NF-κB途径介导Chemerin抑制THP-1源性巨噬泡沫细胞ABCAl表达和降低胆固醇外流

Chemerin Inhibiting ABCAl Expression and Reducing Cholesterol Efflux in THP-1-derived Macrophage Foam Cells Mediated by NF-κB Pathway

目的观察脂肪因子Chemerin是否调节胆固醇流出和巨噬泡沫细胞脂滴蓄积,以及对巨噬泡沫细胞ATP结合盒转运子A1(ATP-binding cassette transporter A1,ABCA1)和酰基辅酶A:胆固醇酰基转移酶-1(Acyl-CoA:choles-terol acyltransferase-1,ACAT1)表达和NF-κB活化的影响。方法佛波酯(phorbol myristate acetate,PMA)诱导THP-1单核细胞分化为巨噬细胞,随后加入胆固醇和ox-LDL培养促进巨噬细胞转化为巨噬泡沫细胞,免疫荧光染色检测巨噬细胞表面抗原CD68的表达,油红O染色后观察泡沫细胞形态,脂肪因子Chemerin干预巨噬泡沫细胞,应用液体闪烁计数仪检测胆固醇流出变化,油红O染色观察细胞内脂滴变化,实时PCR和Western blot检测ABCA1及ACAT1mR-NA和蛋白表达水平。Sandwich ELISA法检测巨噬泡沫细胞NF-κB活化情况。实时PCR检测NF-κB抑制剂四氢化吡咯二硫代氨基甲酸酯(PDTC)预处理后,Chemerin再干预时ABCA1表达的变化。结果 Chemerin抑制巨噬细胞内胆固醇流出,促进脂滴蓄积,下调ABCA1mRNA和蛋白的表达,增加NF-κB活化,差异有统计学意义(均P<0.05),且ABCA1mRNA表达与Chemerin浓度呈负相关,r=-0.936(P<0.05),NF-κB活化程度与ABCA1mRNA表达呈负相关,r=-0.917(P<0.05),但Chemerin对ACAT1mRNA和蛋白的表达无明显影响,PDTC预处理能够逆转ABCA1mRNA表达抑制。结论 Chemerin下调ABCA1mRNA和蛋白的表达,抑制THP-1巨噬泡沫细胞内胆固醇外流,增加细胞内脂滴蓄积,NF-κB活化可能是Chemerin抑制ABCA1mRNA表达的信号通路之一。

Objective To investigate the regulatory effects of Chemerin on cholesterol efflux and lipid droplet accumulation in macrophage foam cells,and explore its effects on ABCA1 and ACAT1 expression and activation of nuclear factor-κB（NF-κB） in macrophage foam cells.Methods Phorbol ester（phorbol myristate acetate,PMA）was used to induce the differentiation of THP-1 monocytes into macrophages,and subsequently the cholesterol and ox-LDL were added to promote transduction of macrophages into macrophage foam cells.Immunofluorescence staining was carried out to test the CD68 antigen on the surface of macrophages,and macrophage foam cells were subjected to oil red O staining.After the macrophage foam cells were treated with Chemerin,the liquid scintillation counter was used to test the changes in cholesterol effloux,the changes in intracellular lipid droplets were observed by using oil red O staining,and real-time PCR and Western blot were used to detect the expression of ABCA1 and ACAT1 mRNA and protein.Sandwich ELISA was used to measure the NF-κB activation in macrophage foam cells.After pretreatment with NF-κB inhibitor PDTC,the changes in ABCA1 expression and NF-κB activation were examined.Results Chemerin inhibited the cholesterol efflux in macrophges,promoted the accumulation of lipid droplets,down-regulated the expression of ABCA1 mRNA and protein,and increased NF-κB activation with the difference being statistically significant（P〈0.05）.ABCA1 mRNA expression was negatively correlated with Chemerin concentration（r=-0.936,P〈0.05）,and NF-κB activation was negatively correlated with the ABCA1 mRNA expression（r=-0.917,P〈0.05）.There were no significant changes in ACAT1 mRNA and protein expression.Pretreatment with PDTC could reverse the Chemerin-induced inhibition of ABCA1 mRNA expression.Conclusion The Chemerin can down-regulate the expression of ABCA1 mRNA and protein,suppress cholesterol efflux from THP-1-derived macrophage foam cells,and increase intracellular lipid droplet accumulation.NF-κB activation may be one of the signaling pathways by which Chemerin inhibits the ABCA1 mRNA expression.