| Objective: i TRAQ proteomics technology was applied to investigate the change of EA. hy926 cell protein expression spectrum with and without the total flavonoids of Folium Apocyni Veneti(TFF) and hyperoside. Through bioinformatics analysis and validation of different proteins, speculated the possible mechanism of TFF protect EA.hy926 cells induced by H2O2. The proteomic profiles and potential biomarkers was investigated in EA.hy926 cells of oxidative damage by H2O2 after treatment of hyp by the same technology to find the main proteins involved in apoptosis and validated on their path, in order to illustrate the potential mechanism of protect the cells and found the biomarkers of thrombotic disease.Methods:(1) The concentration of H2O2, cytotoxicity and the therapeutically effective concentration of TFF and hyp were mersure for subsequent proteomics research by MTT.(2) The proteomic profiles of each group were analysised and identificated by i TRAQ proteomics combined Q- Exactive mass spectrometry. bioinformatics analysis was done with the corresponding software.(3) The expression of different protein and its upstream and downstream protein was tested by western blotting to determine the initial mechanism.Results:(1) the EA.hy926 cells were treated with 0μmol/L, 100μmol/L, 200μmol/L, 300μmol/L, 400μmol/L H2O2 for 4h. Cell apoptosis rate increases with increasing concentrations of H2O2. The apoptosis rate reached 40 % or more with 200μmol/L H2O2for 4h, so it can be as the concentration of model group. Separately given cell 0-72μg / ml TFF and 0-80μmol/L hyperoside role for 24 h, cell activity did not change significantly. The cells were given 200μmol/L H2O2 for 4h after giving different concentrations of TFF and Hyperin for 24 h. 36 ug / ml of TFF and 20 μmol/L hyperin can better reduce the H2O2 induced cell apoptosis.(2) Using an i TRAQ(isobaric tags for relative and absolute quantitation)-based proteomics research approach, 3640 modulated proteins were identified, out of which 250 were found to be altered by hydrogen peroxide. These altered proteins are mainly involved in regulating the biological processes such as cell component organizations, cell growth, response to external stimuli and participating in the pathway such as inositol phosphate metabolism, phosphatidyl inositol signaling pathways, vitamin B6 metabolism. After treatment with TFF, the group showed the tendency to correct the abnormal expressions of 87 proteins, including HBD, TXNDC15, SLC12A6, TFPI, PA1B2 et al. Meanwhile,Hyperin correct the abnormal expressions of 52 proteins, including rootletin, Bid, Mcl-1, TFPI, PA1B2, BAP1 and so on. These proteins were associated with the apoptosis, cell cycle, cytoskeleton organization, etc. Potential target protein interaction can form a large networks and play an anti-apoptotic effect collaboratively.(3) The expression of apoptotic protein Bid and Mcl-1 were verified by western. The results suggest that the expression of each group were consistent with the results of the proteomic experient. Then its upstream and downstream proteins were detected. H2O2 make caspase-3, caspase-8, caspase-9, Fas, Fas L expression increased, however, the expression of each treatment group reduced varying degrees, and ultimately achieve anti-apoptotic effect.Conclusion:(1) TFF and Hyp had no cytotoxicity and could increased the cell viability compared with H2O2 treated cells(2) The function of H2O2 altered proteins and involved in pathways are closely associated with the occurrence of cardiovascular disease, the results show that H2O2 can cause vascular endothelial cell injury model.(3) TFF and Hyp could change the expression of proteins. The altered protein form a huge network of interaction and play a role of anti-apoptotic commonly.(3) The protective effect of hyp against H2O2-induced apoptosis in EA. hy926 cells is jointly work through mitochondrialpathway and death receptor pathway.(4) Bid and Mcl-1might be a potential biomarker for the early diagnosis of thrombotic disease. |
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