| Cardiovascular disease included dyslipidemia, hypertension, coronary heart disease. Atherosclerosis (AS) mainly involving large, medium-sized arteries is the pathogenesis basis of cardiovascular disease. The early lesion of AS is characterized by lipid points and lipid stripes, which is closely related to the macrophage-derived foam cells. Ross believed that the various pathological changes of the AS were the different stages of chronic inflammation of the arteries. The inflammation injuries increase the permeability of endothelial cells and the adhesiveness of leukocytes. These would release the chemokines and promote the monocytes into the intima changing into macrophages. The macrophages absorb OxLDL by the scavenger receptor, then change into foam cells. This macrophage-derived foam cells can increase lipid deposition, reduce cholesterol shipped out and promote the formation of fatty streaks. It is the potential sites of the development of AS. Therefore, to reduce macrophage-derived foam cells is very important for the prevention and treatment of AS.Apigenin is mainly from the celery leaves, which belongs to the umbrella plant. It also exists in many of herbal medicine. It is a flavonoid compound, with the anti-inflammatory, antioxidant, antitumor, neuroprotective and immunoregulatory abilities. The early animal experiments, we found that apigenin can reduce the atherosclerotic plaque area and the MOMA-2expression of ApoE-/-mice. These suggesting that apigenin have a certain effect on the prevention of atherosclerosis and this may be by reducing the quantity of macrophage-derived foam cells.PI3K involved in multiple signaling pathways. The activated PI3K produce the second messenger PIP3in the plasma membrane. The combination of PIP3and PKB singaling proterins in the PH domain activated Akt by the phosphorylation of Thr308and Ser473. This signaling pathway involved in the regulation of cell proliferation, differentiation, and apoptosis. It’s reported that OxLDL could inhibit the apoptosis of foam cells by activating the PI3K/PKB pathway. OxLDL increased the survival of macrophages by increasing glucose uptake. Our results show that, compared to the native LDL, OxLDL could significantly increase the cell viability of macrophages and the phosphorylation of Akt protein expression. apigenin can inhibit all these events, and induced apoptosis of macrophage-derived foam cells.In the two-dimensional electrophoresis detection, we found the differentially expressed protein between the OxLDL stimulated macrophages, with or without apigenin pre-stimulate. The protein identified by mass spectrometry. Plasminogen activator inhibitor-2(PAI-2) is one of the differentially expressed proteins. PAI-2is a serine protease inhibitor protein and an inhibitor of urokinase. It mainly presented in the placenta, also commonly found in monocytes/macrophages, fiber cells and fibroblast-like cells. PAI-2involved in inflammation, cell proliferation, tumor metastasis, and other physiological and pathological activities, the exact mechanism is not yet clear.It’s reported that the activation of Akt phosphorylation could increase PAI-2protein expression. In order to find the relationship between PAI-2and Akt, we build the lentiviral plasmids of Akt overexpression and Akt ser473, thr308phosphorylation sites mutations. The lentiviral plasmids were transfected into peritoneal macrophages of mice to detect the changes of cell viability and PAI-2protein expression. We also use the OxLDL and apigenin to stimulate the transfected macrophages to study the mechanism of the regulation of macrophage-derived foam cells by apigenin.Method1. The effect of apigenin on atherosclerosis in miceHigh fat diet is given to ApoE knockout mice. The mices were randomly divided into three groups(Model group, Apigenin group, Simvastatin group), C57mice as the normal control group, there were4groups of6. Taken medicine by the gavage way when they are8-week-old. After taking medicine for8weeks, one mice of each group was killed, the aortas were took out to detect the formation of atherosclerotic plaque in ApoE-Deficient Mice by the pathomorphological observation. All mice were sacrificed. Sudan III staining and immunohistochemical detection were performed to observe the impact of apigenin on the mouse artery atherosclerotic plaque and the MOMA-2antigen expression in the plaque.2. The impact of OxLDL on macrophagePrimary mouse peritoneal macrophages which were obtained by peritoneal lavage were randomly divided into2groups:OxLDL group, native LDL group; Each group was divided into four concentration groups:control group (control),12.5μg/ml group,25μg/ml group, the50μg/ml group, three holes in each group. Cells were cultured for96hours, used MTT assay to detect cell activity.3. Apigenin on the impact of macrophage derived foam cell viabilityPrimary mouse peritoneal macrophages which were obtained by peritoneal lavage were randomly divided into four major groups:24h,48h,72h,96h, per time group was further divided into three subgroups:control group (control), OxLDL50μg/ml group, apigenin50μM plus OxLDL50μg/ml group, each group with3holes. Cells were cultured for24hours,48hours,72hours,96hours, used MTT assay to detect cell activity.4. The effects of apigenin on the apoptosis of macrophage derived foam cellCells were randomly divided into2groups:DMSO (DMSO as apigenin solvent) group and apigenin group, each group was divided into five subgroups:control group (control), OxLDL12.5μg/ml group, OxLDL25μg/ml group, OxLDL50μg/ml group, OxLDL lOOμg/ml group.We use the Annexin V-FITC Apoptosis Detection Kit, according to the manual operation, the result was detected by the FACSCalibur flow cytometer,We use this way to observe the regulation of apigenin to the apoptosis of macrophage-derived foam cells.5. Two-dimensional Gel Electrophoresis and MALDI-TOF-MS Analysis of the differentially expressed proteinPrimary mouse peritoneal macrophages were obtained by peritoneal lavage method. The cells were randomly divided into three groups:control group (control), OxLDL50μg/ml group, apigenin50μM+OxLDL50μg/ml (Api+OxLDL group) group.Two-dimensional electrophoresis follows the IPGPhor III IEF program guide and IPGstrip operation manual. The identification of differentially expressed proteins were identied by MALDI-TOF-MS Analysis. We use these methods to identify the proteins which may play a regulation role on the apoptosis of macrophages by OxLDL and apigenin stimulated.6. Western blot analysis the effect of apigenin on the Akt phosphorylated protein and PAI-2protein expressionPrimary mouse peritoneal macrophages were obtained by peritoneal lavage method. The cells were randomly divided into three groups:control group (control), OxLDL50μg/ml group, apigenin50μM+OxLDL50μg/ml (Api+OxLDL group) group.We carried out western blot according to the conventional steps of our laboratory. Use Image J analysis software to analyze the gray value of the bands. We use this way to detect apigenin on the regulation of p-Akt ser473, p-Akt thr308and PAI-2protein expression, and detect the transfection effect.7. Rt-PCR analyzes PAI-2mRNA expression in OxLDL stimulated macrophages by the regulation of apigenin.Primary mouse peritoneal macrophages were obtained by peritoneal lavage method. The cells were randomly divided into three groups:control group (control), OxLDL50μg/ml group, apigenin50μM+OxLDL50μg/ml (Api+OxLDL group) group.We carried out Rt-PCR according to the conventional steps of our laboratory. After the reaction, the Dissociation Curve and experimental data were got trough the instrument software. We use this method to analyze PAI-2mRNA expression in OxLDL stimulated macrophages by the regulation of apigenin.8. Molecular cloning to build pcDNA3-flag/Akt plasmidUse PcDNA3-flag as the carrier, to build the pcDNA3-flag/Akt plasmid. This plasmid is for Akt phosphorylation site mutation and for the construction of Akt overexpression lentivirus plasmid, Akt phosphorylation ser473, thr308site mutation lentivirus plasmid.9. The mutation of Akt phosphorylation sitesThe subclone was performed on the basis of the pcDNA3-flag/Akt plasmid. We changed the Akt473site of serine into glycine and Akt308site of threonine into glycine to build S473G, T308G, S473G-T308G sites mutation plasmid.10. The construction of Akt overexpression and Akt phosphorylation site mutation lentivirus plasmidWe used the PHIV-H2BmRFP vector to construct pHIV-Akt overexpression and pHIV-S473G, pHIV-T308G, pHIV-S473G-T308G sites mutation lentivirus plasmid.11. Lentiviral packaging and infection of mouse peritoneal macrophagesConstructed lentivirus plasmid was transfected into293FT cells. After72h viral supernatant was collected by centrifugation. Use the formula titer (TU/ml)=expression of GFP-positive cell count x dilution factor/virus volume to calculate the virus titer, MOI=50TU/Cell control virus pHIV, Akt overexpression (Akt+) lentivirus, sites mutations S473G, T308G, S473G-T308G lentivirus, were infected into mouse peritoneal macrophages, medium was changed after8h. To observed the infection efficiency by Western blot assay.12. Akt overexpression and Akt phosphorylation sites mutation on the impact of macrophage derived foam cell viabilityOn the basis of the effective transfected, the cells which were transfected with different plasmid were randomly divided into three groups:control group (control), OxLDL50μg/ml group, apigenin50μM+OxLDL50μg/ml (Api+OxLDL group) group.we use MTT assay to detect the cell viability.13. Akt overexpression and Akt phosphorylation sites mutation on the impact of PAI-2protein expressionOn the basis of the effective transfected, the cells which were transfected with different plasmid were randomly divided into three groups:control group (control), OxLDL50μg/ml group, apigenin50μM+OxLDL50μg/ml (Api+OxLDL group) group.we used western blot assay to detect the PAI-2protein expression.Then we stimulate the macrophages by apigenin and OxLDL to study the mechanism of the regulation by apigenin to macrophage-derived foam cells.14. Statistical MethodsSPSS13.0statistical soft was used. Measurement data express as x±S. Monothetic analysis use the OneWay-ANOVA, multivariate analysis using factorial analysis, multiple comparisons using LSD method, when the variance of arrhythmia, the use of robust estimation Welch, then the use of T3compared methods. P<0.05indicated statistical significance. Result1. The Apigenin reduce atherosclerotic plaque area in miceCompared with model group, the AS plaque area of aorta was significantly reduced in apigenin group and the simvastatin group (P=0.000). The plaque area and was no significant difference between apigenin group and the simvastatin group (P=0.397).2. the MOMA-2antigen expression in ApoE-/-mouse aortic plaquemorphological image analysis showed that, compared with the control group, the MOMA-2antigen expression were significantly reduced (P=0.000). There was no significant diference between the simvastatin group and the apigenin group (P=0.764).3. MTT assay detects the cell viability of mouse peritoneal macrophages with different concentrations of OxLDL and native LDLDifferent concentrations of OxLDL and native LDL can increase the vitality of mouse peritoneal macrophages compared with the control group, the difference was statistically significant (F=94.501> P=0.000); cell viability between the different drugs were significantly different (F=94.501, P<0.001), in which OxLDL cell viability (X=149.297%) was significantly higher than the native LDL group (X=117.771%); different concentrations of cell viability in a significant difference (F=77.191, P=0.000),when lipoprotein concentrations was50μg/ml, cell viability was significantly higher than other concentrations of cell viability. Combined with the statistical results, the maximum cell viability (206.024%) was OxLDL50μg/ml group.4. MTT assay detects the cell activity of control group, OxLDL group and Api+OxLDL group in different time Cell viability was significantly different (F=525.521, P=0.000) in different time, the highest cell viability was24-hour group(mean109.39%), with the time increment, the cell viability decreased, a minimum cell viability was96-hour group(X=51.12%); cell viability between the different drugs were significantly different (F=1044.646, P=0.000), the highest cell viability was the OxLDL50μg/ml group (X=111.15%), the lowest cell viability was Api+OxLDL50μg/ml group (X=42.21%).5. Flow cytometry analysis of apigenin on apoptosis of the macrophage-derived foam cellThe rate of apoptosis was significant difference (F=4688.481, P=0.000) between different treatment groups, apigenin group apoptosis rate (x=24.734%) was significantly higher than the DMSO group (x=7.87%).6. Two-dimensional gel electrophoresis and MALDI-TOF-MS analysis of differentially expressed proteins between the macrophage-derived foam cells with or without apigenin interfered.By two-dimensional electrophoresis and MALDI-TOF-MS analysis of proteins, OxLDL group and Api+OxLDL group were compared with the control group to identify22differentially expressed proteins. In OxLDL group, there were15protein spots increased expression relative to the control group, of which6protein spots were downregulated in apigenin pre-stimulation group (Api+OxLDL group); relative control group, there were7proteins downregulated in OxLDL group, in which4down-regulated protein spots in the Api+OxLDL group expression is elevated. PAI-2protein is one of the detected differentially expressed proteins, it is highly expressed in the OxLDL group, low expression in the control group and Api+OxLDL group. 7. Western blot analysis of P-Akt ser473and PAI-2protein expression in the macrophage-derived foam cells with apigenin interferenceCompared with the control group, p-Akt ser473and PAI-2protein in OxLDL group significantly increased, the difference was statistically significant (p-Akt ser473:P=0.000; PAI-2:P=0.006); compared with OxLDL group, the expression of p-Akt ser473, PAI-2protein was significantly reduced in the Api+OxLDL group, the difference was statistically significant (P-Akt ser473P=0.000; PAI-2P=0.000).8. Rt-PCR analysis of PAI-2mRNA expression levels of macrophage derived foam cells in apigenin interventionOxLDL for group PAI-2mRNA expression was significantly higher than the control group (P=0.023). Compared with OxLDL group, Api+OxLDL group was significantly lower (P=0.000).9. Lentivirus infection of mouse peritoneal macrophages cellsLentiviral vector pHIV-H2BmRFP with a red fluorescent protein, can expressing red fluorescent when transfected into cells. The lentiviral plasmid pHIV, Akt+, S473G, T308G, S473G-T308G infected into mouse peritoneal macrophages, by the contrast of light and dark vision, the transfection efficiency of up to about90%. By Western blot analysis, p-Akt ser473protein’s expression significant difference (P=0.001), which of Akt+group expression of the highest (mean=0.015). Compare with the pHIV group, the expression of p-Akt ser473protein was no significant differences in S473G and of S473G-T308G group (S473G grop:P=0.012; S473G-T308G group:P=0.013), and there was no significant difference between the S473G group and the S473G-T308G group (P=0.954). In each group, p-Akt thr308protein expression have significant differences (P=0.002). Compare with the pHIV group, the p-Akt308expression was significantly increased in the Akt+group and the S473G group (Akt+group:P=0.042; S473G group:P=0.001). Compared with the pHIV group, there were no significant differences between the T308G, S473G-T308G and pHIV group (T308G组P=0.429; S473G-T308G组P=0.312), suggesting that the lentiviral transfection effectively.10. Western blot detection of the PAI-2protein expression in Lentivirus transfected mouse peritoneal macrophagesThe expression of PAI-2protein was significant differences in different group (P=0.001), by multiple comparison, the expression of PAI-2protein of Akt+group was significantly higher than other groups (P=0.001). Compared with the pHIV group, there was no significant difference between pHIV and S473G, T308G, S473G-T308G group (S473G group:P=0.342; T308G group:P=0.374; S473G-T308G group: P=0.057)11. Cell viability detected by MTT assay after transfection of the LentivirusCell viability between the different drug groups had a significant difference (F=188.848, P=0.000), OxLDL group cell vitality of the highest (mean=85.57%), Api+OxLDL group of cell viability was the lowest (mean=133.46%); cell vitality had a significant difference between different lentivirus infected (F=16.111, P=0.000), Akt+group of cell viability was the highest (mean=117.36%); in the control group, the Akt+group of cells vitality significantly higher than other groups (P=0.019); in OxLDL group, the cell viability of the S473G group was significantly lower than other infection group (P=0.003).12. Western blot analysis of PAI-2protein expression in the different lentivirus infection of macrophages in the apigenin interventionThe expression of PAI-2protein was significant differences in different drug group (F=187.664, P=0.000), the highest PAI-2protein expression was in the OxLDL group (mean=1.11), the minimum was in the Api+OxLDL group (mean=0.60); the PAI-2protein expression had a significant difference in different lentivirus transfected groups (F=25.759, P=0.000), the highest PAI-2protein expression was in Akt+group (mean=1.14); transfected with the the S473G and S473G-T308G lentivirus, PAI-2protein expression was no significant difference to the control group (S473G group:P=0.496, S473G-T308G group:P=0.709), which was not increased by the stimulated with OxLDL.Conclusions1. Apigenin can reduce of ApoE-/-mice artery atherosclerotic lesions.2. Apigenin can inhibit the increase in OxLDL-stimulated macrophage activity and induce apoptosis of macrophage derived foam cell.3. Apigenin can suppression OxLDL-stimulated Akt phosphorylation and PAI-2protein expression.4. Overexpression of Akt can upregulate the expression of PAI-2protein and increase the cell vitality; mutation of the Akt phosphorylation sites can be reduced the PAI-2protein expression.5. Akt phosphorylation ser473site mutation failed to increase the vitality of macrophages and the expression of PAI-2protein, even by stimulated with OxLDL.6. The apigenin can reduce the increased cell viability due to Akt overexpression, and reduce the expression of PAI-2protein.In summary, Apigenin can induce the apoptosis of macrophage-derived foam cell. Its regulation may be mediated by Akt/PAI-2pathway, which inactivated the Akt ser473phosphorylation site, then blocked the regulation of Akt ser473on PAI-2protein expression. This suggests that apigenin has a certain effect on the treatment of atherosclerosis. |
PDF Full Text Request