| ObjectiveThe occurrence of macrophage derived foam cell is the key marker of AS at the early stage. In our study, RAW264.7cells as the research object, we construct the macrophage foam cell induced by ox-LDL, to study the effect of reduced beta2glycoprotein I (R-β2GPI) and β2-GPI on foam cell formation and apotosis in the process of AS, and its possible mechanism.Methods1. Construction of RAW264.7macrophage-derived foam cell model, to explore the suitable concentration of ox-LDL inducing foam cell. In the oxidation of different concentration of low density lipoprotein in solution were incubated with24h, high pressure liquid chromatography combined with mass spectrometry (HPLC-MS) determination of total cholesterol in cell growth, cell viability was detected by MTT.2. RAW264.7macrophage cells were cultivated and divided into four groups:①control group:without any interference factors.②the foam cell group:80mg/Lox-LDL was added.③β2-GPI group:80mg/Lox-LDL was added with100mg/L p2GPI④reduced β2-GPI group:80mg/Lox-LDL was added with100mg/L reduced β2-GPI. Common after24h incubation, cells were observed by oil red O staining; cholesterol content of cells was measured by HPLC-MS assay; the level of apoptosis was detected with flow cytometry. Real-time quantitative PCR method were used to detect the expression level of CD36, SR-B1, ABCA1, ABCG1mRNA of macrophage-derived foam cells induced by ox-LDL.3. the phosphorylation level of p38MAPK signal transduction pathway was detected by Western Blot technology to observe the effect of reduced β2-GPI and β2-GPI on p38MAPK in macrophage derived foam cells induced by ox-LDLResults:1. With ox-LDL concentration inereased, the rate of apoptosis and necrosis rate also inereased. ox-LDL concentrate on80ug/mL macrophagese on strueted expression levels of total cholesterol is better, the optimal on contration for the induction. 2. Oil red O staining, cell detection of cellular cholesterol content display: cholesterol content of β2GPI group and R-β2GPI was significantly lower than that of macrophage foam cells group (P<0.05, P<0.01); cholesterol/total cholesterol (CE/TC) in R-β2GPI were lower than ratio of β2GPI group and foam cell group (P<0.05, p<0.01), and cholesterol content and CE/TC in R-P2GPI group were lower than β2GPI group(P<0.05, P<0.01).3. Apoptosis rate and activity of Caspase3levels significantly increased in foam cell than the control group (P<0.01); while apoptosis rate were significantly decreased in and β2GPI than the foam cells group (P<0.05, P<0.01;); the total cell apoptosis rate and the activity of Caspase3level in R-β2GPI was significantly decreased compared with2GPI group (P<0.05).4. CD36mRNA in R-β2GPI and β2GPI group significantly decreased than in macrophage foam cells group (P<0.05or P<0.01). and it was more lower in R-β2GPI group than β2GPI group (P<0.05). R-β2GPI downregulate p38phosphorylation level copaired with foam cell group (P<0.05).,while β2GPI had no obvious effect.on activation of this pathway induced by ox-LDL.ConclusionBoth β2GPI and reduced β2GPI can inhibit the formation of foam cells and apoptosis induced by ox-LDL and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake ofmacrophages by downregulating CD36as well as protein expression. Reduced β2GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38MAPK and JNK, and the amount of cleaved caspase-3and caspase-9. β2GPI does not inhibit the ox-LDL-induced phosphorylation of p38MAPK. |
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