人载脂蛋白A-I表达上调剂的发现与活性研究

摘要

Objective To find potential small molecules that increase endogenous synthesis of apolipoprotein A-I (ApoA-I), and to identify its ability of increasing ApoA-I at expression and functional levels. Methods Compounds screening was performed with established screening model based on the ApoP-Luc HepG2 cell line. Dose-response relationship of the active compound was achieved in promoter activity. The expression of ApoA-I in HepG2 cells regulated by the compound was detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, ELISA and flow cytometry analysis. The cholesterol efflux assay was applied to investigate the effect of promoted ApoA-I on mediating lipid transfer in RAW 264.7 cells. Results In the present study, compound 5238B-69 was found using the screening model. 5238B-69 increased ApoA-I promoter activity in a dose-dependent manner with EC50 of 0.01 µg/ml, and maximal activity of 408%at 0.30μg/ml. 5238B-69 significantly increased ApoA-I mRNA level and protein level in HepG2 cells. ELISA and flow cytometry analysis showed 5238B-69 could increase ApoA-I protein by 48%and 21.4%compared with control in the media and cells, respectively. The functional efflux assay further illustrated the role of ApoA-I induced by 5238B-69 with RAW 264.7 cells. Conclusion A potential small molecular upregulator was obtained, which could significantly increase the expression of human ApoA-I in liver cells and promote cholesterol efflux from mice macrophages.%目的筛选上调人载脂蛋白 A-I（ApoA-I）基因表达的新型活性化合物，并在表达和功能水平对其作用进行研究。　　方法利用人 ApoA-I 基因表达上调剂筛选模型对化合物库进行筛选；对发现的活性化合物在 ApoA-I 启动子水平进行量效关系研究；利用实时荧光定量 RT-PCR 检测ApoA-I mRNA 的表达水平；利用 Western blot、ELISA 和流式细胞技术检测 ApoA-I 的蛋白表达；利用胆固醇流出试验检测 ApoA-I 介导的巨噬细胞胆固醇外流的情况。结果从20000样次化合物筛选中得到活性化合物5238B-69，在一定浓度范围内存在转录调控活性的量-效关系，当化合物浓度为0.30μg/ml 时，上调 ApoA-I 表达活性达到最高值（408%），EC50为0.01μg/ml。5238B-69能显著上调 HepG2细胞中 ApoA-I mRNA 的水平；同时， Western blot 结果显示5238B-69能增加 HepG2细胞ApoA-I 蛋白的表达。ELISA 结果显示，与对照相比，5238B-69作用48 h 时，胞外 ApoA-I 水平增加了48%，流式细胞检测表明，5238B-69作用24 h 后，胞内 ApoA-I蛋白水平增加了21.4%。功能分析试验显示，5238B-69作用 HepG2细胞上调生成的 ApoA-I，能促进巨噬细胞RAW 264.7内[3H]标记的胆固醇的流出。　　结论得到一个具有上调 ApoA-I 表达的活性化合物，在提高 ApoA-I 表达水平和生物学功能方面均有明显的效果。