| Objective By observe the effect of oxidized low density lipoprotein(ox-LDL)on lipid deposition and cell transformation in vascular smooth muscle cells;we investigate the regulation function of the transcription factor FoxO1 on smooth muscle cells of ox-LDL,and observe the effect of ox-LDL on the transcription factor FoxO1 DNA methylation level.In order to observe the effects of the transcription factor FoxO1 DNA methylation on lipid aggregation in smooth muscle cells,we detect the ox-LDL's regulation on the DNA methylation level of FoxO1.To explain the mechanisms by which the ox-LDL promotes lipid deposition and cell phenotype changes by the transcription factor FoxO1 methylation in smooth muscle cells.Methods Smooth muscle cells was cultured in vitro and replication the cell modle of lipid deposition.The cells were divided into three groups: blank control group(0?g/ml)and ox-LDL intervention group(50?g/ml,100?g/ml).After cultured 48 h with the ox-LDL,Oil Red O staining determined the model was successful and observe the level of lipid deposition.The contents of total cholesterol(TC)and triglyceride(TG)in smooth muscle cells of each group were detected by enzymatic method.Real-time fluorescence quantitative PCR and western blot were used to detect the expression of FoxO1?smoothelin and CON1 in smooth muscle cells.After transfected the FoxO1 over-expressing(Ad-FoxO1)adenoviral vector,we observed and verified its transfection efficiency under fluorescence microscope.qRT-PCR and western blot detected the m RNA and protein expression of FoxO1.Peroxidase method detect the changes of TC and TG in the cell.We use the bioinformatics to analysis of the distributionof CPG islands in the promoter region of FoxO1 gene,and detect the FoxO1 methylation level in smooth muscle cells of each group.After treated with 5-aza-Cd R(10?mol/L methylation inhibitor 5-aza-Cd R)for 48 h,the enzymatic method check the levels of total cholesterol(TC)and triglyceride(TG).We use qRT-PCR and western blot to detect the expression level of smoothelin and CON1 in smooth muscle cells of each group.To clear the effect of methylation on phenotypic conversion.Results1.Oil red O staining of smooth muscle cells shows that,the cell cytoplasm was filled with red lipid particles,which the shape was lipid droplets,and nuclei were stained dark blue,after the cells were cultured with 50 ?g/ml ox-LDL.Compared with 100 ?g/ml ox-LDL group,the increase of intracellular lipid particles was confirmed.Peroxidase assay of ox-LDL intervention cells was used to demonstrate that compared with the normal control group,the TG and TC levels were increased by 1.34 and 1.2 timesin the 50?g/ml ox-LDL group respectively(P<0.001),and the contents of TG and TC were increased by 0.6 and 0.3 times in100?g/ml ox-LDL group(P<0.05).The conclusion show that ox-LDL can promote the accumulation of cholesterol in smooth muscle cells.2.qRT-PCR and western blot were used to detect the m RNA and protein level of smoothelin and CON1,which cultured with different concentrations of ox-LDL in smooth muscle cells for 48 h.Compared with the control group,the m RNA and protein of smoothlin decreased by 31% and 58% in the 50?g/ml ox-LDL group(P<0.05),while the 100?g/ml group decreased by 46% and 68%(P<0.05).Compared with the control group,the m RNA and protein expression of CON1 in the 50?g/ml ox-LDL group increased 0.5 times and 4.1 times respectively(P<0.05),and in the 100 ?g/ml ox-LDL group increased 0.7 times and 6.3 times respectively(P<0.05).3.The results of qRT-PCR and western blot show that,when smooth muscle cells were stimulated with 50?g/ml ox-LDL for 48 h,the relative m RNA expression of FoxO1 was 1.3times higher than that of the control group(P<0.01),and the protein expression trend was similar to that of FoxO1 m RNA,increased by 0.9 times(P<0.05).Compared with 50?g/ml ox-LDL,the expression of FoxO1 m RNA and protein was decreased by 0.39 and 0.65 times in the 100?g/ml group,these results were assayed by qRT-PCR and western blot,and the difference was statistically significant(P<0.01).4.To confirm the role of FoxO1 in ox-LDL-mediated cholesterol accumulation in smooth muscle cells,FoxO1 over-expression(Ad-FoxO1)was used to transfect smooth muscle cells and the transfection efficiency was observed under a fluorescence microscope,we can see that a large amount of green fluorescent protein was observed in both the Ad-GFP group and the Ad-FoxO1 group,no fluorescent protein could be found in the blank control.The m RNA and protein expression of FoxO1 gene was detected by real-time quantitative PCR and western blot.The results showed that the m RNA and protein expression of FoxO1 transfected with Ad-FoxO1 adenovirus was significantly up-regulated in smooth muscle cells,compared with the normal control group and the Ad-GFP group,and the difference was statistically significant(P<0.05).5.With transfected with Ad-FoxO1,the smooth muscle cells was interfered with 50?g/ml ox-LDL for 48 h.Oil red O results showed that more cytoplasmic red lipid particles in Ad-FoxO1 cells compared with 50?g/ml ox-LDL group.The level of TC and TG in smooth muscle cells were assayed by enzymatic,compared with the normal control group,the content of TG and TC in Ad-FoxO1 group increased by 1.4 and 1.9 times(P<0.01),and increased by60% and 63% compared with 50?g/ml ox-LD(P<0.01).6.We use the Bioinformatics Website to predicted the FoxO1 CPG Island.The expression of FoxO1 promoter methylation in smooth muscle cells with or without ox-LDL intervention was compared by n MS-PCR assay.The results showed that compared with the normal control group,the level of FoxO1 methylation was decreased by 26% in 50?g/ml ox-LDL group(P<0.05),and the protein level was increased by 1.3 times(P<0.05).After themethylation inhibitor AZC(10?mol/L)was administered at a concentration of 50?g/m L ox-LDL,the results of n MS-PCR and Western blot showed that AZC could inhibited the methylation level of FoxO1 promoter region significantly.Compared with the control group and 50?g/ml ox-LDL group,the FoxO1 methylation level in AZC group decreased by 50%and 33% respectively(P<0.01,P<0.05),and the protein levels increased by 2.4 and 0.5 times respectively,and the difference was statistically significant(P<0.01,P<0.05).7.The enzymatic methoeds result shows that,after the methylation inhibitor AZC(10?mol/L)interfered with 50?g/ml ox-LDL smooth muscle cells for 48 h,compared with the control group,the TG and TC contents in the AZC group increased by 1.6 and 1.2times,respectively(P<0.01).Compared with the 50?g/ml ox-LDL group,the TG and TC contents increased by50% and 49%(P<0.05).The results show that the lower the level of FoxO1 methylation,the more pronounced the lipid deposition.8.qRT-PCR and wstern blot detect the expression level of smoothelin and CON1 after used the methylation inhibitor AZC in Smooth Muscle Cells.The result showed that the m RNA and protein level of smoothlin in AZC group decreased significant(P<0.05),while the m RNA and protein level of CON1 increased(P<0.05),compared with the normal control group and 50?g/ml ox-LDL.ConclusionsThe phenomenon which ox-LDL promotes lipid deposition in smooth muscle cells and induces the occurrence of osteogenic transformation in cells,is realized by up-regulated the FoxO1 DNA methylation levels in smooth muscle cells.It is one of the most important mechanisms of ox-LDL-induced smooth muscle cell lipid accumulation and eventually induction of vascular calcification. |
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