Hyperin was purchased from the National Institutes for Food and Drug Control (Beijing, China); its chemical structure is shown in Fig. 1. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). iTRAQ reagent was obtained from Applied Biosystems Life Technologies (Foster City, CA, USA). Antibodies against BH3-interacting domain death agonist (Bid; BS1819), myeloid cell leukemia-1 (Mcl-1; BS1220), β-actin (AP0060), Fas (BS6430) and FasL (BS1122) were obtained from Bioworld Technology, Inc., (Nanjing, China). Antibody against truncated tBID (tBID; ab10640) was purchased from Abcam (Cambridge, UK). Antibodies against caspase-3 (#9664), caspase-8 (#8592) and caspase-9 (#9509) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). H₂O₂ was freshly prepared for each experiment from a 3% stock solution.

EA.hy926 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing, China) and were cultured in DMEM supplemented with heat-inactivated FBS (10%), 100 U/ml penicillin, and 100 g/ml streptomycin. The cells were incubated in a humidified incubator aerated with 5% CO₂ at 37°C. Hyperin was dissolved in dimethyl sulfoxide (DMSO), and the DMSO content in all groups was <0.1%. Prior to treatment, the cells were incubated with serum-free medium for 24 h and randomly assigned to three groups: a 'control group', an 'H₂O₂-exposed group', and a 'hyperin-treated group'. The cells in the hyperin group were treated with designated concentrations of hyperin for 24 h prior to 200 μmol/l H₂O₂ exposure for 4 h in fresh medium.

Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, EA.hy926 cells (in logarithmic phase) were seeded into 96-well plates (1×10⁴ cells/well) and cultured for 24 h. The medium was then replaced with fresh medium for the different treatments. Each concentration of reagent was added to the culture fluid of six parallel wells and a blank well was used as a control. Subsequently, 10 μl of 5 mg/ml MTT in phosphate-buffered saline (PBS) was added to each well and the cells were further incubated for 4 h. The culture medium was then carefully removed and DMSO (100 μl/well) was added to dissolve the formazan precipitate. The plates were shaken for 10 min. Optical density was read at 570 nm (490 nm as reference) on a universal microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The viability of the EA.hy926 cells in each well was expressed as a percentage of the viable control cells.

The cells of each experimental group were collected by centrifugation (138 × g for 5 min) and washed twice with PBS. Protein extracts were prepared using lysis buffer (8 mol/l urea, 30 mmol/l HEPES, 1 mmol/l PMSF, 2 mmol/l EDTA, 10 mmol/l DTT). The protein concentration was estimated using a Bradford assay. iTRAQ labeling was performed according to the manufacturer's instructions (Applied Biosystems/MDS SCIEX, Toronto, ON, Canada). Briefly, 100 μg of each protein sample was reduced, alkylated and subjected to trypsin hydrolysis. Each sample was labeled separately with the isobaric tags as follows: control group (114 tags), H₂O₂ group (115 tags), and hyperin group (116 tags). All labeled peptides were pooled and dried in a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA.

The combined iTRAQ-labeled samples were dissolved in 200 μl buffer A [25% acetonitrile (ACN), 10 mmol/l KH₂PO₄, pH 3.0, with phosphoric acid]. The proteins were separated on a Luna SCX column (4.6×250 mm, 100-Å pore size; Phenomenex, Inc., Torrance, CA, USA) with buffers A and B (buffer b: 25% ACN, 2 mol KCl, 10 mmol/l KH₂PO₄, pH 3.0, with phosphoric acid) at a flow rate of 400 nl/min. A solvent gradient system was used as follows: 0–35 min, 0% B; 35–36 min, 0–5% B; 36–56 min, 5–30% B; 56–61 min, 30–50% B; 61–66 min, 50% B; 66–71 min, 50–100% B; 71–81 min, 100% B. Elution was monitored by measuring absorbance at 214 nm and fractions were collected every 1 min. The collected eluents were lyophilized to powder and resuspended in 0.1% (v/v) trifluoroacetic acid (TFA; 40 μl) for further desalting and concentration using a Strata-X C18 cartridge (Phenomenex, Torrance, CA, USA). The desalted samples were lyophilized to powder.

In this study, iTRAQ-labeled samples were redissolved in 6 μl eluent buffer A [0.1% formic acid (FA) in water, v/v]. Each of the fractions was analyzed three times using a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). A flow rate of 400 nl/min was used for protein separation on a C18 capillary column (Michrom Bioresources, Inc., Auburn, CA, USA). A solvent gradient system was used: 0–10 min, 5% B (0.1% FA in ACN); 10–40 min, 5–30% B; 40–45 min, 30–60% B; 45–48 min, 60–80% B; 48–55 min, 80% B; 55–58 min, 80–5% B; 58–65 min, 5% B. A full MS scan (350–2,000 m/z range) was acquired in the Orbitrap at a mass resolution of 70,000. A maximum of 10 precursors per cycle were then chosen for fragmentation by high-energy collision dissociation (HCD) in the C-trap in linear trap quadrupole with an isolation width of 3.0 m/z. Precursor ion activation was performed with an isolation width of 2.5 Da. The ion transfer tube temperature and spray voltage were 320°C and 1.8 kV, respectively.

For protein identification, MS/MS spectra were analyzed using Proteome Discoverer software v. 1.3 (PD; Thermo Fisher Scientific). The precursor ion mass range was set at 350–6,000 Da. The minimum number of peaks in a spectrum was set to 10, and the threshold for the S/N ratio was set to 1.5. Next, the MS spectra were searched using Mascot 2.3.0 (Matrix Science, London, UK) with precursor mass tolerance at 15 ppm, fragment ion mass tolerance at 20 mmu, trypsin enzyme with 1 miscleavage, methyl methanethiosulfonate of cysteine and iTRAQ 8-plex of lysine and the NH₂-terminus as fixed modifications, and deamidation of asparagine and glutamine, oxidation of methionine and iTRAQ 8-plex of tyrosine as variable modifications. Protein identifications were accepted at 95% or higher probability and contained at least two identified peptides with a false discovery rate (FDR) <1%. The peptides were quantified using PD software. Tagged samples were normalized by comparing median protein ratios for the reference channel. Protein quantitative ratios were calculated from the median of all peptide ratios. The proteins with a relative expression of >1.2 or <0.8, and with P<0.05 to ensure up- and downregulation authenticity, were chosen for further analysis. Protein sequences and functional information were retrieved from the UniProt databases (http://www.uniprot.org/). For further analysis, functional annotation analysis of altered proteins was carried out using DAVID annotation software (http://david.abcc.ncifcrf.gov/) and the KEGG database (http://www.genome.jp/kegg/) by importing GenInfo (GI) numbers.

Following treatment with various concentrations of hyperin and/or H₂O₂ as described above, the EA.hy926 cells were harvested and washed with PBS. Protein extracts were prepared with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentration was estimated using a bicinchoninic acid (BCA) protein assay kit (Sangon Biotech, Shanghai, China). For western blot analysis, equal amounts of protein (50 μg) were separated on 12% sodium dodecyl (SDS)-polyacrylamide gels and electrotransferred onto nitrocellulose membranes (PALL Gelman Laboratory, Ann Arbor, MI, USA) that were blocked in 5% non-fat milk. The membranes were incubated overnight at 4°C with primary antibodies. After three washes with Tris-buffered saline containing 0.05% Tween-20 (TBS-T), the membranes were incubated for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody at room temperature. The bands were visualized using an ECL detection kit (CoWin Biotech, Beijing, China). Quantification of the bands was performed by densitometric analysis using the Adobe Photoshop 7.0.1 software (Adobe Systems, Inc., San Jose, CA, USA).

Quantitative detection of apoptotic cells and analysis of cell cycle distribution in the cultures were undertaken using flow cytometry. The EA.hy926 cells treated with different concentrations of hyperin and/or exposed to 200 μmol/l H₂O₂ were collected by centrifugation (138 × g for 5 min) and cell density was adjusted to 1×10⁵ cells/ml. The cells were washed twice with cold PBS and centrifuged (138 × g for 5 min). The pellets were fixed overnight in pre-cooled 70% (v/v) ethanol at 4°C, and then washed with cold PBS. The cells were suspended in 1 ml of propidium iodide solution (20 mg/ml) supplemented with 0.25 mg/ml RNase A and 0.1% (v/v) Triton X-100, and incubated on ice for 30 min in the dark. The samples were analyzed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Each experiment was performed at least 3 times. All data are expressed as the means ± SD. Statistical analysis was performed using a Student's t-test and SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). A P-value <0.05 was considered to indicate a statistically significant difference.

We assessed whether hyperin protects EA.hy926 cells against the effect of H₂O₂ by MTT assay. No obvious cytotoxicity in untreated cells was noted, nor was obvious cytoxicity noted in cells treated with hyperin at concentrations in the range of 2.5–80 μmol/l (Fig. 2A). We also noted that hyperin exerted a protective effect on H₂O₂-injured cells in a dose-dependent manner (Fig. 2B). When the cells were treated with 20 μmol/l hyperin, cell viability was restored to 98%. Therefore, this concentration was selected for subsequent proteomic analysis.

A total of 3,640 proteins were identified using iTRAQ, of which 250 were altered by H₂O₂ (data not shown); the 250 proteins were functionally classified into various relevant categories such as transition metal ion-binding, zinc ion-binding and transferase activity, which are mainly involved in regulating cellular component organization, growth, cytoskeleton organization, and response to stimulus, using the NCBI online database and DAVID software platform; the most significantly up- and downregulated proteins are shown in Fig. 3. Further analysis of the pathways and networks revealed that the regulated proteins were mainly involved in apoptosis, inositol phosphate metabolism and vitamin B6 metabolism.

Following treatment with hyperin, of the 250 proteins that exhibited altered expression upon H₂O₂ exposure, 52 revealed a tendency towards restoration of the expression levels (Table I). Compared with the H₂O₂ group, 28 proteins were downregulated and 24 were upregulated. These proteins were associated with apoptosis, cell cycle and cytoskeleton organization.

An examination of Bid and Mcl-1 expression after the iTRAQ experiment demonstrated marked changes. Therefore, the results were validated by western blot analysis, as shown in Fig. 4. Compared with the H₂O₂-exposed group, Bid expression in the hyperin-treated groups was significantly decreased, while Mcl-1 expression was significantly increased. In both cases, the effect was dose-dependent. The results were in agreement with those of the proteomics analysis.

We further analyzed the effect of H₂O₂ and hyperin on apoptosis and cell cycle distribution by flow cytometry. The group exposed to 200 μmol/l H₂O₂ exhibited a higher rate of apoptosis (30.83%) than the control group (1.32%) (Fig. 5). Treatment with hyperin at 10, 15 or 20 μmol/l resulted in decreased accumulation of apoptotic cells at the sub-G peak compared with that in the H₂O₂-exposed group. The percentage of cells in the G0/G1 phase was increased in the H₂O₂-exposed group; however, it decreased in the hyperin group in a dose-independent manner. The results indicated that the cells were blocked in the G0/G1 phase following H₂O₂ treatment and that hyperin reduced the number of cells in cycle arrest, thereby reducing damage.

Fas, FasL, cleaved caspase-3, -8, -9 play important roles in apoptosis. tBid is the cleaved form of Bid and is involved in the mitochondrial apoptotic pathway (22,23). The expression of these proteins was assayed by western blot analysis to clarify the role of the Bid- and Mcl-1-mediated apoptosis mechanism in H₂O₂-injured EA.hy926 cells and the effect of hyperin. As shown in Fig. 6A–F, hyperin significantly decreased the relative expression of these proteins compared to that in the H₂O₂ group, in a dose-dependent manner.