血管紧张素(1-7)阻断细胞外信号调节激酶1/2、核因子κB信号通路影响泡沫细胞内胆固醇含量
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山西省科学技术厅资助项目(20100311098-4)


An Effect on Cholesterol Content in Foam Cells by Blocking the Extracellular Signal-regulated Kinase 1/2 Signal Pathway and the Nuclear Factor-κB Signal Pathway with Angiotensin-(1-7)
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    摘要:

    目的研究血管紧张素(1-7)对THP-1源性泡沫细胞中细胞外信号调节激酶1/2及核因子κB信号转导通路的影响,以进一步探讨血管紧张素(1-7)促进胆固醇逆转运的调节机制以及细胞外信号调节激酶1/2与核因子κB信号通路之间是否相互影响。方法 采用体外培养的THP-1单核细胞构建泡沫细胞模型, 用不同的干预方法处理细胞72 h,将细胞分为单核细胞(空白对照)组、泡沫细胞组、分别预先经10-6 mol/L血管紧张素(1-7)、10 μmol/L核因子κB特异性阻断剂、10 μmol/L细胞外信号调节激酶1/2特异性阻断剂、10-6 mol/L血管紧张素(1-7)+10 μmol/L核因子κB特异性阻断剂、10-6 mol/L血管紧张素(1-7)+10 μmol/L细胞外信号调节激酶1/2特异性阻断剂干预的泡沫细胞组。油红O染色后显微镜下观察细胞形态;高效液相色谱法检测细胞内胆固醇含量的变化;免疫组化法检测细胞内核因子κB(p65)活性的表达;免疫印迹法检测磷酸化细胞外信号调节激酶1/2蛋白的表达。结果血管紧张素(1-7)显著降低了泡沫细胞内胆固醇的含量,下调了磷酸化细胞外信号调节激酶1/2、核因子κB(p65)活性的表达(p<0.05),细胞外信号调节激酶1/2、核因子κB信号通路被特异性阻断后泡沫细胞内胆固醇含量降低(p<0.05);血管紧张素(1-7)联用细胞外信号调节激酶1/2、核因子κB信号通路的特异性阻断剂后泡沫细胞内胆固醇含量显著降低(p<0.01);核因子κB信号通路被阻断后核因子κB(p65)活性表达显著降低(p<0.01),而磷酸化细胞外信号调节激酶1/2活性表达无明显降低(p>0.05),细胞外信号调节激酶1/2信号通路被阻断后磷酸化细胞外信号调节激酶1/2和核因子κB(p65)活性表达均降低(p<0.05)。结论血管紧张素(1-7)可能通过降低磷酸化细胞外信号调节激酶1/2、核因子κB(p65)的活性,减少细胞内胆固醇的蓄积;细胞外信号调节激酶1/2、核因子κB信号通路被特异性阻断后可减少泡沫细胞内胆固醇的含量;细胞外信号调节激酶可能是核因子κB(p65)信号通路的上游信号。

    Abstract:

    AimTo invstigate the regulation of angiotensin-(1-7) (Ang(1-7)) to the expression of the activity of extracellular signal-regulated kinase-1/2 (ERK1/2) and the activity of nuclear factor-κB (NF-κB) in THP-1 derived-foam cells and to analyze the relationship between the ERK1/2 signal pathway and the NF-κB signal pathway.MethodsTo establish a macrophage-derived foam cell model of human monocylic THP-1 cell line, handle the cells in different conditions for 72 hours, which are divided into control group with medium added nothing, foam cell group, intervention fo-am cell groups advanced respectively by 10-6 mol/L Ang-(1-7), 10 μmol/L PD98059, 10 μmol/L TPCK (tosyl-phenylalanine chloromethyl-ketone), 10-6 mol/L Ang-(1-7)+10 μmol/L TPCK, 10-6 mol/L Ang-(1-7)+10 μmol/L PD98059.The differentiated cells were observed after oil red O staining under light microscope, high performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-MS) was used for quantitative analysis of cellular cholesterol, immunohistochemistry was employed to identify the expression of NF-κB in the differentiated cells, and the expression of p-ERK1/2 protection in the differentiated cells was detected by Western blotting.ResultsAng-(1-7) downregulated the expression of the activity of ERK1/2 and the activity of NF-κB (p<0.05), meanwhile, Ang-(1-7) downregulated the content of cholesterol in THP-1 derived foam cells (p<0.05), specific inhibitor PD98059 and specific inhibitor TPCK was used to reduce the content of total cholesterol in THP-1 derived foam cells (p<0.05), compared with povidone Ang-(1-7) and specific inhibitor PD98059 or specific inhibitor TPCK was proved to significantly decrease the content of total cholesterol in THP-1 derived foam cells (p<0.01).Specific inhibitor TPCK was used to abrogate phosphorylation of NF-κB for further evaluation (p<0.01), but didn't abrogate phosphorylation of ERK1/2 for further evaluation (p>0.05).Specific inhibitor PD98059 was used to abrogate phosphorylation of ERK1/2 and NF-κB for further evaluation (p<0.05).ConclusionsAng-(1-7) can lessen the content of cholesterol in THP-1 derived foam cells by decreasing the the activity of ERK1/2 and the activity of NF-κB.Specific inhibitor PD98059 and specific inhibitor TPCK is used to reduce the content of total cholesterol in THP-1 derived foam cells.ERK1/2 may be the upstream substance before NF-κB in the signaling pathway.

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刘娟,杨志明,边云飞,闫丰.血管紧张素(1-7)阻断细胞外信号调节激酶1/2、核因子κB信号通路影响泡沫细胞内胆固醇含量[J].中国动脉硬化杂志,2012,20(7):593~599.

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  • 收稿日期:2011-11-22
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