Mao Min, Lei Han, Liu Qing, Chen Yaxi, Zhao Lei, Li Qing, Luo Suxin, Zuo Zhong, He Quan, Huang Wei, Zhang Nan, Zhou Chao, Ruan Xiong Z
Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China; Centre for Lipid Research, Key Laboratory of Metabolism on Lipid and Glucose, Chongqing Medical University, Chongqing, P.R. China; Centre for Clinical Research, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China.
Department of Cardiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China; Centre for Lipid Research, Key Laboratory of Metabolism on Lipid and Glucose, Chongqing Medical University, Chongqing, P.R. China.
PLoS One. 2014 Oct 17;9(10):e109722. doi: 10.1371/journal.pone.0109722. eCollection 2014.
The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.
本研究旨在探讨炎性细胞因子是否通过调节THP-1巨噬细胞中的miR-33a-5P来抑制ABCA1/ABCG1介导的胆固醇流出。我们使用白细胞介素-6和肿瘤坏死因子-α，在有或无天然低密度脂蛋白（LDL）存在的情况下刺激THP-1巨噬细胞。THP-1巨噬细胞被对照慢病毒载体或编码miR-33a-5P或反义miR-33a-5P的慢病毒感染。通过油红O染色和细胞内胆固醇定量测定，评估炎性细胞因子、miR-33a-5P和反义miR-33a-5P对细胞内脂质积累和细胞内胆固醇含量的影响。使用荧光固醇（BODIPY-胆固醇）检测载脂蛋白A-I介导的胆固醇流出。使用定量实时聚合酶链反应和蛋白质印迹法检测参与胆固醇转运的分子的基因和蛋白表达。在THP-1巨噬细胞中，无论有无LDL存在，炎性细胞因子或miR-33a-5P均可通过降低ABCA1和ABCG1的表达来增加细胞内脂质积累，并减少载脂蛋白A-I介导的胆固醇流出。然而，反义miR-33a-5P可逆转炎性细胞因子对THP-1巨噬细胞在有无LDL存在情况下的细胞内脂质积累、胆固醇流出以及miR-33a-5P、ABCA1和ABCG1表达的影响。本研究表明，炎性细胞因子通过上调THP-1巨噬细胞中的miR-33a-5P来抑制ABCA1/ABCG1介导的胆固醇流出。
