基于THP-1-HIF-1α-HER-Luciferase细胞模型的抗动脉粥样硬化药物筛选
|
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100μg/mL)的200种化合物预处理THP-1-HIF-1α-HER-Luciferase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m RNA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-1αmRNA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-1αmRNA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。
Objective · To screen the anti-atherosclerosis(AS) activity of the compounds by using 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 screened by luciferase activity evaluation. THP-1 and U937 cells were pretreated with effective compounds, and then induced for 24 h by oxidized low density lipoprotein(OX-LDL). The formation of foam cells was observed by oil red staining. The mRNA level of hypoxia-inducible factor 1α(HIF-1α) was detected by real-time quantitative PCR(qPCR). HIF-1α protein expression was detected by Western 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 induced by hypoxia(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 h by OX-LDL. However, cells were pretreated with C14 for 2 h, which could significantly inhibit the formation of foam cells induced by OX-LDL. Cells were induced for 24 h by OX-LDL, which could significantly increase the expression 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 expression 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.
Objective · To screen the anti-atherosclerosis(AS) activity of the compounds by using 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 screened by luciferase activity evaluation. THP-1 and U937 cells were pretreated with effective compounds, and then induced for 24 h by oxidized low density lipoprotein(OX-LDL). The formation of foam cells was observed by oil red staining. The mRNA level of hypoxia-inducible factor 1α(HIF-1α) was detected by real-time quantitative PCR(qPCR). HIF-1α protein expression was detected by Western 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 induced by hypoxia(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 h by OX-LDL. However, cells were pretreated with C14 for 2 h, which could significantly inhibit the formation of foam cells induced by OX-LDL. Cells were induced for 24 h by OX-LDL, which could significantly increase the expression 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 expression 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.
引文
[1]杨永宗.中国动脉粥样硬化病理生理学研究近况[J].中国动脉硬化杂志,2004,12(4):481-489.
[2]王新,李春阳,苏立平,等.动脉粥样硬化发病机制及治疗的研究进展[J].实用心脑肺血管病杂志,2017,25(2):1-4.
[3]Ross R.Atherosclerosis:an inflammatory disease[J].N Engl J Med,1999,340(2):115-126.
[4]Williams KJ,Tabas I.Lipoprotein retention and clues for atheroma regression[J].Arterioscler Thromb Vasc Biol,2005,25(8):1536-1540.
[5]Sun B,Boyanovsky BB,Connelly MA,et al.Distinct mechanisms for OxLDLuptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI[J].J Lipid Res,2007,48(12):2560-2570.
[6]Yao W,Li K,Liao K.Macropinocytosis contributes to the macrophage foam cell formation in RAW264.7 cells[J].Acta Biochim Biophys Sin(Shanghai),2009,41(9):773-780.
[7]Kruth HS,Jones NL,Huang W,et al.Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein[J].J Biol Chem,2005,280(3):2352-2360.
[8]崔永春,刘晓鹏,张宏,等.不同阶段泡沫细胞模型的建立与鉴定[J].中国分子心脏病学杂志,2014,14(3):953-957.
[9]Lim CS,Kiriakidis S,Sandison A,et al.Hypoxia-inducible factor pathway and diseases of the vascular wall[J].J Vasc Surg,2013,58(1):219-230.
[10]Cavadas MAS,Taylor CT,Cheong A.Acquisition of temporal HIFtranscriptional activity using a secreted luciferase assay[J].Methods Mol Biol,2018,1742:37-44.
[11]Coban D,Milenkovic D,Chanet A,et al.Dietary curcumin inhibits atherosclerosis by affecting the expression of genes involved in leukocyte adhesion and transendothelial migration[J].Mol Nutr Food Res,2012,56(8):1270-1281.
[12]Xu S,Huang Y,Xie Y,et al.Evaluation of foam cell formation in cultured macrophages:an improved method with Oil Red O staining and DiI-oxLDLuptake[J].Cytotechnology,2010,62(5):473-481.
[13]Kaliora AC,Dedoussis GV,Schmidt H.Dietary antioxidants in preventing atherogenesis[J].Atherosclerosis,2006,187(1):1-17.
[14]Gao L,Chen Q,Zhou X,et al.The role of hypoxia-inducible factor 1 in atherosclerosis[J].J Clin Pathol,2012,65(10):872-876.
[15]Parathath S,Mick SL,Feig JE,et al.Hypoxia is present in murine atherosclerotic plaques and has multiple adverse effects on macrophage lipid metabolism[J].Circ Res,2011,109(10):1141-1152.
[16]Parathath S,Yang Y,Mick S,et al.Hypoxia in murine atherosclerotic plaques and its adverse effects on macrophages[J].Trends Cardiovasc Med,2013,23(3):80-84.
[17]Sluimer JC,Daemen MJ.Novel concepts in atherogenesis:angiogenesis and hypoxia in atherosclerosis[J].J Pathol,2009,218(1):7-29.
[18]Mazière C,Mazière JC.Activation of transcription factors and gene expression by oxidized low-density lipoprotein[J].Free Radic Biol Med,2009,46(2):127-137.
[19]Rodríguez JA,Nespereira B,Pérez-Ilzarbe M,et al.Vitamins C and Eprevent endothelial VEGF and VEGFR2 overexpression induced by porcine hypercholesterolemic LDL[J].Cardiovasc Res,2005,65(3):665-673.
[20]Zhu XY,Rodriguez-Porcel M,Bentley MD,et al.Antioxidant intervention attenuates myocardial neovascularization in hypercholesterolemia[J].Circulation,2004,109(17):2109-2115.
[21]Shatrov VA,Sumbayev VV,Zhou J,et al.Oxidized low-density lipoprotein(oxLDL)triggers hypoxia-inducible factor-1α(HIF-1α)accumulation via redox-dependent mechanisms[J].Blood,2003,101(12):4847-4849.
[22]Jiang G,Li T,Qiu Y,et al.RNA interference for HIF-1αinhibits foam cells formation in vitro[J].Eur J Pharmacol,2007,562(3):183-190.
[23]Wang Y,Liang J,Xu J,et al.Circulating exosomes and exosomal lncRNAHIF1A-AS1 in atherosclerosis[J].Int J Clin Exp Pathol,2017,10(8):8383-8388.
[24]Dichtl W,Dulak J,Frick M,et al.HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells[J].Arterioscler Thromb Vasc Biol,2003,23(1):58-63.
[25]Hossain CF,Kim YP,Baerson SR,et al.Saururus cernuus lignans:potent small molecule inhibitors of hypoxia-inducible factor-1[J].Biochem Biophys Res Commun,2005,333(3):1026-1033.
[2]王新,李春阳,苏立平,等.动脉粥样硬化发病机制及治疗的研究进展[J].实用心脑肺血管病杂志,2017,25(2):1-4.
[3]Ross R.Atherosclerosis:an inflammatory disease[J].N Engl J Med,1999,340(2):115-126.
[4]Williams KJ,Tabas I.Lipoprotein retention and clues for atheroma regression[J].Arterioscler Thromb Vasc Biol,2005,25(8):1536-1540.
[5]Sun B,Boyanovsky BB,Connelly MA,et al.Distinct mechanisms for OxLDLuptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI[J].J Lipid Res,2007,48(12):2560-2570.
[6]Yao W,Li K,Liao K.Macropinocytosis contributes to the macrophage foam cell formation in RAW264.7 cells[J].Acta Biochim Biophys Sin(Shanghai),2009,41(9):773-780.
[7]Kruth HS,Jones NL,Huang W,et al.Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein[J].J Biol Chem,2005,280(3):2352-2360.
[8]崔永春,刘晓鹏,张宏,等.不同阶段泡沫细胞模型的建立与鉴定[J].中国分子心脏病学杂志,2014,14(3):953-957.
[9]Lim CS,Kiriakidis S,Sandison A,et al.Hypoxia-inducible factor pathway and diseases of the vascular wall[J].J Vasc Surg,2013,58(1):219-230.
[10]Cavadas MAS,Taylor CT,Cheong A.Acquisition of temporal HIFtranscriptional activity using a secreted luciferase assay[J].Methods Mol Biol,2018,1742:37-44.
[11]Coban D,Milenkovic D,Chanet A,et al.Dietary curcumin inhibits atherosclerosis by affecting the expression of genes involved in leukocyte adhesion and transendothelial migration[J].Mol Nutr Food Res,2012,56(8):1270-1281.
[12]Xu S,Huang Y,Xie Y,et al.Evaluation of foam cell formation in cultured macrophages:an improved method with Oil Red O staining and DiI-oxLDLuptake[J].Cytotechnology,2010,62(5):473-481.
[13]Kaliora AC,Dedoussis GV,Schmidt H.Dietary antioxidants in preventing atherogenesis[J].Atherosclerosis,2006,187(1):1-17.
[14]Gao L,Chen Q,Zhou X,et al.The role of hypoxia-inducible factor 1 in atherosclerosis[J].J Clin Pathol,2012,65(10):872-876.
[15]Parathath S,Mick SL,Feig JE,et al.Hypoxia is present in murine atherosclerotic plaques and has multiple adverse effects on macrophage lipid metabolism[J].Circ Res,2011,109(10):1141-1152.
[16]Parathath S,Yang Y,Mick S,et al.Hypoxia in murine atherosclerotic plaques and its adverse effects on macrophages[J].Trends Cardiovasc Med,2013,23(3):80-84.
[17]Sluimer JC,Daemen MJ.Novel concepts in atherogenesis:angiogenesis and hypoxia in atherosclerosis[J].J Pathol,2009,218(1):7-29.
[18]Mazière C,Mazière JC.Activation of transcription factors and gene expression by oxidized low-density lipoprotein[J].Free Radic Biol Med,2009,46(2):127-137.
[19]Rodríguez JA,Nespereira B,Pérez-Ilzarbe M,et al.Vitamins C and Eprevent endothelial VEGF and VEGFR2 overexpression induced by porcine hypercholesterolemic LDL[J].Cardiovasc Res,2005,65(3):665-673.
[20]Zhu XY,Rodriguez-Porcel M,Bentley MD,et al.Antioxidant intervention attenuates myocardial neovascularization in hypercholesterolemia[J].Circulation,2004,109(17):2109-2115.
[21]Shatrov VA,Sumbayev VV,Zhou J,et al.Oxidized low-density lipoprotein(oxLDL)triggers hypoxia-inducible factor-1α(HIF-1α)accumulation via redox-dependent mechanisms[J].Blood,2003,101(12):4847-4849.
[22]Jiang G,Li T,Qiu Y,et al.RNA interference for HIF-1αinhibits foam cells formation in vitro[J].Eur J Pharmacol,2007,562(3):183-190.
[23]Wang Y,Liang J,Xu J,et al.Circulating exosomes and exosomal lncRNAHIF1A-AS1 in atherosclerosis[J].Int J Clin Exp Pathol,2017,10(8):8383-8388.
[24]Dichtl W,Dulak J,Frick M,et al.HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells[J].Arterioscler Thromb Vasc Biol,2003,23(1):58-63.
[25]Hossain CF,Kim YP,Baerson SR,et al.Saururus cernuus lignans:potent small molecule inhibitors of hypoxia-inducible factor-1[J].Biochem Biophys Res Commun,2005,333(3):1026-1033.