Hyperoside was purchased from the Chinese Food and Drug Inspection Institute (Beijing, China). Dimethyl sulfoxide (DMSO), 4% paraformaldehyde, H₂O₂ and 3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), L-glutamine, penicillin and streptomycin, and TRIzol reagent were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The bicinchoninic acid (BCA) assay kit and cridine orange/ethidium bromide (AO/EB) were purchased from Beyotime Institute of Biotechnology, Inc. (Nanjing, China). Antibodies against phosphorylated (p-)p38 (#9211), p38 (#9212), Bcl-2 (#2876), Bax (#2772) and cleaved-caspase-3 (#9661) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (#CW0103) and β-actin (#CW0097) were purchased from CoWin Biotech Co., Ltd. (Beijing, China). H₂O₂ was prepared freshly for each experiment from a 33% (v/v) stock solution. All other chemicals and reagents were commercially available and of standard biochemical quality.

The HUVEC line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), and 0.1% penicillin/streptomycin at 37°C in 5% CO₂. For all experiments, HUVECs were grown to 70–80% confluence and were subsequently treated as designed for different experiments.

MTT assays were used to evaluate cell viability. HUVECs were seeded into 96-well plates at a density of 1×10⁴ cells/well and cultured at 37°C for 24 h. The culture medium was then removed and fresh medium for different treatments was added to each well. Following treatment, 10 µl of 5 mg/ml MTT solution was added to each well and the cells were incubated for another 4 h. The culture medium was subsequently replaced with 100 µl DMSO. The optical density in each well was determined using a Bio-Rad Microplate Reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 570 nm.

The cells were seeded at 5×10⁵ cells/well in 6-well plates and cultured for 24 h. The cells were subsequently treated with hyperoside for 24 h prior to exposure to H₂O₂ for 4 h. The cells were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 10 min and were washed with ice-cold PBS three times. The cells were stained with AO/EB solution (5 µl) for 30 sec and changes in cell morphology were observed using a fluorescence microscope (Olympus IX-71; Olympus, Tokyo, Japan).

The cells were seeded at a density of 5×10⁵ cells/well into 6-well plates and were cultured for 24 h. The cells were subsequently treated with hyperoside for 24 h prior to exposure to H₂O₂ for 4 h. The Fluo-3/AM fluorescent probe was added to the HUVEC suspension at a final concentration of 10 µM and incubated at 37°C for 40 min. The intracellular Ca²⁺ content in the HUVECs was subsequently measured with laser-scanning confocal microscopy (Olympus FV1000, Olympus) with an excitation wavelength of 488 nm and a measured emission at 530 nm.

The total RNA was extracted from the HUVECs using TRIzol reagent, according to the manufacturer's protocol. The concentration of purified RNA samples were measured spectrophotometrically using a Picodrop (Picodrop Ltd., Walden, UK). The RT reaction was performed using a SuperScript III First-Strand Synthesis system (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Amplification of the RT product by PCR was performed using Promega Taq DNA Polymerase (Promega Co., Madison, WI, USA). All reactions were performed in a thermal cycler (Model 2400; Perkin-Elmer, Norwalk, CT, USA) with primers (Sangon Biotech Co., Ltd., Shanghai, China) specific for Bcl-2, forward: 5′-CTT CGC CGA GAT GTC CAG CCA-3′ and reverse: 5′-CGC TCT CCA CAC ACA TGA CCC-3′; Bax, forward: 5′-TGC TTC AGG GTT TCA TCC AGGA-3′ and reverse: 5′-ACG GCG GCA ATC ATC ATC CTC TG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: 5′-TCT CTG CTC CTC CTG TTC GAC-3′ and reverse: 5′-TTA AAA GCA GCC CTG GTG AC-3′. Thermal cycling conditions involved an initial denaturation step at 94°C for 10 min, followed by 30 cycles of denaturation at 94°C for 10 sec, and primer annealing at 60°C for 1 min, which was followed by agarose gel electrophoresis.

Following treatment with various concentrations of hyperoside prior to H₂O₂ exposure, the HUVECs were washed with ice-cold PBS and harvested by trypsinization. The cells were centrifuged (1,000 rpm for 5 min) and washed with ice-cold PBS three times. The cells were then suspended in 100 µl ice-cold radioimmunoprecipitation lysis buffer (Beyotime Institute of Biotechnology, Inc.), sonicated 10 times for 5 sec with 10 sec pauses in an ice-water bath, and the samples were centrifuged (13,000 rpm for 5 min at 4°C). The supernatants were stored at −80°C. Quantification of the protein was performed using a BCA assay.

Equal quantities of protein extracts (40 µg) were separated on 12% sodium dodecyl sulfate-polyacrylamide gels. The proteins were subsequently transferred onto nitrocellulose membranes (Pall Gelman Laboratory Corporation, Ann Arbor, MI, USA). The membranes were blocked with 5% (w/v) non-fat milk powder in Tris-buffered saline/0.1% Tween-20 (TBST) for 1.5 h at room temperature. Following blocking, the membranes were incubated overnight at 4°C with the primary antibody (dilution, 1:1,000). After three washes with TBST, the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G as the secondary antibody at room temperature (dilution, 1:2,000). After three washes, the proteins were detected using an enhanced chemiluminescence detection kit (CoWin Biotech Co., Ltd., Beijing, China).

The data are presented as the mean ± standard deviation. Statistical comparisons were performed using a Student's t-test, and the differences between multiple groups were assessed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the apoptotic induction of H₂O₂ on HUVECs, HUVECs were incubated with 200 µM H₂O₂ for different durations. As shown in Fig. 2A, the viability of HUVECs decreased ~50% following exposure to 200 µM H₂O₂ for 4 h. Therefore, the exposure of HUVECs to 200 µM H₂O₂ for 4 h was selected to induce apoptosis of HUVECs for the subsequent experiments.

To ensure the suitable concentrations of hyperoside, the cytotoxicity of hyperoside was evaluated by MTT assay. HUVECs were cultured with various concentrations of hyperoside for 24 h and the cell viability was assessed. The result revealed that hyperoside at concentrations of <20 µM caused no affect the viability of HUVECs, as shown in Fig. 2B. Therefore, the concentrations of hyperoside were confirmed as 10, 15 and 20 µM in the subsequent experiments.

As shown in Fig. 2C, the H₂O₂-induced decrease of cell viability was significantly attenuated by hyperoside treatment in a dose-dependent manner (P<0.01), which suggested that hyperoside exhibited protective effect on H₂O₂-induced HUVECs injury.

As shown in Fig. 3, AO/EB staining demonstrated that exposure of HUVECs to 200 µM H₂O₂ for 4 h induced HUVEC apoptosis. However, the treatment of HUVECs with 10, 15 and 20 µM hyperoside significantly attenuated the apoptosis of HUVECs induced by H₂O₂, which demonstrated that hyperoside exhibited anti-apoptosis effects in H₂O₂-induced HUVECs.

As shown in Fig. 4, compared with the control group, intercellular Ca²⁺ content, presented as the fluorescence intensity, was significantly overloaded in the H₂O₂-induced HUVECs (P<0.01). However, the treatment of HUVECs with different concentrations of hyperoside (10, 15 and 20 µM) significantly inhibited the increase of fluorescence intensity induced by H₂O₂ in a dose-dependent manner (P<0.01). This indicated that hyperoside exhibited protective effects on the oxidative stress-induced increase of intercellular Ca²⁺ content.

As shown in Fig. 5, the mRNA expression of Bax was significantly increased in the H₂O₂-induced group. By contrast, the mRNA expression of Bcl-2 was significantly decreased compared with the control group. However, different concentrations of hyperoside (10, 15 and 20 µM) exhibited significant inhibition on the H₂O₂-induced increase of Bax and decrease of Bcl-2 mRNA in a dose-dependent manner (P<0.01).

As shown in Figs. 6 and 7, compared with the control group, the expression of cleaved caspase-3, Bax and p-p38 was significantly increased, while Bcl-2 was significantly decreased, in the H₂O₂-induced group. However, the treatment with different concentrations of hyperoside (10, 15 and 20 µM) revealed a significant inhibition on the H₂O₂-induced increase of cleaved caspase-3, Bax and p-p38, and the decrease of Bcl-2 in a dose-dependent manner (P<0.01). This indicated that hyperoside exhibited antiapoptotic effects on H₂O₂-induced HUVECs.