载脂蛋白嵌合模拟肽对低密度脂蛋白诱导的巨噬细胞凋亡的影响

目的观察模拟肽Ac-hE-18A-NH2对氧化低密度脂蛋白(oxLDL)诱导的RAW264.7巨噬细胞凋亡的影响。方法50 mg.L-1oxLDL处理RAW 264.7细胞48 h后加入不同浓度的Ac-hE-18A-NH2(1、10、50、100 mg.L-1)和β-环糊精(β-CD)或布雷菲得菌素(BFA)共同孵育24 h。通过流式细胞仪检测细胞凋亡,试剂盒检测caspase-3活性及细胞内胆固醇含量,Western blot检测细胞bcl-2蛋白的表达,液体闪烁计数器检测细胞内胆固醇流出。结果 oxLDL诱导的RAW 264.7巨噬细胞的凋亡率随着oxLDL浓度的增加和处理时间的延长而明显提高。Ac-hE-18A-NH2(1、10、50、100mg.L-1)干预后细胞凋亡率以浓度依赖的方式减少。Ac-hE-18A-NH2呈浓度依赖性的促进细胞内胆固醇流出和降低细胞内的胆固醇含量,降低caspase-3的活性,上调bcl-2的蛋白表达。β-CD与Ac-hE-18A-NH2共同作用后胆固醇流出明显增加,细胞凋亡率相应减少。而BFA作用相反。结论Ac-hE-18A-NH2明显抑制oxLDL诱导的巨噬细胞凋亡,其作用可能与促进细胞胆固醇流出、减少细胞内胆固醇蓄积有关。

Effect of Ac-hE-18A-NH2 ,a novel dual-domain apolipoprotein mimetic peptide,on oxLDL-induced macrophage apoptosis

Aim To investigate the effect of the peptide Ac-hE-18A-NH2 on oxLDL-induced apoptosis in RAW264.7 cells and the possible mechanism.Methods RAW264.7 cells were exposed to 50 mg.L-1 oxLDL for 48 hours,and then the cells were incubated with the peptide Ac-hE-18A-NH2 with various concentrations.The apoptosis was detected using Annexin VFITC / PI staining and flow cytometric analysis.The caspase-3 activity and intracellular cholesterol content were measured using commercially available quantitation kits following the manufacturer ’s instructions.Bcl-2 protein expression in macrophages was detected by Western blot analysis.In some experiments,the cells were co-treated with β-cyclodextrin(a cholesterol efflux stimulator) or BFA(a cholesterol efflux blocker) for 24 hours.Results After treated with 50 mg.L-1 oxLDL for various time points,the apoptotic rate of RAW264.7 macrophages increased in a time-dependent manner.OxLDL with increasing concentration induced macrophage apoptosis in a dose-dependent manner.The peptide Ac-hE-18A-NH2 with various concentrations(1 mg.L-1,10 mg.L-1,and 50 mg.L-1) inhibited the oxLDL-mediated apoptosis that was accompanied by an increased rate of intracellular cholesterol efflux,and decreased total cholesterol levels in cells in a concentration-dependent manner.The peptide also decreased the caspase-3 activity and increased Bcl-2 expression in macrophages in a dose-dependent manner.Moreover,blockage of cholesterol efflux by BFA decreased the protective effect of Ac-hE18A-NH2 on oxLDL induced apoptosis,while increase of the cholesterol efflux by β-CD led to a dramatic decrease in the apoptotic rate of cells.Conclusion The mimetic peptide Ac-hE-18A-NH2 exerts a protective effect against macrophage apoptosis,through reducing the accumulation of cholesterol.