole blood relative index (low shear) and the whole blood reduced viscosity (low shear) were significantly increased in group M when compared with group P; whereas the plasma viscosity, the whole blood viscosity at shear rates of 1 s⁻¹ and 5 s⁻¹, the whole blood relative index (low shear) and the whole blood reduced viscosity (low shear) were significantly decreased in group W when compared with group M.