Protective Effect Of Salvianolic Acid B On Ox-LDL Induce Human Umbilical Vein Endothelial Cell Apoptosis

ObjectiveAtherosclerosis（AS） are the incidence of common foundation of coronary heart disease,cerebral infarction and other cardiovascular and cerebrovascular diseases. According to the data of WHO Health Statistical 2007,the mortality rate for cardiovascular diseases in our country in 2002 is 291/10 million people,significantly higher than Cancer’s mortality rate of 148/10 million people.Strengthen the treatment of AS in the development of effective drugs has important practical significance.Endothelial cells（ECs） are the range of flow between blood and vascular tissue as a complete monolayer barrier.ECs has a protective effect of anticoagulant,anti-cell adhesion and many other features.ECs injury was considered as AS pathogenesis of the earliest case.The current study shows that endothelial cell apoptosis is the main form of ECs injury.Apoptosis that is programmed cell death,confirmed that a large number of apoptotic ECs in atherosclerosis of the role:ECs apoptosis can be caused not only the structure of vascular endothelial damage,but also seriously affected the normal function of endothelial cells,weaken the vascular endothelium in the vascular tone,vascular permeability,blood flow,coagulation and the role of thrombolytic, thereby inducing the occurrence of vascular disease. Low density lipoprotein,（LDL） increases are an independent risk factor for coronary heart disease,atherosclerosis at the occurrence and development plays an important role.Increasing evidence shown that oxidized low density lipoprotein （ox-LDL） plays an important role in the endothelial dysfunction.Apoptosis that is programmed cell death,recent research has shown that the endothelial cell apoptosis was the key step of in atherosclerosis.Ox-LDL can promote endothelial cell apoptosis.Apoptosis of endothelial cells can cause vascular endothelial structural damage,and seriously affected the normal function of endothelial cells,weakening the vascular endothelium in the vascular tone,vascular permeability,blood flow, coagulation and on the role of thrombolytic.All over those could induced the occurrence of vascular disease.Salvianolic acid B（Sal B） is the water-soluble components of Danshen, pharmacological studies shown that:Sal B has anti-fibrotic,anti-atherosclerosis, myocardial protection.anti-tumor effect.Sal B has been reported reduce the hydrogen peroxide-induced Human umbilical vein endothelial cells（HUVEC） injury through t intervens PI3K.Sal B can inhibit Vascular endothelial growth factor（VEGF） induced bovine aortic endothelial cells increased permeability.But Sal B’protect effect of ox-LDL-induced endothelial cell injury which was the key step of atherosclerosis are see no coverage at home and abroad.In this experiment,will induce HUVEC injury by oxidized LDL and observe Sal B’protect effect,try to find effectively reduce atherosclerosis incidence of interventions,and Sal B further widely used to provide a scientific basis.Method1 Preparation of oxidized low density lipoproteinWe separated and quantitatified the LDL from blood of healthy individuals by Super high density gradient centrifugation,and adjusted it to a final concentration of 500ug/mL,then incubated with 10mmol/mL CuSO₄.The oxidation was stopped with 100μmol/L EDTA.Finally,we used the method of Bradford to detect the protein level of ox-LDL.2 HUVEC recovery subcultureThe freezing HUVEC lines were inoculated in RPMI-1640 complete medium supplemented with 10%FBS,2 mM L-glutamine,100 U/Lpenicillin and 100μg/ml streptomycin after they were thawed.The culture medium was changed every two days and confluens of HUVEC was typically achieved in 2-3 days with "cobblestone appearance" in optical microscopy,then 0.25%trypase were used in passage of HUVEC at re-cultured to subconfluenceCells of 3～6th passage were initially trypsinized,transferred to 6-well plates with the concentration of 2.0×10⁵ cells/well and re-cultured for 24h in a humidified 95%air and 5%CO₂ atmosphere at 37℃,then they were cultured with serum-free medium and rolled into the experiment.3 Sal B Mother liquor preparationDissolved Crude drug of Sal B（10mg） in 5ml RPMI1640 Medium.Sal B Mother liquor（200μg/ml） dissolved with RPMI1640 complete medium or RPMI1640 medium according to different need.4 Observation of different concentrations of Sal B on human umbilical vein endothelial cell activity by MTT colorimetricAdjusted the density of HUVEC to 2×10⁴/ml with 10%FBS containing RPMI1640 medium,inoculated with the 96-well plates,each plant hole 200μl.After incubate 24h,cells were randomly divided into 6 groups:control group（control）,Sal B 5μg/ml Group,Sal B 10μg/ml Group,Sal B 20μg/ml Group,Sal B 40μg/ml Group, Sal B 80μg/ml group,each hole 6 hole.MTT cell viability assay was measured after incubate 48 hours,5 Observation of different concentrations of ox-LDL on human umbilical vein endothelial cell activity by MTT colorimetric:Adjusted the density of HUVEC to 2×10⁴/ml with 10%FBS containing RPMI1640 medium,inoculated with the 96-well plates,each plant hole 200μl.After incubate 24h.cells were randomly divided into 6 groups:control group（control）, ox-LDL 10μg/ml Group,ox-LDL 20μg/ml Group,ox -LDL 40μg/ml Group,ox-LDL 80μg/ml Group.ox-LDL 160μg/ml group,each hole 6 hole.MTT cell viability assay was measured after incubate 24 hours.6 Observation of Sal B on the ox-LDL-induced human umbilical vein endothelial cell injury intervention role by MTT colorimetricAdjusted the density of HUVEC to 2×10⁴/ml with 10%FBS containing RPMI1640 medium,inoculated with the 96-well plates,each plant hole 200μl.After incubate 24h.cells were randomly divided into 6 groups:control group（control）. ox-LDL 50μg/ml Group,ox-LDL 50μg/ml + Sal B 5μg/ml Group,ox-LDL 50μg/ml+ Sal B 10μg/ml Group,ox-LDL 50μg/ml + Sal B 20μg/ml each hole 6 hole.MTT cell viability assay was measured after incubate 24 hours.7 The use of flow cytometry to observe Sal B of ox-LDL-induced apoptosis of human umbilical vein endothelial cells of the intervention effectAdjusted the density of HUVEC to 2×10⁵/ml with 10%FBS containing RPMI1640 medium,inoculated with the 6-well plates,each plant hole 1500μl.After 24h.cell incubate with serum-free RPMI1640 medium for 24h.Cells were randomly divided into 5 groups:control group（control）,ox-LDL 50μg/ml Group,ox-LDL 50μg/ml + Sal B 5μg/ml Group.ox-LDL 50μg/ml + Sal B10μg/ml Group,ox-LDL 50μg/ml + Sal B 20μg/ml each hole add 1ml drug-containing medium.After 12h Dosing based on the cell culture medium to a suitable suction centrifugal tube,PBS washing adherent cells once digested with trypsin to digest cell cells.Cells after digestion down,transferred to centrifuge tubes,PBS wash three times（1000g centrifugation 10 minutes）.Add 200μl binding buffer,re-suspended cells.Add 10μl Annexin V-FITC,10μl PI Staining Solution mixing gently.Dark incubation at room temperature for 15 minutes,30 minutes on machines to detect cell apoptosis.8 Statistical MethodsSPSS 13.0 statistical soft was used.Measurement data express as（?）±S.Comparison of test results using one-way ANOVA（One-way ANOVA）,multiple comparisons using LSD method,when the variance of arrhythmia,the use of robust estimation Welch,then the use of T3 compared methods.P＜0.05 indicated statistical significance.Result1 HUVEC identificationIn the inverted microscope,HUVEC were single-layer growth with "cobblestone appearance" after 2-3d.Ⅷfactor monoclonal antibody indirect immunofluorescence staining showed that the cultured cells express a higher density ofⅧfactor related antigen,the positive rate was 100%,demonstrate that the cultured cells for endothelial cells.2 Oxidized low density lipoprotein oxidation degree of identification:Aetermination the concentration of MDA in Cu⁺²-induced oxidation of LDL solution.Results showed that the concentration of MDA 7 times heiger than before.3 Different concentrations of Sal B on HUVEC cell viability impact:Different concentrations of Sal B on endothelial cells has different effects.Low doses of the drug concentration（5-20μg/ml） cell activity has no statistical difference with the control group.The ceil viability of 40μg/ml group decreased approximately 50%,80μg/ml group decreased by 70%（P＜0.01 VS.control group）.So the concentration of Sal B we used in this experiment was 5,10,20μg/ml,for ensure that drugs on the cells,no significant poisoning effect. 4 Different concentrations of ox-LDL on human umbilical vein endothelial cell viabilityDifferent concentrations of ox-LDL has different activity effecte on HUVEC cell.Compared with the control group,10μg/ml group trend to promote HUVEC cell activity but there was no significant difference compared with the control group（P＞0.05）.Cell activity of 20-100mg/l group decreased with the concentration（P＜0.05 or P＜0.01）.In this experiment we used 50μg/ml as the concentration of oxidized low density lipoprotein.5 Sal B of ox-LDL-induced human umbilical vein endothelial cell injury intervention roleCell activity of ox-LDL group decreased compared with the control group（P＜0.01）.Ox-LDL group had a significant reduction in cell activity.Cell activity of Sal B groups in different doses were higher than the ox-LDL group,compared with ox-LDL group,the differences were significant（P＜0.01）.The three dose groups of cell activity was significantly higher than ox-LDL group,suggesting that Sal B could inhibit the ox-LDL-induced HUVEC injury.Cell activity of Sal B 10μg/ml and Sal B 20μg/ml group heiger than Sal B 5μg/ml group.the difference is significant（P=0.01 or P＜0.01）.Cell activity of Sal B 20μg/ml tended to heiger than Sal B 10μg/ml group. but no significant difference（P=0.185）.Sal B has the endothelial protective effect at a certain scale with the dose-effect relationship.6 Sal B of ox-LDL-induced injury in human umbilical vein endothelial cell apoptosisThe results showed that flow cytometry.the apoptosis rate of each group was significantly difference（P＜0.01）.Ox-LDL group was significantly increased apoptosis rate.compared with the control group（P＜0.01）.The apoptosis rate of Sal B groups in different doses were lower than the ox-LDL group,the differences were significant（P＜0.01）,indicating that the three-dose group of the apoptosis rate was significantly lower than ox-LDL group,suggesting that Sal B could inhibit the ox-LDL-induced apoptosis in HUVEC.The apoptosis rate of Sal B group reduced gradually with the increasing of dose.The difference between three groups was statistically significant（P＜0.01）.Sal B has the endothelial protective effect at a certain scale with the dose-effect relationship.ConclusionsSal B could inhibit the ox-LDL-induced HUVEC apoptosis,the endothelial protective effect of Sal B has dose-effect relationship.