The human umbilical vein endothelial cell line EA.hy926 was obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Ginsenoside Rb1 was purchased from The China Food and Drug Administration. Mycoplasma testing was performed on the cells to ensure they were not contaminated. Cells were cultured in DMEM supplemented with 10% FBS and incubated with 5% CO₂ at 37˚C. Adherent cells were harvested by digestion with trypsin and centrifuged (1,000 x g for 5 min at 37˚C).

The experiments were divided into the following groups: Control group, ginsenoside Rb1 control group, EA.hy926 cells treated with 160 mg/l ginsenoside Rb1 alone for 48 h; MCT group, cells treated with 7.5 mM MCT for 48 h; and ginsenoside Rb1+MCT group, treated with 40, 80 or 160 mg/l ginsenoside Rb1 for 2 h prior to treatment with 7.5 mM MCT for 48 h. All cell culture results shown are based on at least three individual experiments.

In the preliminary experiments, the protective effect of different concentrations of ginsenoside Rb1 (40, 80, 160 or 200 mg/l) against MCT-induced damage of umbilical vein endothelial cells were assessed, and the results showed that there was no statistically significant difference between 160 and 200 mg/l ginsenoside Rb1 (data not shown); thus, the maximum effective concentration of ginsenoside Rb1 was deemed to be 160 mg/l. When deciding on the concentration of ginsenoside for the Rb1 control group, the maximum concentration of 160 mg/l was used in order to exclude the harmful effect of ginsenoside Rb1 on umbilical vein endothelial cells.

In order to visually show that MCT induced cell apoptosis, and that ginsenoside Rb1 protected against MCT-induced apoptosis, Hoechst 33258 staining was used. The cells were cultured in 6-well plates until they were 80% confluent and then incubated with the aforementioned drugs with 5% CO₂ at 37˚C. The cells were washed with PBS, then fixed with 4% formaldehyde for 10 min at room temperature, and subsequently stained with 10 µg/ml Hoechst 33258 at room temperature for 10 min. Finally, after the cells were washed with PBS, morphological changes were observed under a fluorescence microscope.

Annexin V-FITC-propidium iodide (PI) double staining assay was used to quantify apoptosis in EA.hy926 cells using FACScan flow cytometer (BD Biosciences). After 48 h of drug treatment, cells were harvested, washed with ice-cold PBS, resuspended in 250 µl binding buffer and incubated with 5 µl Annexin V-FITC for 10 min at room temperature in the dark. Subsequently, samples were washed with binding buffer, resuspended in PBS, counterstained with 5 µg/l PI for 10 min at room temperature in the dark and analyzed by flow cytometry to identify apoptotic cells. The extent of early apoptosis was determined as the percentage of Annexin V⁺/PI^- cells.

EMPs were isolated as described previously (17,26). Cell culture supernatants were collected and centrifuged for 10 min at 3,000 x g at 4˚C to remove any detached cells and fragments. The supernatants were then ultracentrifuged at 16,000 x g for 1 h at 4˚C. The EMP-rich pellets were resuspended in PBS and ultracentrifuged again at 16,000 x g for 1 h at 4˚C, and then resuspended in 50 µl PBS for use in subsequent experiments.

First, 50-µl aliquots of EMP suspensions were incubated with 20 µl phycoerythrin-labeled anti-CD62E (BD Pharmingen; BD Biosciences) at room temperature in the dark for 30 min, and the reaction was stopped with 1.5 ml PBS, then ultracentrifuged at 16,000 x g for 1 h at 4˚C. The samples were resuspended in 50 µl PBS, mixed with 2 µl fluorescent beads (Sigma-Aldrich; Merck KGaA) 1-µm in diameter, then added to the microscope slide and immediately analyzed using confocal laser scanning microscopy (magnification, x300; Olympus FV500; Olympus Corporation) with excitation and emission wavelengths of 549 and 565 nm, respectively. Differential interference contrast images and fluorescent confocal images were obtained simultaneously.

As described above, EMP-rich pellets were suspended in 250 µl PBS and analyzed on a FACScan flow cytometer. EMPs were defined as particles ≤1.0 µm in size positive for the endothelial cell antigen CD62E. Fluorescent beads, 1-µm in diameter, were used as the internal standard and for gating the particles.

Detection of apoptosis related proteins was detected in whole-cell lysates. Following the collection of EMPs, the TF bound to EMPs and the soluble TF were separated and preserved in EMP suspensions or the supernatant, respectively. The cells and EMPs were homogenized and lysed separately, in order to further detect the TF proteins bound to each. Subsequently, proteins were loaded on an 8 or 12% SDS-gel, resolved using SDS-PAGE and transferred to nitrocellulose membranes. The blots were probed with mouse anti-TF (cat. no. 553014; 1:500; BD Pharmingen), and anti-β-actin (cat. no. sc-8432; 1:1,000; Santa Cruz Biotechnology, Inc.) at 4˚C overnight. Immunoblots were washed and then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 016-030-084; 1:4,000; Pierce; Thermo Fisher Scientific, Inc.) at room temperature for 2 h, and signals were visualized using SuperSignal™ enhanced chemiluminescence detection (Thermo Fisher Scientific, Inc.) and detected using a chemiluminescence detection system (Bio-Rad Laboratories, Inc.).

A commercial ELISA kit (cat. no. RPN1231; Imubind Tissue Factor, American Diagnostica Inc.) was used to detect soluble TF protein in the supernatant medium, according to the manufacturer's protocol. TF protein levels are expressed as pg/ml using a reference curve created with TF standards provided with the kit.

All data were analyzed using SPSS version 22.0 (IBM Corp.). All results are expressed as the mean ± standard deviation. A Student's t-test or a one-way ANOVA followed by a Tukey's post-hoc test were used to compare quantitative data populations with normal distributions and equal variances. P<0.05 was considered to indicate a statistically significant difference.

As shown in Fig. 1A, after EA.hy926 cells were treated with 7.5 mM MCT for 48 h, the percentage of early apoptotic cells was significantly increased compared with the control group (4.5±0.71 vs. 2.6±0.42%; P<0.05). There was no significant difference between the percentage of apoptotic cells in the Rb1 control group and the control group, showing the low toxicity of ginsenoside Rb1. However, the percentage of early apoptotic cells was significantly lower in 80 (2.95±0.35%) or 160 mg/l (2.05±0.07%) ginsenoside Rb1+MCT groups compared with the MCT group (4.5±0.71%; both P<0.05; Fig. 1A).

`In addition, similar protective effects of ginsenoside Rb1 were observed on MCT-induced apoptosis in EA.hy926 cells using fluorescent Hoechst 33358 staining (Fig. 1C-F). Cells treated with 7.5 mM MCT for 48 h appeared shrunken and dark, and chromatin condensation, marginalization or nuclear beading was observed in the nuclei. Fragmentation of apoptotic nuclei dividing into smaller structures was observed frequently as well (Fig. 1E). However, in the Rb1 control group, only a few typical morphological features of apoptotic nuclei, including pyknotic nuclei and formation of apoptotic bodies, were observed (Fig. 1F).

EMPs are cell membrane vesicles derived from endothelial cells. The morphological features of EMPs were confirmed using confocal microscopy. Fluorescent beads 1 µm in diameter were used as an internal standard and appeared as blue particles. EMPs appeared as red, rounded vesicular structures with a diameter of ~1 µm and were positive for CD62E. The proportion of EMPs in the MCT group was significantly higher compared with control group (Fig. 2B and C).

To further investigate the effect of ginsenoside Rb1 on MCT-induced EMP secretion, the percentage of CD62E⁺-EMPs in culture medium in different groups using flow cytometry was determined (Fig. 2A). Cells in the Rb1 control group showed similar levels of CD62E⁺-EMPs secretion when compared with the control group (P>0.05). MCT significantly increased CD62E⁺-EMPs production compared with the control group (12.8±2.18 vs. 1.93±0.86; P<0.05). However, exposure of EA.hy926 cells to 40, 80 or 160 mg/l ginsenoside Rb1 prior to treating with MCT resulted in a significant decrease in CD62E⁺-EMPs production in a dose-dependent manner compared with the MCT group (40 mg/l, 9.67±3.57; 80 mg/l, 4.08±0.46; 160 mg/l, 3.00±0.40; MCT group, 12.8±2.18; all P<0.05; Fig. 2A).

TF is considered an integral membrane protein expressed at the plasma membrane of endothelial cells that are not exposed to blood. In the present study, the TF protein expression levels on cells were determined by western blotting. The results showed that the levels of TF protein in the MCT group were significantly higher compared with the control group (0.387±0.03 vs. 0.243±0.09; P<0.05; Fig. 3A and B). When cells were treated with ginsenoside Rb1 (40, 80 and 160 mg/l) and MCT, the protein expression levels of TF protein were significantly decreased in a dose-dependent manner compared with the MCT group (40 mg/l, 0.288±0.04; 80 mg/l, 0.238±0.06; 160 mg/l, 0.128±0.07; MCT group, 0.387±0.03; all P<0.05; Fig. 3A and B).

Recently, significantly higher levels of soluble TF proteins were observed in several endothelial related diseases, and these soluble TF proteins possess functions in numerous pathological processes, including hemostasis, thrombosis and inflammation (27,28). In the present study, the supernatant medium of EA.hy926 cells treated with ginsenoside Rb1 and MCT were collected, and the sTF levels were detected using ELISA. The results showed that the levels of sTF in the MCT group were significantly higher compared with the control groups (52.574±2.571 vs. 18.121±0.969 pg/ml, respectively; P<0.05; Fig. 3C). When cells were treated with 40, 80 or 160 mg/l ginsenoside Rb1 combined with MCT, and the levels of sTF were significantly decreased in each group compared with the MCT group (MCT+Rb1.40 group, 42.549±0.954 pg/ml; MCT+Rb1.80 group, 43.368±1.712 pg/ml; MCT+Rb1.160 group 27.538±1.152 pg/ml; MCT group, 52.574±2.571 pg/ml; all P<0.05; Fig. 3C).

To clarify the molecular mechanism underlying the biological effects of EMPs in endothelial damage, the expression of EMP-bound TF proteins were determined by western blotting analysis (Fig. 3D). The results showed that in cells treated with 7.5 mM MCT for 48 h, the expression of EMP-bound TF proteins was significantly increased compared with the control groups (0.704±0.024 vs. 0.355±0.0495; P<0.05; Fig. 3D). However, when cells were treated with 160 mg/l ginsenoside Rb1 for 2 h prior to exposing cells to 7.5 mM MCT for 48 h, the levels of EMP-bound TF protein were significantly lower compared with the MCT group (0.536±0.01 vs. 0.704±0.024; P<0.05; Fig. 3D).