Protective Effects Of High-density Lipoprotein On Injury Of Human Umbilical Vein Endothelial Cells Induced By H₂O₂

Background and objective: The common cause of morbidity and mortality in the world is cardiovascular disease,it affects millions of people every year,and the prevalence will increase exponentially.The root cause of most cardiovascular diseases is atherosclerosis,which causes many morbidity and mortality worldwide,including most myocardial infarctions and many strokes,and disabling peripheral artery diseases.When oxidative stress stimulates the body,it will make body be in a condition of imbalance between oxidation and anti-oxidation,cells will overproduce Reactive oxygen species,bringing about stock of ROS and related metabolites in the cells,causing tissue damage.In recent years,a lot of studies have indicated that the growth,proliferation,migration and apoptosis of endothelial cells and smooth muscle cells,oxidative modification of lipoproteins can be affected by oxidative stress,and promotion of inflammation play a key role in atherosclerosis.Therefore,what is ectremely crucial for the prophylaxis and cure of cardiovascular diseases is inhibiting the damage of oxidative stress to endothelial cells.High density lipoprotein is a complex group of macromolecules composed of lipids and proteomes,which has a variety of physical and chemical properties and metabolic functions.HDL has anti-atherosclerotic and cardioprotective effects.In addition to the classic action of reverse cholesterol transport,HDL also plays an antioxidant and anti-inflammatory role.It has been figured out by lots of studies that HDL can alleviate oxidative stress injury and play a protective role in cardiovascular disease.Thus,exploring whether HDL can reduce the damage of endothelial cell induced-by oxidative stress will provide more thinking for cardiovascular diseases. This study detected the cell survival rate,senescence state,cell migration ability,tubeforming ability and protein expression,to study that whether it can cut out the H₂O₂-induced damage of HUVECs under the intervention with different concentrations of HDL.Methods: 1.Culture of HUVECs: DMEM medium + 10% fetal bovine serum,cultured at 37 ℃,5%CO2 cell incubator,cell adherent growth,passage every 2-3 days.2.The effect of H₂O₂ on the survival rate of HUVECs: HUVECs was stimulated with different concentrations of H₂O₂（100,200,300 μmol/L）for 24 hours,and the cell survival rate was detected by CCK-8 method.3.Effects of different concentrations of HDL on cell survival: The experimental groups were as follows: blank group（untreated）,H₂O₂（300 μ mol / L）group,HDL 50 μ g / m L group,HDL 100 μ g / m L group,H₂O₂（300 μ mol / L）+ HDL 50 μ g / m L group,H₂O₂（300 μ mol / L）+ HDL 100 μ g / m L group,intervened for 24 hours,and the cell survival rate was detected by CCK-8 method.4.Using cell senescence β-galactosidase staining kit to estimate the senescence of cells stimulated by H₂O₂.5.Using the transwell assay and scratch test to estimate the migration ability of HUVECs.6.Using the tube formation test to evaluate the angiogenic ability of HUVECs.7.The expression of Bcl-2,Bax and Caspase-3 and VEGF in each group was estimated by Western Blot.Results: 1.The survival rate of HUVECs stimulated by different concentrations of H₂O₂: HUVECs was stimulated by different concentrations（100,200,300 μ mol /L）of H₂O₂ for 24 hours.Compared with the blank group（96.31 ±0.94%）,the cell survival rate was in inverse proportion to the concentration of H₂O₂.The survival rates were follows:86.46 ±2.12%,76.93 ±1.53% and 65.56 ±1.75%（Figure 1）.2.HDL increased the survival rate of HUVECs stimulated by H₂O₂: The survival rate was no apparent difference in HDL50 μ g / m L group and HDL 100 μ g / m L group between the blank group（Figure 2）.H₂O₂ obviously cut out the survival rate of HUVECs,while 50 μ g / ml and 100 μ g / ml HDL could improve that of HUVECs stimulated by H₂O₂.（Figure 3）.3.Using cell senescence β-galactosidase staining kit to observe H₂O₂-stimulated cell senescence: The turquoise staining of the cytoplasm was positive for SA-β-gal staining,and then observed the cells of each group under microscope.The rates of positive cell in the blank group,H₂O₂ group,H₂O₂+ HDL50 μ g / m L group and H₂O₂+ HDL100 μ g / m L group were follows:10.43 ±2.99%,79.63 ±2.96,47.51 ±3.79% and 34.12 ±2.49%.The difference in the positive cell rate among the four groups was sharp（P < 0.05）（Figure 4）.4.Using transwell assay and scratch test to assess the migration effect of HDL on HUVECs.Transwell test showed that H₂O₂ obviously weakened the cell migration of HUVECs,while 50 μ g / ml and 100 μ g / ml HDL could improve that of HUVECs stimulated by H₂O₂（Figure 5）.Similar results were observed in scratch test（Figure 6）.5.The angiogenic ability of HUVECs was evaluated by tube formation test: Compared with the blank group,H₂O₂ significantly weakened the angiogenic ability of HUVECs,and the angiogenic ability of HUVECs in H₂O₂+ HDL50 μ g / m L group and H₂O₂ + HDL100 μ g / m L group was higher than that in H₂O₂ group（Figure 7）.6.The expression of apoptotic protein stimulated by H₂O₂ was decreased by HDL and the expression of VEGF was increased by HDL: Drawing a comparision with the blank group,the expression of Bax and Caspase-3 in H₂O₂ group increased,the expression of Bcl-2 decreased,and the difference was statistically significant（P < 0.05）.Compared with H₂O₂ group,Bax and Caspase-3 expression in H₂O₂+ HDL50 μ g / m group and H₂O₂ + HDL 100 μ g / m L group decreased significantly,the expression of Bcl-2 increased（Figure 8）.Drawing a comparision between H₂O₂ group and blank group,the expression of VEGF in H₂O₂ group cut out,and the difference was statistically significant（P < 0.05）.Compared with H₂O₂ group,the expression of VEGF in H₂O₂ + HDL50 μ g / m L group and H₂O₂ + HDL 100 μ g / m L group was significantly higher,and the difference was statistically significant（P < 0.05）（Figure 9）.Conclusion: 1.A certain concentration of HDL could increase the survival rate of HUVECs stimulated by H₂O₂.2.A certain concentration of HDL can improve the senescence of HUVECs stimulated by H₂O₂.3.H₂O₂ weakens the migration and angiogenesis of HUVECs,and HDL at a certain concentration can improve the migration and angiogenesis of HUVECs stimulated by H₂O₂.It may have a bearing on the expression of VEGF.4.The expression of anti-apoptotic protein could be increased by HDL and the expression of pro-apoptotic protein could be inhibited by HDL in HUVECs stimulated by H₂O₂,indicating that the protective effect of HDL on oxidative stress injury may be related to the inhibition of apoptosis.