Endometrial tissues were obtained from the hysterectomies of 20 patients with uterine fibroids in the Affiliate Hospital of Hebei Engineering University (Handan, China) between September 2013 and December 2014. The procedure was approved by the Ethics Committee of Hebei Engineering University, and informed consent was obtained from the patients with uterine fibroids prior to the hysterectomies. The patients were aged between 30 and 45 years old, with menstrual cycles from 24–35 days (mean, 28 days). No patient received any hormonal treatment for at least 3 months prior to the hysterectomies. Endometrial tissues were confirmed as in hyperplasia period and disease-free by postoperative pathology. Endometrial tissue was scraped immediately from the uterus under sterile conditions in vitro, and placed in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F12) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), including 10% fetal bovine serum, penicillin and streptomycin (100 mg/ml; Gibco; Thermo Fisher Scientific, Inc.) in an ice bath, and transported to the laboratory within 2 h.

LBPs were purchased from Shanghai Organic Chemistry Institute (Shanghai, China). A stock solution was prepared of 5,000 mg/l in basal medium DMEM/F12 and maintained at −20°C. For the following experiments, the final concentration of LBPs was 100 µg/ml produced by diluting the stock solution with DMEM/F12.

Following several washes with phosphate buffered saline (PBS), the tissue was cut into 1–2 mm3 pieces with sterile scissors and incubated with 5 ml DMEM/F12 containing 0.2% collagenase I (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) in an incubator with atmosphere of 5% CO₂ at 37°C for 60 min. During the incubation, the tissue pieces were pipetted gently to disperse the cells. The whole cell suspension was centrifuged at 500 × g for 5 min at room temperature. The supernatant fluid, which contained ESCs, was centrifuged at 1,200 × g for 5 min at room temperature. The supernatant fluid was discarded, while the precipitate was resuspended in a culture bottle with 3 ml complete cell-culture medium [DMEM/F12+10% fetal bovine serum (FBS)+1% penicillin and streptomycin]. The ESCs attached to the culture bottle were washed several times with serum-free DMEM/F12 to remove red blood cells.

A trypan blue exclusion assay was performed to assess the proportion of living cells. Then, 1 ml cell suspension (1×10⁵ cells/ml) was placed into a six-well plate containing coverslips and cultured in atmosphere of 5% CO₂ at 37°C for 5 days for the identification and assessment of the purity of the cells.

ESCs were cultured for 0 and 5 days and then stained with hematoxylin and eosin (H&E) following fixing in 4% paraformaldehyde for 1–2 h at 4°C and dehydration with 70, 80, 90, 95 and 100% ethanol and 100% xylene. The morphology and structure of the ESCs were observed with an inverted microscope equipped with phase-contrast apparatus and with a light microscope.

Immunocytochemical staining was performed to identify the ESCs and assess their purity. PBS was used instead of a primary antibody as a negative control. Cells cultured on coverslips were fixed with 4% paraformaldehyde and treated with 0.25% Triton X-100. After blocking with 5% normal goat serum for 20 min at 37°C, the rabbit anti-human primary antibody vimentin (1:100; cat. no. MA3-745) and cytokeratin (1:100; cat. no. PA5-14263; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) were incubated with the cells at 4°C overnight. The cells were subsequently incubated with goat anti-rabbit IgG (1:100; Boster Corporation, Wuhan, China) for 20 min at 37°C and stained with 3,3′-diaminobenzidine (5 mg/ml; Sigma Aldrich; Merck Millipore) for 5 min at room temperature. The specimens were washed with PBS for 5 min three times and observed using light microscopy.

ESCs were divided into four groups according to the different intervention factors. Control group: Serum-free DMEM/F12; H₂O₂ group: H₂O₂ and serum-free DMEM/F12; LBP group: LBPs and serum-free DMEM/F12; H₂O₂ + LBP group: H₂O₂, LBPs and serum-free DMEM/F12. The final concentration of H₂O₂ and LBPs was 10–4 M/l and 100 µg/ml respectively.

ESCs (4×104/ml) were seeded in 6-well plates and were divided into the above four groups and cultured for 12 h. Morphology and structure of the ESCs were observed with an inverted microscope equipped with phase-contrast apparatus.

Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ESCs (4×104/ml) were seeded in 96-well plates and cultured with serum-free DMEM/F12 for 12 h for synchronization. They were divided into the four groups described previously, each group into 6-well plates and cultured for 24 h. Thereafter, MTT (5 mg/ml) was added to each well and the plates were incubated at 37°C for 2 h. The medium was then replaced with 150 µl DMSO and agitated for 10 min at room temperature. The absorbance at 560 nm was measured using a microplate reader (Packard Instrument Company, Inc., Meriden, CT, USA).

The four groups of cells were cultured for 12 h, absorbing the culture supernatant fluid, according to the manufacturer's protocol of SOD assay kit and MDA assay kit (R&D Systems, Minneapolis, MN, USA), the activity of SOD was detected by autoxidation of pyrogallic acid, as previously described (13) and the content of MDA was detected by the thio-barbituric acid method, as previously described (14).

Cells from the above four groups were grown in 10 cm dishes and cultured for 12 h, followed by washing with PBS. The cells were subsequently lysed with lysis buffer (pH 7.4; 1 M Tris-HCl, 1% Triton X-100, sodium deoxycholate, 10% SDS). Solubilized proteins were centrifuged at 14,000 × g at 4°C for 30 min. Extracted proteins were quantified by Coomassie Protein assay reagent (Sigma-Aldrich; Merck Millipore). Western blot analysis of Bcl-2 and caspase-3 were performed. In brief, 30 mg isolated protein was electrophoresed on 8% sodium dodecyl sulphate-polyacrylamide gel (100 V for 1.5 h), and transferred onto PVDF membranes. The membranes were treated with blocking solution [Tris-buffered saline (TBS) pH 7.2, 0.1% Tween, 5% milk] for 1 h at room temperature and incubated for 12 h at 4°C with rabbit anti-human monoclonal Bcl-2 (cat. no. 13-8800) and caspase-3 (cat. no. 700182) antibodies (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), diluted 1:1,000 in TBS (pH 7.2) 0.1% Tween. After four washings for 15 min with TBS (pH 7.2) 0.1% Tween, the membranes were incubated with the goat anti-rabbit IgG antibody horseradish peroxidase conjugate (cat. no. 35552) (Invitrogen; Thermo Fisher Scientific, Inc.), diluted 1:1,000 in TBS pH 7.2, 0.1% Tween for 1 h at room temperature. They were then washed four times for 15 min with TBS (pH 7.2), treated with an enhanced chemiluminescent method according to the protocols of an enhanced chemiluminesence detection kit (eECL western blot detection kit) (Invitrogen; Thermo Fisher Scientific, Inc.) and exposed to Kodak X-ray film for 0.5–20 min as necessary to detect the signals. The relative intensity of the immunoreactive bands on the films was quantified by a computer-assisted densitometry program (SmartView; Major Science, Saratoga, CA, USA). Proteins expression was quantified by comparison with internal control β-actin.

Total RNA was extracted from the cells in the four groups by culturing for 12 h with the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and the RNA was dissolved in RNase-free water. The integrity of the RNA was assessed by ethidium bromide agarose gel electrophoresis and the quantity of RNA was determined by the relative absorbance at 260 vs. 280 nm. cDNA (2 µg) was synthesized in a volume of 10 µl with a cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. The cDNA was stored at −20°C.

RT-qPCR, primers (Invitrogen; Thermo Fisher Scientific, Inc.) were derived from the GeneBank database. β-actin was used as the housekeeping gene. The primers used were as follows: Caspase-3 forward, 5′-TATCCTGAGATGGGTTTA-3′ and reverse, 5′-TGTTTCCCTGAGGTTTGC-3′; Bcl-2 forward, 5′-CTGGGAGAACAGGGTACGATAA-3′ and reverse, 5′-CCCACCGAACTCAAAGAAGG-3′; β-actin forward, 5′-CGGGAAATCGTGCGTGAC-3′ and reverse, 5′-CAGGAAGGAAGGCTGGAAG-3′.

The RT-qPCR reactions were performed using Brilliant SYBR-Green QRT-PCR Master Mixture according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). RNA for caspase-3 and Bcl-2 was amplified using an ABI Prism 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.). For all RT-qPCR studies, synthesizing by PCR procedure was performed with the following time courses: 94°C for 10 min, 40 cycles at 94°C for 15 sec, 59°C for 30 sec, and 72°C for 32 min for amplification. The amplified products were subjected to a stepwise increase in temperature from 59–94°C and dissociation curves were constructed.

Target mRNA was quantified by measuring the threshold cycle and reading against a calibration curve. The relative amount of each mRNA was normalized to the housekeeping gene, β-actin mRNA. Results were analyzed using the relative standard curve method of analysis/2^−ΔΔCq method of analysis (15).

All data are presented as bar graphs with the mean ± standard deviation of six independent experiments with samples from different subjects. Data were analyzed using SPSS for windows, version 15.0 (SPSS Inc., Chicago, IL, USA). Statistical analysis was performed by one-way analysis of variance, Student-Newman-Keuls was used to compare individual groups. P<0.05 was considered to indicate a statistically significant difference.

The ESCs were observed using an inverted microscope equipped with phase-contrast apparatus. Differential centrifugation identified the purity of the isolated ESCs as 92–95%. Trypan blue staining demonstrated that the viability of the EECs was 96–98%. Recently isolated ESCs exhibited mostly round morphology (Fig. 1A), later growing as spindle and polygon-shaped cells with long cytoplasmic processes, usually reaching confluence after 5 days culture, exhibiting a single-cell monolayer growth pattern (Fig. 1B). The ESCs were stained by H&E staining after being cultured for 5 days (Fig. 1C) to indicate the cell morphology.

The ESCs were detected by immunocytochemical staining. The cytoplasm of positive ESCs was stained as brownish/yellow granules. The ESCs were positive for vimentin (Fig. 1D).

After the four groups of ESCs had been cultured for 12 h, the changes in the ESCs morphology and structure were observed using an inverted microscope equipped with phase-contrast apparatus. No significant changes were observed in the morphology and structure of ESCs in the LBPs group compared with the control group, while those in the H₂O₂ group became irregular in shape and declined in number. The H₂O₂ + LBP group demonstrated significantly less deterioration compared with the H₂O₂ group (Fig. 2A).

After the cells had been cultured for 12 h, the change in the number of the ESCs was observed by detecting the purple crystals in each culture well at an absorbance value of 550 nm. The result demonstrated that compared with the control group, the number of ESCs in the LBP group had no significant change (P=0.7976), while H₂O₂ significantly decreased the proliferation of ESCs in a time dependent manner (P<0.0001), the H₂O₂ + LBP group demonstrated significantly less deterioration compared with the H₂O₂ group (Fig. 2B).

The result of colorimetric analysis demonstrated that compared with the control group, no significant change was observed in the content of MDA and the activity of SOD in the LBP group, while the content of MDA increased and the activity of SOD decreased significantly in H₂O₂ group (P<0.0001 and P=0.0002, respectively). Compared with the H₂O₂ group, the content of MDA decreased and the activity of SOD increased significantly in the H₂O₂ + LBP group (P<0.0001 and P<0.0001; Fig. 3).

After cells were cultured for 12 h, the results of western blot analysis demonstrated that the control group cells had positive expression levels of caspase-3 and Bcl-2, and that the LBP group had similar expression levels of caspase-3 and Bcl-2 to the control group (P>0.05). The expression levels of caspase-3 in the H₂O₂ group increased significantly compared with that of the control group (P<0.0001), and decreased significantly in the LBP + H₂O₂ group compared with that of the H₂O₂ group (P<0.05); while the expression levels of Bcl-2 increased significantly in the H₂O₂ group compared with that of the control group (P<0.0001), and decreased in the LBP + H₂O₂ group compared with that of the H₂O₂ group (P<0.0001; Fig. 4A and B).

After cells were cultured for 12 h, the result of RT-qPCR demonstrated that the control group cells had positive expression of caspase-3 and Bcl-2, as did the LBP group. Compared with the control group, the expression level of caspase-3 of the H₂O₂ group was increased significantly (P<0.0001) and the expression level of Bcl-2 was decreased significantly (P<0.05). Compared with the H₂O₂ group, the expression levels of caspase-3 in the LBP + H₂O₂ group was decreased significantly (P<0.0001), although the expression of Bcl-2 increased significantly (P<0.0001; Fig. 4C).