| Objective:Diabetes, especially type2diabetes is the independent risk factors of the progression of cardiovascular disease. There is80%of death from cardiovascular complications in diabetic patients, the average life expectancy by around10years. oxLDL/β2-GPI complexes, CRP/oxLDL complexes, CRP/oxLDL/β2-GPIcomplexes have been shown predominantly found in sera of DM. CRP/oxLDL complexes, CRP/oxLDL/p2-GPIcomplexes were predominantly found in sera of DM patients with atherosclerosis. To explicit the effects of these complexes on the occurrence and development of atherosclerosis. In vitro, Pre-treatment of macrophages with oxLDL/β2-GPI complexes, CRP/oxLDL complexes and CRP/oxLDL/β2-GPI complexes, we observatived the accumulation of cholesterol and explore its mechanism.Methods:To RAW264.7macrophage of mice as the research object, treating the cells with80ug/ml oxLDL,80ug/ml CRP,80ug/mlβ2-GPI,80ug/ml CRP/oxLDL complexes,80ug/ml oxLDL/β2-GPI complexes,80ug/ml CRP/oxLDL/β2-GPIcomplexes. On the morphology, we observed the accumulations of lipid droplet in macrophage by Oil red staining. And detected the total cholesterol content of cells quantitatively with a kit; The expression of mRNA that relate to lipid engulfed such as Scavenger receptor class A(SRA), Scavenger receptor class B (CD36), scavenger receptor class B typel (SR-BI) and ATP-Binding Cassette Transporter Al (ABCA1) were detected by real-time quantitative PCR(RT-QPCR); The level of p38MAPK and ERK phosohorylation in the MAPK pathway were measured by Western blot.Results:1. Oil red O staining results:after24hours intervention, there is lipid accumulation in the macrophages in oxLDL group in which red is evident and cells become into bubble shape, the model of foam cell were builded up; CRP/oxLDL group, oxLDL/β2-GPI group, CRP/oxLDL/p2-GPIgroup all have foam cells formation. Compared with oxLDL group, CRP/oxLDL group had more oil red staining indicating that intracellular accumulation of lipid droplets was more; Inversely, red cells obviously decreased in oxLDL/β2-GPI group, so visible lipid accumulation is less; CRP/oxLDL/β2-GPI had no difference between the amount, but the number of cells decresed.2. Cells total cholesterol assay kit showed that the total cholesterol in the oxLDL group was obviously increased, its content is1.99times that of the control group (P<0.01); Compared with the group of control, intracellular cholesterol content of CRP/oxLDL group, oxLDL/β2-GPI group and CRP/oxLDL/β2-GPI group, was4.3times (P<0.01),1.53times (P<0.05) and1.94times (P<0.01)respectively. Compared with oxLDL group, CRP/oxLDL promoted foam cells formation and content of total cholesterol was2.16times of oxLDL group (P<0.05); oxLDL/β2-GPI inhibited oxLDL induced lipid droplet accumulation in macrophage, the content of total cholesterol decreased23.5%(P<0.05); CRP/oxLDL/β2-GPI has no obvious effect on the formation of oxLDL induced foam cells compared with oxLDL (P>0.05).3. RT-PCR showed that oxLDL promoted expression of CD36mRNA and decreased SR-BI, ABCA1mRNA; Compared with oxLDL group, the CRP/oxLDL group promoted SRA and CD36mRNA expression; OxLDL/β2-GPI group promoted SR-BI, ABCA1mRNA expression. CRP/oxLDL/β2-GPIgroup had no statistical significance between oxLDL group.4. Western blot showed that oxLDL promoted the expression of p38MAPK and ERK phosphorylation; compared with oxLDL group, CRP/oxLDL group, the phosphorylation of p38MAPK and ERK raised obviously, the difference was statistically significant (P<0.05); in oxLDL/β2-GPI group, the phosphorylated p38MAPK increased, but there was no statistically significant difference (P>0.05), but phosphorylation of ERK had been reduced, although there was no statistically significant difference (P>0.05); in CRP/oxLDL/β2-GPI group, p38MAPK phosphorylation levels increased, the difference was statistically significant (P<0.05), but ERK phosphorylation level there was no statistically significant difference (P>0.05).Conclusions:oxLDL promoted the accumulation of lipids within the cells by promoting the expression of CD36mRNA and reducing the SR-BI, ABCA1mRNA, and the p38MAPK and ERK were involved in the formation of foam cells; CRP/oxLDL significantly promoted the expression of SRA and CD36mRNA, leading to the intracellular lipids accumulation and increasing the formation of foam cells, at the same time promoted p38MAPK and ERK phosphorylation level; oxLDL/β2-GPI inhibited macrophage phagocytosis of lipid by promoting the SR-BI, ABCA1mRNA, compared with oxLDL, the level of p38MAPK and ERK phosphorylation had no obvious change; CRP/oxLDL/p2-GPI has no obvious change in phagocytosis of lipid, and the ERK phosphorylation had no obvious change, but p38MAPK phosphorylation levels increased, so we can infered that both the p38MAPK and ERK are all invovled in the occurrence and development of atherosclerosis, and p38MAPK may mainly relates to the generation of inflammation, ERK may mainly invovled in the process of lipid phagocyte in mice. |
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