| Atherosclerosis is the chronic inflammation of large and medium vessels. Many studies shows that the serum level of secreted phospholipase A2 typeⅡA (sPLA2-ⅡA) is elevated in cardiovascular diseases, which is proportional to the severity of the disease. It is proved by many studies that sPLA2-ⅡA is not only the risk factor of atherosclerosis and predictive factor of incidence of coronary event or coronary attack but also playing role in the occurance and progression of atherosclerosis. The mechanism of sPLA2-ⅡA in atherosclerosis development is still not fully understood, though the sPLA2-ⅡA inhibitor(Varespladib) is already undergoing phaseⅡclinical trail. The counter transport of cholesterol whose initial step is the outflow of cholesterol plays key role in the development of atherosclerosis. So the aim of this study is to investigate the influence of sPLA2-ⅡA on cholesterol outflow and its mechanism.1. Investigate the effect of sPLA2-ⅡA in different concentrations on ABCA1, ABCG and SRB1 mRNA and protein expression in human peripheral mononuclear macrophage.2. We examined the effect of sPLA2-ⅡA in different concentrations on the cellular cholesterol efflux from peripheral MDM to HDL and apoA1.1. Peripheral blood mononuclear cells (PBMC) were isolated from perapheral blood of healthy people through Ficoll-Paque density gradient centrifugation which were subjected to adherence selection for monocytes. Then the monocytes were were allowed to differentiate with human macrophages colony stimulant factor (hM-CSF). Then CD68 IHC stain was used to identify macrophage cells.2. Quantitative Real-time PCR was used to measure the effect of sPLA2-ⅡA of different concentration on the expression of mRNA expression of ABCA1,ABCG1 and SRB1.3. Western blot was used to detect the effect of sPLA2-ⅡA of different concentration on protein expression of ABCA1,ABCG1 and SRB1. 4. Cholesterol assays-sPLA2-IIA incubated the MDM for 24h, the Cells were radio labeled with 1μCi/ml of [3H] cholesterol for 24 hours in the presence of 10% FBS medium and then equilibrated for 2 hours in the presence of serum free medium containing 0.2% fatty acid free bovine serum albumin (BSA). HDL, apoAl were then used to induce cholesterol efflux from the labeled cells for 4 hours.5. Statistic analysis-Data for all experiments were analyzed using the SPSS 13.0 software program. Comparisons between groups were performed using ANOVA methods. Data are graphically represented as mean±S.E.1. 1)The data of real-time PCR illustrated that the expression of ABCA1mRNA gradually decreased along with the increase of sPLA2-IIA concentration. When macrophage cells were interfered with sPLA2-IIA of 0.01μg/ml,0.1μg/ml and 0.5μg/ml, ABCA1mRNA level was obviously lower than that of negative control (P< 0.01) but there was no obvious difference in ABCA1mRNA level among the three groups. SPLA2-ⅡA in lug/ml showed strongest inhibition to ABCA1 mRNA expression which was obviously different than that of other groups (P<0.01).2) When the concentrations of sPLA2-IIA were 0.01μg/ml,0.1μg/ml and 0.5μg/ml, the expression of ABCG1mRNA showed no obvious change. However, 1μg/ml sPLA2-IIA could significantly inhibit ABCG1 expression(p=0.033).3) sPLA2-IIA gradually showed inhibitory effect on SRB1mRNA expression, when sPLA2-IIA concentrations were 0.5μg/mland 1μg/ml, it showed strong inhibition on SRB1mRNA level.2. The protein expression level of ABCA1, ABCG1 and SRB1 by western blot showed no significant difference among groups which were challenged by sPLA2-IIA in different concentration.3. Although there was no significant variance among groups in Cholesterol effluxes to HDL, ApoA1 (p>0.05), sPLA2-ⅡA gradually showed inhibitory effect on Cholesterol efflux to ApoAl and HDL, when sPLA2-ⅡA concentration was continuously increased.4. Cholesterol effluxes to HDL, autoserum and pool serum were decreased in macrophages from type 2 diabetic subjects compared with healthy controls. Cholesterol efflux to lipid-poor ApoA1 was unchanged between diabetes without CHD and controls and. However, the significant difference occurred between the diabetes subjects with CHD and healthy controls.1. In this study, human macrophages derived from monocytes were processed with sPLA2-ⅡA in different concentrations for 24 hours. All the sPLA2-ⅡA concentration could obvoiously inhibit ABCA1 mRNA expression level. The ABCG1 mRNA expression level was significantly decreased when sPLA2-ⅡA was 1μg/ml. And SRB1 expression level was decreased when sPLA2-IIA concentrations were 0.5μg/mland 1μg/ml.2. ABCA1, ABCG1 and SRB1 protein expression were not changed after macropahges were treated by sPLA2-IIA2 different concentrations for 24 hours.3. Cholesterol efflux from macrophages to HDL and ApoAl were gradually impaired, when sPLA2-IIA concentration was continuously increased. But each group compared with the control group,there are no significant differences.
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