Human trophoblast HTR8/SVneo cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's Medium (DMEM)/F12 nutrient mixture (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin in a humidified incubator containing 5% CO₂ at 37°C. Cells in the logarithmic growth phase were used in subsequent experimentation.

LBP is the main active component of the Chinese wolfberry (21). LBP was purchased from Qinghai General Health Bio-science Co., LLC (Xining, China) and the purity of LBP was >50%. HTR8/SVneo cells were treated with different concentrations (100, 200 and 400 µg/ml) of LBP dissolved in PBS for 6 h. H₂O₂ (250 µmol/l; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was subsequently added to treat cells for 6 h. There were five experimental groups in total: LBP1 + H₂O₂ (cells treated with 100 µg/ml LBP and 250 µmol/l H₂O₂), LBP2 + H₂O₂ (cells treated with 200 µg/ml LBP and 250 µmol/l H₂O₂), LBP3 + H₂O₂ (cells treated with 400 µg/ml LBP and 250 µmol/l H₂O₂), H₂O₂ (cells treated with 250 µmol/l H₂O₂) and control (without treatment) (22–24).

HTR8/SVneo cell viability was determined following treatment with LBP and H₂O₂, with Cell Counting kit-8 (CCK-8) assay (Beyotime Institute of Biotechnology, Haimen, China). Cells were treated with 250 µmol/l H₂O₂ for 1, 4 and 6 h. Briefly, following cells treatments, cells were seeded into a 96-well plate at an initial density of 5×10³ cells/well and incubated in DMEM media with FBS for 24 h at 37°C. Then cells were treated with 100 µl 250 µmol/l H₂O₂ for 1, 4 and 6 h. At total of 20 µl CCK-8 reagent was added in and incubated for 1 h in a 5% CO₂ incubator at 37°C. Finally, the optical density values were acquired with a microplate reader at 450 nm.

ROS were detected in different groups (Control, H₂O₂, LBP1 + H₂O₂, LBP2 + H₂O₂, LBP3 + H₂O₂) with a 2′,7′-dichlorofluorescin diacetate (DCFH-DA) assay (Beyotime Institute of Biotechnology). After 6 h of 250 µmol/l H₂O₂ treatment, cells, treated with different concentrations (100, 200 and 400 µg/ml) of LBP for 6 h, were seeded into wells of 6-well plate. DCFH-DA (10 µmol/l) was subsequently added into each well. After incubation for 20 min at 37°C, cells were rinsed with PBS and analyzed by flow cytometry. ROS levels were analyzed by CellQuest software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA) and results were calculated relative to the control group.

Alterations in the MMP of each group treated with LBP (100, 200 and 400 µg/ml) for 6 h and 250 µmol/l H₂O₂ for another 6 h were determined by aJC-1 assay (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), using the cationic dye to detect potential-dependent accumulation in mitochondria. Briefly, 5×10⁵ cells were collected and resuspended in 500 µl PBS with JC-1 (10 µmol/l) for 20 min at 37°C. MMP alterations were reflected by a fluorescence emission shift from 550 nm (red) to 525 nm (green). Cells were analyzed by flow cytometry with Cell Quest software version 5.1 (BD Biosciences).

The cell apoptosis proportion in each group treated with LBP (100, 200 and 400 µg/ml) for 6 h and 250 µmol/l H₂O₂ for another 6 h were determined by an Annexin-V/propidium iodide (PI) double-stain assay, according to the manufacturer's protocol (Roche Diagnostics, Indianapolis, IN, USA). Briefly, both floating and trypsinized adherent cells (5×10⁵) of the five experimental groups were collected and resuspended in 500 µl PBS containing 0.5 µg/ml Annexin-V-fluorescein isothiocyanate for 20 min. Subsequently, 400 µl PBS with 50 µg/ml PI was added to cells for 5 min at room temperature in the dark. The analysis of cell apoptosis rate was immediately conducted with a flow cytometer and CellQuest software version 5.1 (BD Biosciences).

The cell supernatant of each group was collected. The activity of the antioxidant enzyme SOD was determined using the total SOD assay kit with WST-8 (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. During the reaction process of SOD detection, 2-iodophenyl-3-nitrophenyl tetrazolium chloride was catalyzed to formazin, which could be detected by a microplate reader. The activity of LDH was detected with a LDH assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

The mRNA expression levels of survivin, hypoxia inducible factor 1-α (HIF1-α) and Bcl-2 apoptosis regulator (Bcl-2), Bcl-2 associated X apoptosis regulator (Bax) in each group was measured by RT-qPCR. Total RNA was extracted from cells using an RNeasy kit, and 1 µg RNA was reverse transcribed to cDNA using a Quantiscript Reverse Transcriptase kit (both Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's protocol. PCR amplification was performed for 15 sec at 95°C, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 25 sec in an ABI 7300 Thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) with Fast SYBR-Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). GAPDH was used as the reference gene; primer sequences are displayed in Table I. The quantification was identified by 2^−ΔΔCq method (25).

Cells were harvested, washed twice with PBS, lysed using lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.02% NaN2, 100 µg/ml phenylmethanesulfonyl fluoride, 1 µg/ml aprotinin, and 1% Triton X-100) and centrifuged at a speed of 12,000 × g for 30 min at 4°C. The supernatant was subsequently collected from the lysate and protein concentration was determined by a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). A total of 10 µg proteins were boiled and separated by 10% SDS-PAGE, followed by transfer onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% non-fat dry milk in PBS for 1 h at 37°C and incubated with the following primary antibodies at 4°C overnight: Anti-survivin (cat. no. ab76424; 1:5,000), anti-HIF1-α (cat. no. ab51608; 1:1,000), anti-Bax (cat. no. ab53154; 1:1,000), anti-Bcl-2 (cat. no. ab59348; 1:1,000), anti-GAPDH (cat. no. ab9485; 1:2,500; all Abcam, Cambridge, MA, USA). Subsequently, proteins were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. ab6721; 1:5,000; Abcam). PVDF membranes were exposed to X-ray film and immunoreactive bands were detected with an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences). Finally, protein density was detected by Bio-Rad ChemiDoc XRS+ System with Image Lab Software version 4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data is expressed as the mean ± standard deviation of three independent experiments. Statistical analyses were performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA) and significance was calculated with one-way analysis of variance followed by Dunnett's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To identify the effects of LBP on the proliferation of HTR8/SVneo cells injured by H₂O₂, a CCK8 assay was conducted in each experimental group (control, H₂O₂, LBP1 + H₂O₂, LBP2 + H₂O₂ and LBP3 + H₂O₂ groups). The results revealed that the cell proliferation ability of the H₂O₂ group was significantly reduced by 250 µmol/l H₂O₂ in a time-dependent manner, compared with the control group (P<0.01). When pre-treated with LBP for 6 h prior to the addition of H₂O₂, proliferation in the LBP1 + H₂O₂, LBP2 + H₂O₂ and LBP3 + H₂O₂ groups significantly increased in a dose-dependent manner, compared with the H₂O₂ group (P<0.05), indicating that LBP may protect the cell proliferation ability of HTR8/SVneo cells injured by H₂O₂ (Fig. 1).

Intracellular ROS levels were measured using the oxygen-sensitive fluorescent dye DCTH-DA. The assay results revealed that the accumulation of ROS in HTR8/SVneo cells was significantly increased in the H₂O₂ group, compared with the control (P<0.01). Furthermore, a significant decrease in ROS formation was detected in the LBP1 + H₂O₂, LBP2 + H₂O₂ and LBP3 + H₂O₂ groups, in a dose-dependent manner, compared with the H₂O₂ group (P<0.05; Fig. 2A).

LDH leakage reflects cytotoxicity and mitochondrial damage (26). Levels of antioxidant enzyme SOD and leaked LDH in the cell supernatant were determined by the corresponding assay kits. Decreased SOD activity and increased LDH activity was detected in the H₂O₂ group, compared with the control group (P<0.01). LBP increased SOD activity and decreased LDH activity in dose-dependent manner, compared with H₂O₂ group (P<0.05; Fig. 2B and C).

The percentage of cells with MMP disruption was measured by JC-1 assay. The percentage of cells with MMP disruption in the H₂O₂ group was significantly increased compared with the control group (P<0.01). The percentage of cells with MMP disruption in the LBP1 + H₂O₂, LBP2 + H₂O₂ and LBP3 + H₂O₂ groups decreased significantly in a dose-dependent manner, when pretreated with different concentrations of LBP (100, 200 and 400 µg/ml) prior to H₂O₂ injury, compared with the H₂O₂ group (P<0.05; Fig. 3A). These results indicate that LBP may reduce MMP disruption in HTR8/SVneo cells with H₂O₂-induced injury.

An Annexin-V/PI double-stain assay was performed to detect the apoptosis status of each group. The apoptosis rate of the H₂O₂ group significantly increased compared with control group (P<0.01); apoptosis in the LBP1 + H₂O₂, LBP2 + H₂O₂ and LBP3 + H₂O₂ groups was significantly decreased in a dose-dependent manner, compared with the H₂O₂ group (P<0.05; Fig. 3B). This indicated that LBP may inhibit H₂O₂-induced apoptosis in HTR8/SVneo cells.

To detect whether the protective function of LBP on HTR8/SVneo cells injured by H₂O₂ was via regulation of apoptosis-associated factors, RT-qPCR (Fig. 4A) and western blot analysis (Fig. 4B and C) was performed to detect the expression of survivin, HIF1-α, Bax and Bcl-2 in TR8/SVneo cells treated with H₂O₂ and/or LBP. The results revealed that the mRNA and protein levels of survivin, HIF1-α and Bcl-2 decreased significantly in H₂O₂ group compared with control group, and significantly increased in the LBP treated groups in a dose dependent manner (P<0.05). The mRNA and protein levels of Bax exhibited the opposite trend (P<0.05).