Oxidative Stress And Apoptosis In Endothelial Cells Exposed To PM2.5 And The Protective Effect Of Sodium Tanshinone IIA Sulfonate

Objective: Umbilical vein endothelial cells(EA.hy926)were used as a model to evaluate the toxic effects of PM2.5 on the oxidative stress and apoptosis of vascular endothelial cells.The results revealed that PM2.5 might induced the apoptosis of EA.hy926 cells;and to observed the protective effect of sodium tanshinone IIA sulfonate(STS)on the oxidative stress and apoptosis of endothelial cells induced by PM2.5,to explore the protection mechanism of traditional Chinese medicine Tanshinone IIA by its direct impact on vascular endothelial cellsMethod :1)The cell viablity of EA.hy926 after PM2.5 treatment.EA.hy926 cells were logarithmic growth phase and in good condition,with 8 x 103 per well in 96 wells plate,divided into control group and different doses of PM2.5 group(final concentration of PM2.5 were 12.5,25,50,100 and 200 ?g/mL),with 6 wells in each group.After 24 h,add 10 ?L per well reagent according to the CCK-8 kit instructions,incubated 2 h,with a microplate absorbance of each well at 450 nm,calculated cell survival rate and median lethal dose,to determine the concentrations of the follow-up experiments.The cell survival rate(control %)=exposure group OD/ average value of control OD x 100%.2)The detection of ROS after PM2.5 treatment.The cells were divided into control group and different doses of PM2.5 group(choice the concentration of PM2.5 as 25,50 and 100 ?g/mL according to the results of cell viablity)and N-acetylcysteine(N-acetyl-L-cysteine,NAC)treatment group(NAC 10 mmol/m L pretreated for 1 h than added PM2.5 100 ?g/ml).with 1 x 106 cells per well in 6 wells plate,removed the medium after 24 h,adding DCFH-DA probe(10 ?mol/L)and were incubated for 90 min,with warm PBS rinsed 2 times,and then cultured for 30 min with medium without serum,useing the fluorescence microscope camera observed and took photos.3)Flow cytometry to detect the apoptosis after exposed to PM2.5.The cell groups and treatment were as above.after incubated for 24 h,incubated with trypsin without EDTA,cold PBS centrifugal washed the cells 2 times,and then used 400 ?L Binding Buffer cell to resuspended cells.Added Annexin V-FITC 5 ?L lucifugal incubation for 15 min in 4?,then add propidium iodide(PI)10 ?L lucifugal incubation for 5 min,and than detected cell apoptosis by flow cytometry.4)The apoptosis related signal molecules test after PM2.5 treatment.The cell groups and treatment were as above.after incubated for 24 h,washing 3 times with cold PBS,Lysis cells on ice with RIPA cell lysate containing PMSF for 30 min,then centrifugated cells for 5 min with 12500 g in 4?.The supernatant was collected for measurement of protein concentration by BCA kit.30 g protein was obtained by SDS-PAGE electrophoresis,rotating film and Western blot.The main steps of Western blot were as follows: 5 % skim milk incubated at room temperature for 1 h,TBST cleaned samples for 10 min * 3 times,added antibody I(1:1000),incubated them in 4 ? overnight,than added enzyme labeled antibody II(1:3000)after TBST cleaning,incubated samples at room temperature for 1 h,finally washed them with TBST and took pictures by ECL gel imaging system analysis software,analysised the gray value of bands by Image-J,the ratio of target protein and ?-actin gray values to represent the relative expression levels of protein.5)Effect of STS on the viablity of EA.hy926 cells EA.hy926 cells were logarithmic growth phase and in good condition,with 8 * 103 per well in 96 wells plate,divided into control group and different doses of STS group(final concentrations were 12.5,25,50 ?g/m L,STS pretreatment for 2 h,than added PM2.5 100 ?g/m L),with 6 wells in each group.According to the experimental group,contaminated cells.After 24 h,add 10 ?L per well reagent according to the CCK-8 kit instructions,incubated 2 h,with a microplate absorbance of each well at 450 nm,calculated cell survival rate and median lethal dose,to determine the concentration of the follow-up experiments.The cell survival rate(control %)=exposure group OD/ average value of control OD x 100%.6)Effects of STS on oxidative stress and apoptosis in EA.hy926 cells exposed to PM2.5 The groups were as above,with 1 x 106 per well in 6 wells plate.The cells were incubated for 24 h,than removed the medium,using DCFH-DA fluorescent probe method to moniter ROS generation after pretreated by PM2.5 in EA.hy926 cells which exposed to PM2.5;using FITC/PI double staining monitoring cell apoptosis;Western Blot method was used to monitor the apoptosis related protein Caspase-9 and Caspase-3 protein expression in EA.hy926 cell after exposed to PM2.5 for 24 h.Result :1)When the PM2.5 concentration is 12.5 ?g/mL,the vibality of EA.hy926 cells was decreased,but no significant difference with control group;when the PM2.5 concentration is higher than 25 ?g/mL,the survival rate of EA.hy926 cells were decreased significantly compared with the control group,the difference was significantly.2)The fluorescent staining results showed that compared with control cells,visible fluorescence after PM2.5 stimulation in cells was strong intensity,and by the concentration of PM2.5 gradually increasing,showed a clear dose-effect relationship.The intracellular fluorescence intensity was significantly decreased after pretreatment with antioxidant NAC,which indicated that NAC could reduce ROS production induced by PM2.5.3)The flow cytometry results showed that the EA.hy926 after exposured to PM2.5,the number of apoptotic cells were increased,and increased in a concentration dependent manner;The results also showed that pretreatment with NAC after exposed to PM2.5 can decrease the cell apoptosis,and compared with PM2.5 in the same concentration group,the apoptosis rate decreased significantly.4)Compared with the control group,PM2.5 can induced the content of Cyt-C in EA.hy926 cells increased significantly,while the activated Caspase-3 and activated caspase-9 increased.When the groups were pretreated by NAC,the Cyt-C protein was significantly decreased,while the activated Caspase-3 and activated caspase-9 expression decreased,indicating that NAC can downregulate the expression of apoptosis protein.5)The viablity of the cells was decreased when exposed to 100 ?g/mLPM2.5,which was significantly decreased compared to the control group.The cell vibality was significantly higher than that of PM2.5 group after STS pretreated,which indicated that STS could reduce the toxicity of PM2.5 on vascular endothelial cells.6)PM2.5 fluorescence staining showed that the cells in the control group only had a small amount of green fluorescence,while observed green fluorescence intensity increased significantly after PM2.5 stimulated;while in different concentrations of STS groups,the intracellular fluorescence intensity was significantly lower than PM2.5 group;Flow cytometry showed that compared with the control group,after exposed to PM2.5,the apoptosis rate of EA.hy926 increased significantly,but the apoptosis of vascular endothelial cells was significantly lower compared to PM2.5 group if pretreated with STS,P <0.5;PM2.5 in EA.hy926 cells can increase the actived Caspase-9 and actived Caspase-3 expression;The cells pretreated wih STS can inhibit signal molecule activation induced by PM2.5 and decease the expression of actived Caspase-9 and actived Caspase-3.Conclusion: 1)PM2.5 induced oxidative stress and apoptosis in the vascular endothelial cells,which may be one of the mechanisms that PM2.5 influences the function of cardiovascular system.2)STS could protect vascular endothelial cells by reducing oxidavtive stress and cell apoptosis induced by PM2.5.