孤儿核受体Nur77通过转录激活功能调控巨噬细胞脂质沉积

Lipid loading in macrophages is regulated by Nur77 nuclear import and its DNA binding activity

目的:探讨孤儿核受体Nur77调控巨噬细胞脂质沉积与其转录激活功能的关系。方法:建立稳定表达GFP、GFP-Nur77、GFP-Nur77/ΔDBD和GFP-Nur77/ΔTAD c DNA的小鼠巨噬细胞RAW264.7单克隆细胞系,40μg/ml氧化型低密度脂蛋白(ox-LDL)刺激24 h后,油红"O"染色定性观察细胞内脂质沉积,液相色谱-质谱联用法(LC-MS)定量检测细胞内胆固醇酯,荧光实时定量PCR(RT-PCR)检测CD36和ABCA1 mRNA水平的变化,流式细胞仪检测CD36蛋白表达,Western blot方法检测ABCA1蛋白表达。结果:与对照GFP-RAW264.7相比,高表达Nur77能够显著减少ox-LDL诱导引起的巨噬细胞内脂质沉积,同时降低CD36的mRNA及蛋白表达,上调ABCA1的mRNA及蛋白表达。但GFP-Nur77/ΔDBD RAW264.7和GFP-Nur77/ΔTAD RAW264.7细胞内胆固醇都明显升高,并伴随CD36的mRNA及蛋白表达的降低和ABCA1的mRNA及蛋白表达的升高。结论:孤儿核受体Nur77通过减少脂质摄取和增加脂质排出而减轻巨噬细胞内脂质沉积,这一作用与Nur77 DNA结合能力以及转录活性有关。

AIM：To investigate the role of Nur77 and its transcriptional function in lipid loading in macrophages. METHODS： We established four stable RAW264.7 strains expressing GFP, GFP-Nur77, GFP-Nur77/ΔDBD and GFP-Nur77/ΔTAD, respectively, by transfecting with corresponding plasmids. After 24 h stimulation by ox-LDL, intracellular lipid loading of each strain was observed by Oil red O dye. The intracellular cholesterol level was measured by liquid chromatographic-mass spectrometry （LC-MS）. Transcriptional changes of CD36 and ABCA1 were monitored by real-time quantitative-PCR, and the expressions of these two proteins were assayed by flow cytometry and Western blot, respectively. RESULTS： Compared with GFP RAW264.7 cells, the high expression of Nur77 lowered the intracellular lipid loading in macrophages induced by ox-LDL, and downregulated CD36 and upregulated ABCA1 mRNA transcription and protein expression. However, the intracellular lipid loading in GFP-Nur77/ΔDBD and GFP-Nur77/ΔTAD RAW264.7 cells increased significantly with the increasing of CD36 mRNA and protein and the decreasing of ABCA1 mRNA and protein. CONCLUSION： Orphan nuclear receptor Nur77 reduces intracellular lipid loading in macrophages through decreasing cholesterol influx and increasing cholesterol efflux. The anti-atherosclerotic effect of Nur77 may be associated with its nuclear import and its DNA binding activity.