Sprague-Dawley female rats (age, 9 weeks; weight 180–220 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All rats were housed under specific pathogen-free conditions with a 12-h day/night cycle, and ad libitum access to food and water. All animals were maintained at a temperature of 22±1°C and humidity of 55±5%. All the experimental operations for rats conformed to the Guide for the Care and Use of Laboratory Animals (19). This study was approved by the ethics committee of the People's Hospital of Wuhan University. A total of 55 rats were used, of which five rats were used in the preliminary experiments, and 50 rats were used in the current study.

WJ-MSCs were acquired from Professor Chen Yantian (20) (Cell Culture and Bioprocess Engineering Lab, School of Pharmacy, Shanghai Jiao Tong University). The cells were cultured in complete medium, comprising α-minimum essential medium (cat. no. 12571063; Gibco; Thermo Fisher Scientific, Inc.), 10% fetal bovine serum (FBS; cat. no. 16140071; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. B540734; Sangon Biotech Co., Ltd.), and maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The phenotype of WJ-MSCs was determined by flow cytometry (FACSCalibur; BD Biosciences). Flow cytometric analysis was conducted with fluorochrome-conjugated antibodies against human CD73, CD90, CD105, CD34 and CD43. Briefly, the cells in the logarithmic phase were suspended in Hank's Balanced Salt Solution (HBSS) supplemented with 2% FBS, and the cell density was adjusted to 1×107 cells/ml. Then, cells (100 µl) were transferred to a 1.5-ml Eppendorf tube with antibodies against CD73 (cat. no. 550257; BD Biosciences), CD90 (cat. no. 555596; BD Biosciences), CD105 (cat. no. 560839; BD Biosciences), CD34 (cat. no. 550761; BD Biosciences) and CD45 (cat. no. 555483; BD Biosciences) (20 µl), and incubated on ice for 30 min. After washing three times, cells were transferred to a flow tube and resuspended in HBSS for flow cytometry on a BD FACSCalibur flow cytometer (BD Biosciences). The data were analyzed using FlowJo 10 software (FlowJo LLC).

The IUA rat model was constructed using the ethanol damage method (21). Briefly, rats were anesthetized with 1% pentobarbital sodium (30 mg/kg) through intraperitoneal injection, and the abdominal wall and cavity was surgically opened to expose the uterus under sterile conditions. The upper and lower ends of the bilateral uterine walls were clamped with bulldog clamps and 0.5 ml 95% ethanol was injected into the bottom of the uterine wall. After maintaining it for 3 min, saline solution was injected into the uterine wall twice for washing. In addition, saline solution was used to replace the ethanol injection in the control group following the same operation. Finally, the abdominal cavity and wall were closed in layers, and 80,000 U/100 mg penicillin (North China Pharmaceutical Co., Ltd) was intramuscularly injected to prevent infection 3 days after the operation. After 14 days, IUA was confirmed by evaluating the histopathological changes in the uterine specimens using hematoxylin and eosin (H&E) staining.

A total of 50 Sprague Dawley female rats were used in the present study. The rats were randomly divided into five groups (n=10 rats/group): Control group; IUA model group; WJ-MSCs treatment group; estrogen treatment group; and WJ-MSCs + estrogen treatment group. IUA model rats were prepared as aforementioned. In the WJ-MSCs treatment group, 2 weeks after IUA model construction, a relaparotomy was performed and 2×10⁶ WJ-MSCs at middle passage (P3-P7) were injected into the left uterine horn of IUA rats. In total, the intraperitoneal injections of WJ-MSCs were conducted three times at 5-day intervals. In the estrogen treatment group, 2 weeks after model rats were successfully prepared, IUA rats received 0.2 mg/kg estrogen (cat. no. E8875; Sigma-Aldrich; Merck KGaA) through intragastric administration, once every 2 days for 8 weeks. In the WJ-MSCs + estrogen treatment group, 2 weeks after IUA model construction, model rats were treated with WJ-MSCs injection and estrogen intragastric administration together. During the period, the health and behavior of rats were observed every other day. After 8 weeks of intervention, the rats were anesthetized with 1% pentobarbital sodium (30 mg/kg) by intraperitoneal injection prior to sacrifice by decapitation (animal death was verified by observing no behavior after 5 min), and the uterine tissues of the experimental rats were resected.

The pathology of the specimens in each group was evaluated by H&E staining under an inverted microscope (IX73; ×40 magnification; Olympus Corporation). In brief, uterine tissues were fixed at room temperature for 48 h in 10% formalin and dehydrated in ascending concentrations of ethanol: 70% for 3 h, 80% for 40 min, 95% two times for 40 min each, 100% for 40 min, and 100% again for 30 min. Then, the dehydrated tissues were cleared by xylene and filtered into wax for 1 h three times consecutively. After embedding in paraffin, the specimens were cut into 4-µm sections and dried at 60°C for 30 min. Next, the slides were deparaffinized and rehydrated prior to staining with H&E. The uterine thickness and the number of glands were observed under a light microscope (magnification, ×40).

The expression levels of pan-keratin, vimentin, TGF-β1, Rho subfamily members (RhoA, RhoB and RhoC), ROCKI and ROCKII in uterine tissue were examined using immunohistochemistry. After slide preparation as aforementioned, antigen retrieval was performed by inhibiting endogenous peroxidase by incubation with 3% H₂O₂ for 10 min at room temperature, followed by microwave heating for 3 min to expose antigenic sites. Non-specific binding was blocked with PBS containing 10% goat serum (Wuhan Boster Biological Technology, Ltd.) for 30 min at 37°C prior to transfer to an incubator (DHG-9053A; Shanghai Jinghong Experimental Equipment Co., Ltd.) for 30 min at 37°C. The slides were incubated with primary antibodies against pan-keratin (1:10; cat. no. ab8068; Abcam), vimentin (1:500; cat. no. ab8069; Abcam), TGF-β1 (1:100; cat. no. ab92486; Abcam), RhoA (1:1,000; cat. no. ab54835; Abcam), RhoB (1:500; cat. no. bs-11142R; BIOSS), RhoC (1:100; cat. no. ab180785; Abcam), ROCKI (1:100; cat. no. ab45171; Abcam), and ROCKII (1:200; cat. no. ab71598; Abcam) overnight at 4°C. Then, the slides were incubated with a secondary antibody for 30 min at 37°C (1:1,000; cat. no. PV-9000; OriGene Technologies, Inc.) followed by washing with PBS three times (5 min/wash). Finally, the detection of the bound antibody was performed using the avidin-biotin complex reagent for 30 min at 37°C. Diaminobenzidine solution was added and incubated for 2 h at room temperature with the slides, then the slides were counterstained with hematoxylin for 1 min at room temperature, rinsed with sterile water and air-dried. Finally, the slides were sealed with neutral resin and visualized under a fluorescent microscope (TE2000-E; Nikon Corporation). The expression of proteins was calculated based on the average gray value from six randomly selected fields at ×400 magnification. Briefly, the area of positive staining and tissue area were analyzed by Image-Pro Plus 6.0 (Media Cybernetics, Inc.). Light yellow, brown-yellow or dark brown were regarded as positive staining. The percentage of positive expression was calculated using the following equation: Area of positive staining/ area of tissue.

All data are presented as the mean ± standard deviation (SD). GraphPad Prism 5 (GraphPad Software, Inc.) was used for data analysis and graphics. All experiments were repeated three times. Statistical analyses were performed using one-way ANOVA, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The morphology of WJ-MSCs was observed as fusiform in shape (Fig. 1A). The cell surface markers (CD34, CD45, CD73, CD90 and CD105) were detected in WJ-MSCs by flow cytometry (Fig. 1B). As a result, WJ-MSCs presented positive expression of CD73, CD90 and CD105, and negative expression of CD34 and CD45. CD34 and CD45 are not markers for WJ-MSCs; therefore, this confirmed the cells were WJ-MSCs.

Histopathological changes in uterine specimens were evaluated by H&E staining. The results revealed that uterine thickness of the model rats was thinner and gland numbers were decreased compared with rats in the control group. In addition, the uterine orifice was closed. After WJ-MSCs or estrogen treatment, the uterine thickness of rats recovered to near normal, and the number of glands increased. The uterine tissue of rats in the WJ-MSCs + estrogen treatment group presented with increased uterine thickness and gland numbers, as well as an improvement in IUA symptoms; however, the degree of IUA restoration was less than in the WJ-MSCs or estrogen treatment groups (Fig. 2).

The expression levels of pan-keratin, vimentin, TGF-β1, RhoA, RhoB, RhoC, ROCKI and ROCKII were detected by immunohistochemistry. In the IUA model group, the expression of pan-keratin was markedly reduced compared with the control group (P=0.0011), and its expression was gradually increased after WJ-MSCs (P=0.0386), estrogen (P=0.0480) and combined treatment (P=0.0032) (Fig. 3) compared with the IUA model group. The expression of vimentin showed no significant difference among the groups (Fig. 3). In contrast with the control group, the expression levels of RhoA (P=0.0243), RhoB (P=0.0044) and RhoC (P=0.0463) were significantly higher in the IUA model group (Figs. 4 and 5). However, their expression levels varied after different treatment interventions. As compared with the IUA model group, the expression of RhoA was significantly decreased in WJ-MSCs (P=0.0248) and WJ-MSCs + estrogen treatment groups (P=0.0247), but was not significantly changed (P=0.4299) after estrogen treatment alone (Fig. 4); however, the expression of RhoB was only significantly decreased (P=0.0004) after WJ-MSCs + estrogen treatment (Fig. 4) and the expression of RhoC was not obviously changed in all treatment groups (Fig. 5). In addition, the expression levels of ROCKI (P=0.0080; Fig. 5), ROCKII (P=0.0446; Fig. 6) and TGF-β1 (P=0.0013; Fig. 6) were significantly increased in the IUA model group compared with the control group. Notably, the expression level changes in ROCKI and TGF-β1 were similar in all treatment groups; these markers were significantly downregulated in WJ-MSCs (ROCKI, P=0.0002; TGF-β1, P=0.0013), estrogen (ROCKI, P=0.0012; TGF-β1, P=0.0045) and WJ-MSCs + estrogen treatment groups (ROCKI, P=0.0008; TGF-β1, P=0.0020) compared with the IUA model group. The expression of ROCKII was significantly reduced in estrogen (P=0.0204) and WJ-MSCs + estrogen treatment groups (P=0.0222) compared with that in the IUA model group, but no significant difference was detected between the model group and WJ-MSCs treatment group (P=0.9998).