Objective To determine the effect and possible mechanism of an apolipoprotein (apo) A-I mimetic peptide, D-4F, on cholesterol effiux in RAW264.7 macrophages. Methods RAW264.7 macrophages were incubated in the medium containing 8-bromo cAMP (8-Br-cAMP,0.5 mmol/L) and ox-LDL (50 μg/mL) for 24 h. Then various concentrations of D-4F (0 - 100 μg/mL) or H89 (20 μmol/L, a protein kinase A inhibitor) were added for the purpose of interference. The intracellular cyclic AMP (cAMP) level was determined by enzyme-linked immunoabsobant assay ( ELISA ). ATP binding cassette transporter A 1 ( ABCA 1 ) expression in the macrophages was quantitated by real-time PCR and Western blot. Results D-4F significantly increased the cholesterol efflux in both concentration and time-dependent manner accompanied by the increase in the intracellular cAMP level, ABCA 1 mRNA and protein expression. The effect of D-4 F on cholesterol efflux ABCAI expression was enhanced by 8-Br-cAMP. Although H89 did not affect the basal cholesterol efflux and ABCA1 expression, it could attenuate the effect of 8-Br cAMP. Conclusion D-4F affects cholesterol efflux, cAMP level, and ABCA 1 expression in macrophages, which is likely involved in the pathway of cAMP/PKA/ABCA1.%目的:观察载脂蛋白A-I(apoA-I)模拟肽D-4F对RAW264.7巨噬细胞胆固醇流出的影响,并探讨其机制.方法:巨噬细胞种植于24孔板,用1.85×107 Bq/孔 3H -胆固醇和含50 μg/mL氧化型低密度脂蛋白(ox-LDL)共同孵育 24 h后,给予不同浓度的D-4F(0～100 μg/mL)干预24 h,收集细胞用液体闪烁计数法检测胆固醇流出.采用酶联免疫吸附试验(ELISA)测定细胞内环磷酸腺苷(cAMP)含量,采用实时荧光定量PCR及Western印迹检测三磷酸腺苷结合盒转运体A1(ABCA1)的mRNA及蛋白表达.结果:D-4F呈浓度依赖及时间依赖性促进巨噬细胞内胆固醇流出,增加细胞内cAMP 水平,上调ABCA1 mRNA和蛋白表达.8-Br-cAMP显著增加D-4F介导的胆固醇流出和ABCA1表达,而蛋白激酶A(PKA)抑制剂虽然对基础胆固醇流出及ABCA1表达几乎无作用,却可以抑制8-Br-cAMP对胆固醇流出和ABCA1表达的促进作用.结论:D-4F 促进巨噬细胞胆固醇流出,cAMP释放及ABCA1表达,可能与激活cAMP-PKA-ABCA1通路有关.
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