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

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)

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10-6mol/L血管紧张素(1-7)、10μmol/L核因子κB特异性阻断剂、10μmol/L细胞外信号调节激酶1/2特异性阻断剂、10-6mol/L血管紧张素(1-7)+10μmol/L核因子κB特异性阻断剂、10-6mol/L血管紧张素(1-7)+10μmol/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。

Aim To 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.Methods To 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.Results Ang-（1-7） downregulated the expression of the activity of ERK1/2 and the activity of NF-κB（P0.05）,meanwhile,Ang-（1-7） downregulated the content of cholesterol in THP-1 derived foam cells（P0.05）,specific inhibitor PD98059 and specific inhibitor TPCK was used to reduce the content of total cholesterol in THP-1 derived foam cells（P0.05）,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（P0.01）.Specific inhibitor TPCK was used to abrogate phosphorylation of NF-κB for further evaluation（P0.01）,but didn＇t abrogate phosphorylation of ERK1/2 for further evaluation（P0.05）.Specific inhibitor PD98059 was used to abrogate phosphorylation of ERK1/2 and NF-κB for further evaluation（P0.05）.Conclusions Ang-（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.