目的 测定巨噬细胞ABCG1介导的氧化固醇外流情况.方法 利用ABCG1基因小干扰RNA慢病毒表达载体感染THP-1细胞,经有限稀释法筛选出敲低单克隆稳转株;用PMA诱导THP-1为巨噬细胞,ox-LDL(50μg/ml)刺激24 h后,通过液相色谱串联质谱测定细胞氧化固醇7β-羟基胆固醇(7β-OHC)和7-酮基胆固醇(7-KC)外流情况.结果 对待测样品进行了氧化固醇定性与定量分析,发现与无外流接受体的空白对照组相比,诱导氧化型胆固醇外流率增加(7-KC:28.6％±2.3％比4.1％±0.7％,P<0.001;7β-OHC:35.5％±5.7％比3.7％±1.1％,P<0.001).与阴性对照组相比,ABCG1敲低组氧化型胆固醇的外流率降低(7-KC:16.1％±3.1％比24.7％±2.3％,P<0.05;7β-OHC:15.7％±0.7％比33.1％±3.7％,P<0.001).胞内氧化固醇沉积量通过细胞总蛋白进行校正,结果显示,与对照组比较,ABCG1低表达巨噬细胞胞内氧化固醇沉积增加[7-KC:(6.61±1.28)μg/mg比(2.13±0.08)μg/mg,P<0.05;7β-OHC:(1.61±0.51)μg/mg比(0.65±0.20)μg/mg,P<0.05].结论 巨噬细胞ABCG1低表达可抑制氧化固醇7β-OHC及7-KC外流,增加胞内氧化固醇沉积.

Background: Cigarette smoking disturbs plasma lipid level and lipoprotein metabolism; however, the effects of smoking on the functional state of high density lipoprotein (HDL) are still not clear. This study aimed to determine the antioxidant and antichemotactic properties of HDL and HDL-mediated cholesterol efflux in healthy subjects after cigarette smoking. Materials and Methods: Healthy male subjects, including nonsmokers (n = 16) and chronic smokers (n = 8), were enrolled. After smoking 8 cigarettes within 2 hours, plasma HDL was isolated and tested. Copper-induced low density lipoprotein (LDL) oxidation was used to determine the antioxidant ability of HDL. The concentration of serum amyloid A was measured by Enzyme Linked Immunosorbent Assay. Chemotaxis was detected by transwell assay. HDL-mediated cholesterol efflux was measured using fluorescent cholesterol analog. Results: LDL baseline oxidation state was higher in chronic smokers than that in nonsmokers. Meanwhile, HDL-induced cholesterol efflux in macrophages in chronic smokers was significantly enhanced compared with that in nonsmokers. After acute smoking, both the antioxidant and antichemotactic ability of HDL declined in nonsmokers. However, in healthy chronic smokers, the effect of HDL on the susceptibility of LDL to oxidation was compensatorily enhanced. Nevertheless, their bodies were still in a higher oxidation state. Also, acute smoking did not affect HDL-mediated cholesterol efflux significantly in both nonsmokers and chronic smokers. Conclusions Our data suggest that acute smoking attenuates the antioxidant and antichemotactic abilities of HDL in nonsmokers. Chronic smokers are in a higher oxidative state, although the antioxidant function of their HDL is compensatorily enhanced.

Aims: The previous studies on ABCG1 using genetically modified mice showed inconsistent results on atherosclerosis. The aim of this study was to determine whether accurate target knockout of ABCG1 would result in transcriptional changes of other atherosclerosis-related genes. Methods: ABCG1 knockout mouse model was obtained by precise gene targeting without affecting non-target DNA sequences in C57BL/6 background. The wildtype C57BL/6 mice were regarded as control group. 12-week-old male mice were used in current study. We performed whole transcriptome analysis on the peripheral blood mononuclear cells obtained from ABCG1 knockout mice (n = 3) and their wildtype controls (n = 3) by RNA-seq. Results: Compared with wildtype group, 605 genes were modified at the time of ABCG1 knockout and expressed differentially in knockout group, including 306 up-regulated genes and 299 down-regulated genes. 54 genes were associated with metabolism regulation, of which 13 were related to lipid metabolism. We also found some other modified genes in knockout mice involved in cell adhesion, leukocyte transendothelial migration and apoptosis, which might also play roles in the process of atherosclerosis. 7 significantly enriched GO terms and 19 significantly enriched KEGG pathways were identified, involving fatty acid biosynthesis, immune response and intracellular signal transduction. Conclusions: ABCG1 knockout mice exhibited an altered expression of multiple genes related to many aspects of atherosclerosis, which might affect the further studies to insight into the effect of ABCG1 on atherosclerosis with this animal model.

Introduction: ATP-binding cassette transporter G1(ABCG1)is a transmembrane protein mediated efflux of cellular cholesterol to HDL, which plays a vital part in macrophage lipid homeostasis, but the role of ABCG1 in atherosclerosis still remains controversial. Our previous study demonstrated that human ABCG1 -367 G>A polymorphism in promoter region, which leads to lower ABCG1 protein expression, was associated with a reduced risk of CAD. However, the mechanism behind is not fully understood. Hypothesis: Oxysterols, the oxidized derivatives of cholesterol, whose efflux are mediated by ABCG1, exert cytotoxic effect on macrophage and accelerate cell apoptosis. Low ABCG1 expression induces excess oxysterols accumulation in macrophage, which augments the inflammatory response and accelerates apoptosis. Since defective apoptotic macrophage clearance in late lesions promotes atherosclerosis progression, we hypothesize that macrophage with lower ABCG1 expression may remove apoptotic cells more efficiently, thereby generating an antiatherogenic effect. Methods: To investigate the effect of ABCG1 expression on macrophage, we established ABCG1 knockdown cell line by means of lentivirus mediated shRNA. Apoptosis was determined by flow cytometry (FCM). Fluorescence labeled apoptotic model was generated and macrophage phagocytosis was evaluated. The inflammatory factors level and phagocytosis associated gene expression were measured by ELISA and realtime PCR. Results: Compared with control group, macrophage with lower ABCG1 expression had a higher secretion level of Inflammatory factors, such as TNF-α and IL-1β, showing an increased expression of classic inflammatory M1 phenotype markers,such as CD86 and TGF-β, and a down-regulation of CD163,Arg and IL-6 expression, which are markers of anti-inflammatory M2 phenotype macrophage. The apoptosis was significantly accelerated and the phagocytic clearance was enhanced after ABCG1 expression decreased, which means apoptotic cells could be removed more quickly. Both CD36 and Mertk showed a higher mRNA level in ABCG1 knockdown group. Conclusions: Decreased expression of macrophage ABCG1 may exert antiatherogenic function through accelerating apoptosis and enhancing phagocytosis.