| ObjectiveHUVECs were injured by hyperlipidemic serum.The MTT method,Nitric acid reductase method,Western blotting and RT-PCR were used to observe the protective effects of Pae on HUVECs injured by hyperlipidemic serum and explore the antiatherosclerotic mechanism further.Methods1. Culture and identification of HUVECs:HUVECs were obtained using the complex enzyme of 0.1% I collagenase and 0.25%trypsin,0.02%EDTA by the technique of irrigative digestion,and were cultured with DMEM which contained 20ng/ml bFGF,20%fetal calf serum in the carbon dioxide incubator,and then were passaged using 0.125% trypsin,0.01%EDTA digestion.The cultured cells were identified by morphological observation and immunohistochemistry detectingⅧfactor.2. The preparation of hyperlipidemic serum:Twenty four healthy rabbits (weight 2±0.5kg) were randomly divided into two groups: the control group(six), the model group(eighteen) . The control group was fed with ordinary forage, the model group with high lipid forage, each with 100 g, sustained for ten weeks.Cardiac blood was obtained under sterile conditions to prepare sersum, part of which was used to detect TC,TG and LDL-C. (the ingredients of high lipid forage were 1% cholesterol,5% egg yolk,10% lard and 84%common feedstuff)3. Establishment of damaged HUVECs model injured by hyperlipidemic serum:3.1 Cell viability was detected by the method of MTT. HUVECs were cultured in vitro. They were divided into normal group, hyperlipidemic serum groups with different concentrations(5%,10%,15%,20%). After incubated for 24h, cell viability was detected by the method of MTT.3.2 The morphological changes of HUVECs were observed with inverted microscope. HUVECs were cultured in vitro.They were divided into normal group, hyperlipidemic serum groups with different concentrations(5%,10%,15%,20%). After incubated for 24h, the morphological changes of HUVECs were observed in each group respectively with inverted microscope.4. Determination of Safety Concentrations of Pae to HUVECs: HUVECs were cultured in vitro.They were divided into normal group, Pae groups with different concentrations(3.125μg/ml,6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,75μg/ml,100μg/ml,150μg/ml,250μg/ml). After incubated for 24h, cell viability was detected by the method of MTT.5. Experimental group: select the cells in the exponential phase of growth and groups are divided as follows:①control group : 20%blank serum+80%DMEM②model group : 20%hyperlipidemic serum+80%DMEM③the low-dose group of Pae : 20%hyperlipidemic serum+25μg/ml Pae+80%DMEM④the middle-dose group of Pae : 20%hyperlipidemic serum+50μg/ml Pae+80%DMEM⑤the high-dose group of Pae : 20%hyperlipidemic serum+100μg/ml Pae+80%DMEM Each group was continued to culture for 24h.6. Cell viability was detected in HUVECs in each group by the method of MTT. The morphological changes of HUVECs were observed in each group respectively with inverted microscope.7. NO was detected in the cultured supernate of HUVECs in each group by the method of nitric acid reductase.8. Western blotting was used to detect the expression of ICAM-1,E-selectin protein in HUVECs in each group.9. RT-PCR was used to measure the expression of NF-κB p65,eNOS mRNA in HUVECs in each group.Results1. Culture and identification of HUVECs:HUVECs could adhere to the plate completely after 24 hours and got a monolayer 3～4 days later.Morphological changes and growth manner of subcultured HUVECs were similar to primary HUVECs. Above ninety percent of cultured cells were covered with CytosolicⅧfactor by immunohistochemistry.2. The preparation of hyperlipidemic serum:After ten weeks, The content of TC,TG,LDL-C of rabbits in model group was(33.50±6.87)mmol/L,(5.01±2.15)mmol/L,(11.32±5.32) mmol/L,respectively.3. Establishment of damaged HUVECs model injured by hyperlipidemic serum:3.1 Cell viability was detected by the method of MTT.According to the method of MTT, The OD of hyperlipidemic serum groups (5%,10%) had no significant differences compared with cells in nomal control group(P>0.05);The OD of hyperlipidemic serum group(15%,20%) decreased obviously compared with cells in nomal control group(P<0.01);The OD of hyperlipidemic serum group(20%) was obviously lower than that of hyperlipidemic serum group(15%)(P<0.01).3.2 The morphological changes of HUVECs observed were as follows in each group respectively with inverted microscope.After 24h, the cells of hyperlipidemic serum groups (5%,10%) were in good condition. The cells were oval in shape and the border was clear. The morphology of cells in hyperlipidemic serum group (15%)changed a lot compared with cells in nomal control group. Enlarged space occurred among cells and part of cells became round,so hyperlipidemic serum group (15%) began to show cytotoxic effect. Most cells in hyperlipidemic serum group (20%) shaped like a lobe and came off ,so cytotoxic effect of hyperlipidemic serum group (20%) was more serious.4. Determinationof Safety Concentrations of Pae to HUVECs:According to the method of MTT, The OD of Pae groups (3.125μg/ml,6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,75μg/ml,100μg/ml) had no significant differences compared with cells in nomal control group(P>0.05);The OD of Pae groups (150μg/ml,250μg/ml) decreased obviously compared with cells in nomal control group(P<0.01). Safety Concentration of Pae to HUVECs was below 150μg/ml.5. Cell viability was detected in HUVECs in each group by the method of MTT.compared with nomal control group, The OD of model group decreased significantly(p<0.01).The OD of the low-dose,the middle-dose and the high-dose group of Pae was remarkably higher than that of model group(p<0.01)and they are in a concentration-dependent manner(p<0.01).The morphological changes of HUVECs observed were as follows in each group respectively with inverted microscope.compared with nomal control group, most cells in model group shaped like a lobe and came off . Enlarged space narrowed down among cells and the morphology was tending to normal after intervention with Pae. 6. NO was detected in the cultured supernate of HUVECs in each group by the method of nitric acid reductase.compared with nomal control group, the content of NO in the cultured supernate of model group decreased significantly(p<0.01). The content of NO of the low-dose,the middle-dose and the high-dose group of Pae was remarkably higher than that of model group(p<0.01)and they are in a concentration-dependent manner(p<0.01).7. The expression of ICAM-1,E-selectin protein in HUVECs in each group detected by Western blotting.7.1 ICAM-1 protein:compared with cells in nomal control group, ICAM-1 protein expression in the cells of model group increased significantly(p<0.01). ICAM-1 protein expression in the cells of the low-dose,the middle-dose and the high-dose group of Pae was remarkably lower than that of model group(p<0.01)and they are in a concentration-dependent manner(p<0.01).7.2 E-selectin protein:compared with cells in nomal control group, E-selectin protein expression in the cells of model group increased significantly(p<0.01). E-selectin protein expression in the cells of the low-dose,the middle-dose and the high-dose group of Pae was remarkably lower than that of model group(p<0.01)and they are in a concentration-dependent manner(p<0.01).8. The expression of NF-κB p65,eNOS mRNA in HUVECs in each group detected by RT-PCR.8.1 NF-κB p65 mRNA:compared with cells in nomal control group, NF-κB p65 mRNA expression in the cells of model group increased significantly(p<0.01). NF-κB p65 mRNA expression in the cells of the low-dose,the middle-dose and the high-dose group of Pae was remarkably lower than that of model group(p<0.01)and they are in a concentration-dependent manne(rp<0.01).8.2 eNOS mRNA:compared with cells in nomal control group, eNOS mRNA expression in the cells of model group decreased significantly(p<0.01). eNOS mRNA expression in the cells of the low-dose,the middle-dose and the high-dose group of Pae was remarkably higher than that of model group(p<0.01)and they are in a concentration-dependent manner(p<0.05).conclusionsThe above results indicate:1. The primary culture and subculture of HUVECs was successfully performed and damaged HUVECs model injured by hyperlipidemic serum was established.2. Pae inhibits the expression and activation of NF-κB, downgrades the expression of ICAM-1,E-selectin , therefore it alleviates the adhesion to leukocyte. In addition, Pae can improve the gene expression of eNOS and increase the content of NO, which shows Pae has a strong protective effect on damaged HUVECs injured by hyperlipidemic serum. |
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