Inhibitory effects of extracellular cholesterol and lipopolysaccharide on cellular cholesterol efflus

doi:10.3969/j.issn.2095-4344.2016.07.012

Abstract

Abstract:

BACKGROUND: Cholesterol is closely linked to the occurrence and progression of atherosclerosis. Current approaches to study cellular cholesterol dynamics have their own limitations.
OBJECTIVE: To measure the cholesterol efflux rate of RAW 264.7 mouse macrophages by BODIPY-Cholesterol labeling and to explore the effects of extracellular cholesterol and lipopolysaccharide on the cholesterol efflux rate.
METHODS: RAW 264.7 cells were cultured in vitro with DMEM containing 10% fetal bovine serum, and labeled with BODIPY-Cholesterol for 1, 2, 4, 8 hours. Then, the cells were rinsed with serum-free DMEM and inoculated for 6, 12, 24, 48, 96 hours to optimize the labeling time and incubation time. We measured and compared the cholesterol efflux rates after cultured cells were treated with cholesterol, lipopolysaccharide, human sera with high cholesterol or human sera with normal cholesterol.
RESULTS AND CONCLUSION: The best labeling time for BODIPY-Cholesterol was 2-8 hours. Cholesterol efflux rates were gradually decreased after the cells that were labeled for 2 hours were incubated with increasing concentrations of cholesterol (0.1, 0.5, 2.5 mmol/L, P < 0.01). Treating cells with lipopolysaccharide also decreased the cholesterol efflux rate (P < 0.05). Furthermore, the cholesterol efflux rate was decreased after cells were treated with human sera with high cholesterol (P < 0.05). These findings indicate that BODIPY-Cholesterol can be used to measure cellular cholesterol efflux rate and to study the effects of extracellular cholesterol and lipopolysaccharide on the cholesterol efflux rate.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Liu Jie, Zheng Yun-mei, Tian Zhi-hui, Chang Guang-ming, Li Hai-dong. Inhibitory effects of extracellular cholesterol and lipopolysaccharide on cellular cholesterol efflus[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(7): 1000-1005.

Figures/Tables 4

References

[1] Mao M, Lei H, Liu Q,et al.Effects of miR-33a-5P on ABCA1/G1-mediated cholesterol efflux under inflammatory stress in THP-1 macrophages. PLoS One.2014;9(10): e109722.
[2] Pisetsky DS, Spencer DM. Effects of progesterone and estradiol sex hormones on the release of microparticles by RAW 264.7 macrophages stimulated by Poly(I:C). Clin Vaccine Immunol.2011;18(9): 1420-1426.
[3] 王智.他汀与动脉粥样硬化斑块逆转[J].临床荟萃.2013, 28(7): 831-834.
[4] Iizuka M, Ayaori M, Uto-Kondo H,et al.Astaxanthin enhances ATP-binding cassette transporter A1/G1 expressions and cholesterol efflux from macrophages. J Nutr Sci Vitaminol (Tokyo).2012;58(2): 96-104.
[5] Yvan-Charvet L, Wang N, Tall A R. Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses. Arterioscler Thromb Vasc Biol.2010; 30(2): 139-143.
[6] Li Z,Mintzer E,Bittman R.First synthesis of free cholesterol- BODIPY conjugates. J Org Chem. 2006; 71(4):1718-1721.
[7] 黄柳倩. BODIPY基荧光传感器对半胱氨酸的检测[J]. 杭州师范大学学报:自然科学版, 2014, 13(5): 499-502.
[8] Gao S, Wang L, Liu W, et al.The synergistic effect of homocysteine and lipopolysaccharide on the differentiation and conversion of raw264.7 macrophages. J Inflamm (Lond).2014;11:13.
[9] Cho W, Kang JL, Park YM. Corticotropin-Releasing Hormone (CRH) Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1). PLoS One.2015; 10(6): e0130587.
[10] 洪雪华,生瑜. BODIPY类荧光染料的研究进展[J].广州化工, 2012, 40(7): 65-68.
[11] Solanko LM, Honigmann A, Midtiby HS, et al. Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure. Biophys J. 2013;105(9): 2082-2092.

[12] Ariola FS, Li ZG, Cornejo C, et al. Membrane Fluidity and Lipid Order in Ternary Giant Unilamellar Vesicles Using a New Bodipy-Cholesterol Derivative. Biophys J. 2009;96(7): 2696-2708.
[13] Holtta-Vuori M, Uronen RL, Repakova J, et al. BODIPY-Cholesterol: A New Tool to Visualize Sterol Trafficking in Living Cells and Organisms. Traffic. 2008;9(11): 1839-1849.
[14] Wustner D, Solanko L, Sokol E, et al. Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY- cholesterol. Chemistry and Physics of Lipids.2011; 164(3):221-235.
[15] 宋玮.荧光标记胆固醇用于测定人单核巨噬细胞脂质外流[J].中国动脉硬化杂志, 2012, 20(8):749-754.
[16] Zhang J,Cai S,Peterson BR,et al.Development of a cell-based, high-throughput screening assay for cholesterol efflux using a fluorescent mimic of cholesterol. Assay Drug Dev Technol. 2011;9(2): 136-146.
[17] Kanerva K, Uronen RL, Blom T, et al. LDL cholesterol recycles to the plasma membrane via a Rab8a- Myosin5b-actin-dependent membrane transport route. Dev Cell. 2013;27(3): 249-262.
[18] 张晔.细胞骨架改构在LPS作用后VE-Cad胞吞途径转化中的作用[J].中国普外基础与临床杂志, 2015, 22(3): 269-273.
[19] 贝克曼库尔特商贸(中国)有限公司对尿酸检测试剂盒、肌酐测定试剂盒、乳酸测定试剂盒、脂肪酶测定试剂盒、胆固醇测定试剂盒主动召回[J].中国医疗设备, 2015, 30(3):120.
[20] Ladeiras-Lopes R, Agewall S, Tawakol A, et al. Atherosclerosis: Recent trials, new targets and future directions. Int J Cardiol.2015;192:72-81.
[21] 吴先杰.动脉粥样硬化发生机制研究现状及思路[J].中华实用诊断与治疗杂志, 2012, 26(7): 629-631.
[22] Meiler S, Baumer Y, Toulmin E, et al. MicroRNA 302a Is a Novel Modulator of Cholesterol Homeostasis and Atherosclerosis. Arterioscl Throm Vas.2015; 35(2): 323-331.
[23] Li X H, Li Y, Cheng ZY, et al.The Effects of Phellinus linteus Polysaccharide Extracts on Cholesterol Efflux in Oxidized Low-Density Lipoprotein-Loaded THP-1 Macrophages. J Invest Med.2015;63(5): 752-757.