摘要/Abstract

摘要： 目的 ·基于 THP-1-HIF-1α-HER-Luciferase高通量筛选模型，对化合物的抗动脉粥样硬化（ atherosclerosis，AS）活性进行筛选，并对有效化合物的抗 AS功能进行验证。方法 ·利用不同浓度（ 1、10和 100 μg/mL）的 200种化合物预处理 THP-1-HIF-1α-HERLuciferase细胞 2 h，再低氧培养 24 h，收集细胞并检测荧光素酶活性，通过荧光素酶活性的变化评价化合物抗 AS活性。利用有效化合物预处理单核巨噬细胞 THP-1和 U937，再经氧化低密度脂蛋白（ oxidized low density lipoprotein，OX-LDL）诱导 24 h，通过油红染色观察泡沫细胞形成，实时荧光定量 PCR（real-time quantitative PCR，qPCR）检测缺氧诱导因子 1α（hypoxia-inducible factor 1α， HIF-1α）的 mRNA含量，蛋白印迹法（ Western blotting）检测 HIF-1α的蛋白表达，并分析有效化合物的抗 AS活性。结果 · 200种化合物中，有 11种化合物能够有效抑制由低氧引起的 THP-1-HIF-1α-HER-Luciferase细胞内荧光素酶活性升高（均 P<0.05）；其中，化合物 C14抑制作用最为显著。THP-1和 U937细胞由 OX-LDL诱导 24 h可出现泡沫化，化合物 C14对其预处理 2 h则能够明显抑制由 OX-LDL引起的单核细胞泡沫化。qPCR和 Western blotting检测显示，OX-LDL诱导 THP-1和 U937细胞 24 h可明显增强胞内 HIF-1α mRNA含量及其蛋白的表达（ P<0.05），而 C14预处理则能梯度依赖地抑制由 OX-LDL诱导的胞内 HIF-1α mRNA含量及其蛋白表达的增强（ P<0.05）。结论 ·抗 AS化合物的高通量筛选模型 THP-1-HIF-1α-HER-Luciferase具有较高的可靠性，化合物 C14具有良好的抗 AS活性特征。
关键词: 缺氧诱导因子1&, alpha, THP-1细胞, U937细胞, 泡沫细胞, 动脉粥样硬化
Abstract:
Objective · To screen the anti-atherosclerosis (AS) activity of the compoundsusing THP-1-HIF-1α-HER-Luciferase high-throughput model, and to verify the anti-AS function of the effective compounds. Methods · THP-1-HIF-1α-HER-Luciferase cells were pretreated with different concentrations of compounds (1, 10, and 100 μg/mL) for 2 h, then cultured under hypoxia for 24 h. Luciferase activity of cells was detected and compounds with anti-AS activity were screenedluciferase activity evaluation. THP-1 and U937 cells were pretreated with effective compounds, and then induced for 24 hoxidized low density lipoprotein (OX-LDL). The formation of foam cells was observedoil red staining. The mRNA level of hypoxia-inducible factor 1α (HIF-1α) was detectedreal-time quantitative PCR (qPCR). HIF-1α protein was detectedWestern blotting. Anti-AS activity of effective compounds were evaluated. Results · Among the 200 compounds, 11 compounds could significantly inhibit the increase of luciferase activity in THP-1-HIF-1α-HER-Luciferase cells inducedhypoxia (all P<0.05), and compound numbered 14 (C14) had the most significant inhibitory effect. THP-1 and U937 cells formed foam cells induced for 24 hOX-LDL. However, cells were pretreated with C14 for 2 h, which could significantly inhibit the formation of foam cells inducedOX-LDL. Cells were induced for 24 hOX-LDL, which could significantly increase the of HIF-1α mRNA and protein (all P<0.05), while cells pretreated with C14 could significantly inhibit the increase of HIF-1α mRNA and protein in a gradient-dependent manner (all P<0.05). Conclusion · THP-1-HIF-1α-HER-Luciferase high-throughput model can be reliability used in screening of compounds with anti-AS activity. C14 has the good anti-AS activity characteristics.
Key words: hypoxia-inducible factor 1&, alpha, (HIF-1&, alpha, ), THP-1 cell, U937 cell, foam cell, atherosclerosis (AS)


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