Vaccarin was purchased from Shanghai Shifeng Technology Co., Ltd. (Shanghai, China). Sulforhodamine B (SRB) was purchased from Sigma-Aldrich (St. Louis, MO, USA). An Annexin V-Fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kit (cat. no. KGA104) was purchased from KeyGEN Biotechnology, Co., Ltd. (Nanjing, China). A DAB kit (cat. no. P0203) was purchased from Beyotime Institute of Biotechnology (Jiangmen, China). Rabbit polyclonal notch1 (cat. no. ab52627; 1:500), rabbit polyclonal Hes1 (cat. no. ab108937; 1:500), rabbit polyclonal caspase3 (cat. no. ab32351; 1:1,000) and rabbit polyclonal β-tublin (cat. no. ab6046; 1:1,000) antibodies were purchased from Abcam (HongKong, China). Polyclonal goat anti-rabbit IgG secondary antibody (cat. no. ab10058; 1:2,000) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Kits used for the measurement of lactate dehydrogenase (LDH; cat. no. 20130620), methane dicarboxylic aldehyde (MDA; cat. no. 20130608), super oxygen dehydrogenase (SOD; cat. no. 20130618) and bicinchoninic acid (BCA; cat. no. 20130619) concentrations were purchased from the Institute of Jiancheng Bioengineering (Nanjing, China). M-PER Mammalian Protein Extraction Reagent was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA).

Human EA·hy926 endothelial cells (cat. no. CRL-2922; American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37°C in a humidified air containing 5% CO₂.

Prior to induction with high glucose, the cells were grown to 80–90% confluence and placed in 2% serum-containing media for 12 h to achieve cell synchronization. The vaccarin solution was diluted (3.44, 6.88 or 13.76 µM) with culture medium immediately prior to the experiment. The cells were treated with glucose in the absence or presence of vaccarin. The cell monolayers were pretreated with vaccarin (3.44, 6.88 or 13.76 µM) at 37°C for 24 h, followed by being induced by glucose. Following treatment with glucose, the cells were maintained in 10% serum-containing media in a 5% CO₂ atmosphere at 37°C and used for further experiments.

An SRB assay was performed to assess EA·hy926 cell viability (20). The EA·hy926 cells were seeded into 96-well culture plates, with four replicates for each concentration. Culture of the cells in medium was continued for 24 h, following which vaccarin solution at three final concentrations (3.44, 6.88 and 13.76 µM), dissolved in serum-free medium, were added to each well. Following 24 h incubation at 37°C, glucose solution (90, 180 or 270 mM) dissolved in serum-free medium were added to each well and continued to culture for 12 and 24 h. The medium was then removed and 5% trichloroacetic acid (TCA; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to each well for 200 µl to fix the cells for 40 min at 4°C. The TCA solution was then removed and replaced with 100 µl SRB, and the cells were incubated at 30°C for 30 min. Following removal of the SRB, the samples were washed with deionized water twice. Finally 10% tris hydroxymethyl aminomethane (Tris; Sinopharm Chemical Reagent Co., Ltd.) was used to dissolve the SRB, and the samples were shaken for 30 sec twice at room temperature. The results were determined at 540 nm using a Multiskan MK3 reader (Thermo Fisher Scientific, Inc.), with the cell viability expressed as an optical density (OD) value. In addition, the cell morphology was observed under an inverted/phase contrast microscope, and images were captured at 200× magnification using a Nikon Eclipse Ti microscope (Nikon, Toyko, Japan).

The migration rate of the cells was measured using a wound healing assay (21). Briefly, the EA•hy926 cells (8×10⁴/well) were seeded into wells and were cultured at 37°C in a saturated humidity containing 5% CO₂ for 24 h. When the cells had attached completely, a line was scaped through the middle of the cell plate, measuring ~1 mm in width, following treatment with vaccarin (6.88 and 13.76 µM). The cells were incubated, and images of randomly-selected fields were captured at 100× magnification under a microscope video system (Nikon Eclipse Ti; Nikon). The mean distances between the two ends of the scratch were quantified by manual measurements, and the migration rate was calculated with the control defined as 100%.

LDH, an indicator of cell injury, was detected using an assay kit, according to the manufacturer's protocol, with the activity of the enzyme expressed as units per liter, and the absorbance was read at 450 nm using a Multiskan MK3 microplate reader (Thermo Fisher Scientific, Inc.). As described previously (22), the activities of SOD and MDA were determined using commercially available kits, according to the manufacturer's protocol, with enzyme activities expressed as units/mg protein. A single unit of SOD activity was defined as the quantity, which reduced the absorbance at 450 nm by 50%. The measurement of BCA was performed prior to determining the SOD. Levels of MDA were measured at 532 nm by its reaction with thiobarbituric acid to form a stable chromophoric product, with MDA levels expressed as nmol/mg protein.

The EA.hy926 cells were prepared for analysis, according to the manufacturer's protocol of the Annexin V-FITC/PI Apoptosis Detection kit. The stained cells were quantitatively detected using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) in the FL1-H and FL2-H channels. Data were analyzed using CellQuest Pro software version 6.0 (BD Biosciences), with a total of 10,000 cells analyzed.

In a 24-well plate with cover slips, 6×10⁴ EA•hy926 cells were seeded into each well and cultured for 24 h, following treatment with different doses of vaccarin (6.88 and 13.76 µM) for 24 h prior to being subjected to 24 h glucose (180 mM) induction. Following removal of the culture medium, the cells were fixed with 0.5 ml 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.), and then washed twice with phosphate-buffered saline (PBS). Following treatment with Hoechst33342 (Wuhan Boster, China) for 10 min, the cells were rinsed twice with PBS. The stained cells were immediately observed under a fluorescence microscope (Nikon Eclipse Ti, Nikon, Tokyo Japan).

Protein levels were analyzed using Western blot analysis, as described previously (23). Briefly, total protein was extracted using the BCA kit and 25 µg total protein/well was loaded onto SDS-PAGE gels (Beyotime Institute of Biotechnology) following denaturing in loading buffer at 100°C for 5 min. The protein extracts were subjected to 8–12% SDS-PAGE and transferred onto a nitrocellulose membrane (EMD Millipore, Billerica, MD, USA). Following the transfer, the membranes were blocked at room temperature for 2 h in 5% nonfat dry milk/Tris-buffered saline with Tween 20 (TBST; Sinopharm Chemical Reagent Co., Ltd.) and then incubated at 4°C overnight with the following primary antibodies: Notch1 (1:500), Hes1 (1:500), caspase3 (1:1,000) and β-tublin (1:1,000). The following day, the membranes were washed three times with TBST for 10 min at room temperature, and subsequently incubated in secondary antibody (anti-rabbit IgG; 1:2,000) conjugated to horseradish peroxidase for 2 h at room temperature. Following incubation, the membranes were washed, as above, and the protein bands were visualized using a DAB Advanced Western Blotting Detection kit (Beyotime Institute of Biotechnology). β-tublin was used as the protein loading control following measurement of the integral optical density.

The results are expressed as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance with SPSS 20.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

In order to investigate the effect of high glucose on EA·hy926 cells in the present study, the cells were induced by high glucose (90, 180 or 270 mM) for 12 and 24 h, and cell viability was examined using an SRB assay. As shown in Fig. 1B, treatment with high glucose alone significantly reduced cell viability by >50% following 12 h treatment at 270 mM, and following 24 h treatment with 180 and 270 mM glucose. The OD values in the control group were 0.885±0.005 and 1.022±0.020 following 12 and 24 h in culture, respectively. All the groups had a significant decrease in viability, compared with these normal control groups (P<0.01). These results resulted in the selection of 24 h treatment with 180 mM glucose for the subsequent experiments. In addition, the present study investigated the expression levels of Notch1 and Hes1 following 24 h high glucose culture and the results suggested that high glucose treatment significantly increased the expression levels of Notch1 and Hes1 in a dose-dependent manner (Fig. 1C).

The effects of vaccarin on EA·hy926 cell proliferation were examined following 12 and 24 h treatment with 180 mM glucose. As shown in Fig. 2A, the cell viability in the presence of vaccarin increased significantly, compared with the groups without vaccarin treatment, respectively (P<0.01). Vaccarin afforded dose-dependent protection against the reduction in cell viability induced by high glucose concentrations between 3.44 and 13.76 µM. As observed under the microscope, high glucose treatment resulted in significant cell shrinkage, compared with the control group. However, pretreatment with three different vaccarin concentrations (3.44, 6.88 and 13.76 µM) attenuated high glucose-injured cell shrinkage (Fig. 2B). Based on these results, pretreatment with 6.88 and 13.76 µM vaccarin and 180 mM glucose for 24 h was selected for use in the subsequent experiments. As shown in Fig. 2C, following treatment with high glucose, the migratory ability of cells decreased, resulting in a migration ratio of 23.16±2.87% (P<0.01), compared with the normal cells. However, vaccarin at concentrations of 6.88 and 13.76 µM significantly increased the migration ratio (50.71±7.33 and 82.00±1.95%, respectively, compared with the high glucose group (P<0.01).

The induction of apoptosis was measured by Annexin V/PI double staining. The results of the flow cytometric analysis in the high glucose group showed increase apoptosis. The ratio of prophase and late apoptosis reached 8.66±0.30 and 13.35±1.11%, respectively (P<0.01), compared with the control group. However, the apoptotic ratio following treatment with vaccarin in the 6.88 and 13.76 µM groups significantly declined (P<0.01), compared with the high glucose group (Fig. 3A).

In the Hoechst staining experiment, it was also found that apoptosis was markedly higher in the high glucose group (Fig. 3B). Following treatment with vaccarin (6.88 and 13.76 µM), the apoptotic index was significantly decreased (P<0.01), compared with the high glucose group.

Treatment of the cells with high glucose for 24 h decreased the levels of SOD, but increased LDH release and the levels of MDA (P<0.01), compared with the control group. As shown in Fig. 4, incubation of the EA·hy926 cells in the presence of vaccarin (6.88 and 13.76 µM) with high glucose significantly increased SOD activity (Fig. 4A) and decreased the level of MDA and release of LDH, respectively (Fig. 4B and C). According to these results, vaccarin significantly altered SOD activity, LDH leakage and MDA levels in the high glucose-induced endothelial cells in a concentration-dependent manner.

To further investigate the effect and mechanism of vaccarin of high glucose-injured EA·hy926 cells, the presented study examined the expression levels of Notch1, Hes1 and caspase 3 using Western blot analysis. As shown in Fig. 4D, treatment with high glucose significantly increased the expression levels of Notch1, Hes1 and caspase 3, relative to the control group (P<0.01). By contrast, in the cells were pretreated with vaccarin (6.88 and 13.76 µM), the expression levels of Notch1, Hes1 and caspase 3 decreased significantly (P<0.01), compared with the high glucose group.