Contributed equally
Endothelial injury has been implicated in the pathogenesis of many cardiovascular diseases, including thrombotic disorders. Hyperin (quercetin-3-O-galactoside), a flavonoid compound and major bioactive component of the medicinal herb
Endothelial dysfunction has been implicated in the pathogenesis of many cardiovascular diseases, including thrombotic disorders (
Flavonoids are polyphenolic compounds that are widespread in many plants, and exert various biochemical and pharmacological effects (
Isobaric tags for relative and absolute quantitation (iTRAQ) (
In the present study, iTRAQ-based proteomic analysis was applied to investigate the effect of hyperin against H2O2-induced injury in human endothelium-derived EA.hy926 cells, and to elucidate the potential protective mechanism of hyperin in oxidative stress-induced injury.
Hyperin was purchased from the National Institutes for Food and Drug Control (Beijing, China); its chemical structure is shown in
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
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
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
The combined iTRAQ-labeled samples were dissolved in 200
In this study, iTRAQ-labeled samples were redissolved in 6
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 (
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
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
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
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
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 (
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
We further analyzed the effect of H2O2 and hyperin on apoptosis and cell cycle distribution by flow cytometry. The group exposed to 200
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 (
In our previous study (
In the present study, mitogen-activated protein kinase kinase kinase 7 (MAP3K7) and receptor-type tyrosine-protein phosphatase-kappa (PTPRK) expression was upregulated following hyperin treatment (
By contrast, the expression of macrophage migration inhibitory factor (Mif), and cofilin 1 (CFL1) in response to H2O2 was downregulated in hyperin-treated cells (
iTRAQ proteomic analysis revealed marked changes in the expression of the anti-apoptotic protein, Mcl-1, and the pro-apoptotic protein, Bid. However, the mechanism underlying Bid- and Mcl-1-mediated apoptosis remains unclear and requires further study.
Mcl-1 is an outer mitochondrial membrane-bound protein of the Bcl-2 family that has a BH3-like domain, and plays an important role in the anti-apoptotic process (
In conclusion, we systematically examined and compared the proteomic profiles of untreated, H2O2-exposed, and hyperin pre-treated EA.hy926 cells for the first time, to the best of our knowledge. The results demonstrate that hyperin effectively prevents H2O2-induced cell injury through regulation of the Mcl-1- and Bid-mediated anti-apoptotic mechanism. Our findings suggest that hyperin is a promising candidate for use in the treatment of thrombotic diseases.
This study was financially supported by the National Natural Science Foundation of China (grant nos. 81274132 and 81172938). We are grateful to Editage for providing editorial assistance.
Chemical structure of hyperin.
Cell viability determined by MTT assay. (A) EA.hy926 cells were incubated with increasing concentrations of hyperin (2.5–80
Schematic representation of the altered proteomic pathways in response to hyperin treatment. The altered proteins in the hyperin + H2O2 group are labeled with (↓) and (↑), representing the downregulated and upregulated proteins compared with the H2O2-exposed group.
Western blot analysis of the effects of hyperin on the expression of (A) myeloid cell leukemia sequence-1 (Mcl-1) and (B) BH3-interacting domain death agonist (Bid) in H2O2-exposed EA.hy926 cells. EA.hy926 cells were treated with hyperin for 24 h and then treated with 200
Apoptosis of EA.hy926 cells was measured by flow cytometry. (A) Control cells (no treatment). (B) Cells exposed to 200
Western blot analysis of the effects of hyperin on expression of (A) tBid, (B) cleaved caspase-3, (C) Fas, (D) cleaved caspase-9, (E) FasL, (F) caspase-8 and cleaved caspase-8 in H2O2-exposed EA.hy926 cells. EA.hy926 cells were treated with hyperin for 24 h and then exposed to 200
List of differentially expressed proteins.
Accession |
Description | MW [kDa] |
Calc. pI |
115/114 |
116/115 |
---|---|---|---|---|---|
Q5TZA2 | Rootletin | 228.4 | 5.5 | 4.464 | 0.244 |
P68402 | Platelet-activating factor acetylhydrolase IB subunit β | 25.6 | 5.92 | 1.767 | 0.610 |
P33981 | Dual specificity protein kinase TTK | 97 | 8.16 | 1.733 | 0.647 |
Q6NSJ5 | Leucine-rich repeat-containing protein 8E | 90.2 | 6.96 | 1.653 | 0.555 |
O60518 | Ran-binding protein 6 | 124.6 | 5.01 | 1.639 | 0.603 |
Q6PJG6 | BRCA1-associated ATM activator 1 | 88.1 | 5.27 | 1.462 | 0.801 |
P55957 | BH3-interacting domain death agonist | 22 | 5.44 | 1.416 | 0.691 |
Q9BVK2 | Probable dolichyl pyrophosphate Glc1Man9GlcNAc2 α-1,3-glucosyltransferase | 60 | 9.14 | 1.416 | 0.691 |
Q8IUC8 | Polypeptide N-acetylgalactosaminyltransferase 13 | 64 | 6.83 | 1.385 | 0.791 |
P02795 | Metallothionein-2 | 6 | 7.83 | 1.361 | 0.685 |
P04732 | Metallothionein-1E | 6 | 7.96 | 1.294 | 0.798 |
Q71U36 | Tubulin α-1A chain | 50.1 | 5.06 | 1.290 | 0.654 |
P62328 | Thymosin β-4 | 5 | 5.06 | 1.271 | 0.780 |
P51858 | Hepatoma-derived growth factor | 26.8 | 4.73 | 1.263 | 0.817 |
P20962 | Parathymosin | 11.5 | 4.16 | 1.261 | 0.736 |
P14174 | Macrophage migration inhibitory factor | 12.5 | 7.88 | 1.241 | 0.808 |
P23528 | Cofilin-1 | 18.5 | 8.09 | 1.236 | 0.790 |
Q92688 | Acidic leucine-rich nuclear phosphoprotein 32 family member B | 28.8 | 4.06 | 1.232 | 0.810 |
O60232 | Sjoegren syndrome/scleroderma autoantigen 1 | 21.5 | 5.24 | 1.230 | 0.717 |
Q3MJ13 | WD repeat-containing protein 72 | 123.3 | 6.67 | 1.230 | 0.821 |
Q3YEC7 | Rab-like protein 6 | 79.5 | 5.22 | 1.230 | 0.783 |
P39687 | Acidic leucine-rich nuclear phosphoprotein 32 family member A | 28.6 | 4.09 | 1.220 | 0.768 |
Q00535 | Cyclin-dependent kinase 5 | 33.3 | 7.66 | 1.218 | 0.755 |
Q15147 | 1-Phosphatidylinositol 4,5-bisphosphate phosphodiesterase β-4 | 134.4 | 6.9 | 1.217 | 0.729 |
P58546 | Myotrophin | 12.9 | 5.52 | 1.209 | 0.769 |
Q8NCW5 | NAD(P)H-hydrate epimerase | 31.7 | 7.66 | 1.206 | 0.807 |
P00338 | L-lactate dehydrogenase A chain | 36.7 | 8.27 | 1.202 | 0.798 |
O43318 | Mitogen-activated protein kinase kinase kinase 7 | 67.2 | 7.11 | 0.833 | 1.244 |
Q9H6Y7 | E3 ubiquitin-protein ligase RNF167 | 38.3 | 5.63 | 0.824 | 1.233 |
Q04756 | Hepatocyte growth factor activator | 70.6 | 7.24 | 0.820 | 1.284 |
O00255 | Menin | 68 | 6.55 | 0.811 | 1.243 |
Q9UP83 | Conserved oligomeric Golgi complex subunit 5 | 92.7 | 6.6 | 0.806 | 1.224 |
Q8IWE4 | DCN1-like protein 3 | 34.3 | 5.12 | 0.796 | 1.280 |
Q9UBR2 | Cathepsin Z | 33.8 | 7.11 | 0.794 | 1.219 |
P0CG39 | POTE ankyrin domain family member J | 117.3 | 5.97 | 0.783 | 1.311 |
Q9Y676 | 28S ribosomal protein S18b, mitochondrial | 29.4 | 9.38 | 0.779 | 1.259 |
Q9UMR5 | Lysosomal thioesterase PPT2 | 34.2 | 6.33 | 0.769 | 1.227 |
Q9BT40 | Inositol polyphosphate 5-phosphatase K | 51.1 | 6.54 | 0.747 | 1.200 |
P42356 | Phosphatidylinositol 4-kinase α | 231.2 | 6.87 | 0.733 | 1.404 |
P39210 | Protein Mpv17 | 19.7 | 9.47 | 0.730 | 1.201 |
Q9H330 | Transmembrane protein 245 | 100.9 | 8.87 | 0.713 | 1.369 |
Q68CQ4 | Digestive organ expansion factor homolog | 37.3 | 5.66 | 0.705 | 1.234 |
Q9P2K3 | REST corepressor 3 | 87 | 5.88 | 0.692 | 1.428 |
Q6PJF5 | Inactive rhomboid protein 2 | 55.5 | 8.27 | 0.676 | 1.349 |
Q8NF91 | Nesprin-1 | 96.6 | 8.82 | 0.657 | 1.522 |
Q9UHW9 | Solute carrier family 12 member 6 | 1010.5 | 5.53 | 0.611 | 1.366 |
Q63HN8 | E3 ubiquitin-protein ligase RNF213 | 127.5 | 7.08 | 0.604 | 1.515 |
Q86UB9 | Transmembrane protein 135 | 591 | 6.48 | 0.587 | 1.958 |
Q92560 | Ubiquitin carboxyl-terminal hydrolase BAP1 | 52.3 | 9.45 | 0.453 | 1.938 |
Q86WA8 | Lon protease homolog 2, peroxisomal | 80.3 | 6.84 | 0.442 | 1.352 |
Q07820 | Induced myeloid leukemia cell differentiation protein Mcl-1 | 94.6 | 7.3 | 0.408 | 2.215 |
Q8TE02 | Elongator complex protein 5 | 34.8 | 4.97 | 0.772 | 1.217 |
Differentially expressed proteins.
Swiss-Prot accession number;
MW, molecular weight of the matched protein in kDa;
pI, isoelectric point of the matched protein;
fold-changes of the proteins in the H2O2 vs. control groups;
fold-changes of the proteins in the hyperin vs. H2O2 groups.