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A reliable and reproducible assay for determining the effect of natural product on macrophages lipid uptake and cholesterol efflux: A case study of maslinic acid

Bee Kee Ooi, Nafees Ahemad, Wei Hsum Yap Abstract - 141 PDF - 96


Macrophage foam cell formation represents a key feature that contributes to the development of atherosclerotic lesions. Assessment of cardioprotective natural compounds targeting macrophage foam cell formation processes including lipid uptake and cholesterol efflux could lead to the identification of potential lead compounds for development into novel anti-atherosclerotic drugs. In this case study, maslinic acid, a natural product was used to study the effect on lipid uptake and cholesterol efflux in THP-1-derived macrophages.  Oil red O (ORO) staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled oxidized low-density lipoprotein (Dil-labeled oxLDL) uptake assays were performed to determine lipid uptake by macrophages while cholesterol efflux was assessed using 3-hexanoyl-NBD labeled cholesterol. ORO-stained images were further analyzed using ImageJ analysis software to determine intracellular lipid droplets accumulation and flow cytometric analysis of mean fluorescence intensity were obtained to quantify Dil-labeled oxLDL uptake by macrophages. Meanwhile, 3-hexanoyl-NBD labeled cholesterol uptake and efflux from THP-1-derived macrophages were characterized. The fluorescence intensity values obtained from the medium and cell lysates were then converted into percentage of cholesterol efflux. The results have shown that incubation with maslinic acid suppressed oxLDL-induced macrophage foam cell formation which may be contributed from its effect in reducing lipid uptake and enhancing cholesterol efflux. In conclusion, the optimized ORO staining, Dil-labeled oxLDL uptake, and fluorescent-labeled cholesterol efflux assays provide reproducible and reliable results for assessment of foam cells formation.

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Levitan I, Volkov S & Subbaiah PV. Oxidized ldl: Diversity, patterns of recognition, and pathophysiology. Antioxid Redox Signal 2010; 13(1): 39-75.

Maiolino G, Rossitto G, Caielli P, et al. The role of oxidized low-density lipoproteins in atherosclerosis: The myths and the facts. Mediators Inflamm 2013; 2013: 13.

Nagy L, Tontonoz P, Alvarez JGA et al. Oxidized ldl regulates macrophage gene expression through ligand activation of PPARγ. Cell 1998; 93(2): 229-240.

Moore KJ & Freeman MW. Scavenger receptors in atherosclerosis. Arterioscler Thromb Vasc Biol 2006; 26(8): 1702-1711.

Stephen SL, Freestone K, Dunn S, et al. Scavenger receptors and their potential as therapeutic targets in the treatment of cardiovascular disease. Int J Hypertens 2010; 2010.

Ooi BK, Goh BH & Yap WH. Oxidative stress in cardiovascular diseases: Involvement of Nrf2 antioxidant redox signaling in macrophage foam cells formation. Int J Mol Sci 2017; 18(11): 2336.

Ghosh S. Macrophage cholesterol homeostasis and metabolic diseases: Critical role of cholesteryl ester mobilization. Expert Rev Cardiovasc Ther 2011; 9(3): 329-340.

Chapman MJ, Laplaud PM, Luc G, et al. Further resolution of the low density lipoprotein spectrum in normal human plasma: Physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation. J Lipid Res 1988; 29(4): 442-458.

Murtola T, Vuorela TA, Hyvönen MT, et al. Low density lipoprotein: structure, dynamics, and interactions of apoB-100 with lipids. Soft Matter 2011; 7(18): 8135-8141.

Griffin BA, Caslake MJ, Yip B, et al. Rapid isolation of low density lipoprotein (ldl) subfractions from plasma by density gradient ultracentrifugation. Atherosclerosis 1990; 83(1): 59-67.

Vieira OV, Laranjinha JA, Madeira VM, et al. Rapid isolation of low density lipoproteins in a concentrated fraction free from water-soluble plasma antioxidants. J Lipid Res 1996; 37(12): 2715-2721.

Dong J, Guo H, Yang R, et al. Serum ldl- and hdl-cholesterol determined by ultracentrifugation and HPLC. J Lipid Res 2011; 52(2): 383-388.

Xu S, Huang Y, Xie Y, et al. Evaluation of foam cell formation in cultured macrophages: An improved method with oil red o staining and diI-oxLDL uptake. Cytotechnology 2010; 62(5): 473-481.

Zhou YD, Cao XQ, Liu ZH, et al. Rapamycin inhibits oxidized low density lipoprotein uptake in human umbilical vein endothelial cells via mTOR/NF-κB/LOX-1 pathway. PloS one 2016; 11(1): e0146777.

Song W, Wang W, Wang Y, et al. Characterization of fluorescent NBD-cholesterol efflux in THP-1-derived macrophages. Mol Med Rep 2015; 12(4): 5989-5996.

Song YS, Lee SH, Park BH, et al. Cholesterol efflux monitoring in macrophage form cells by using fluorescence lifetime imaging. SPIE 2015.

Gonzlez-Reyes S, Guzmn-Beltrn S, Medina-Campos ON et al. Curcumin pretreatment induces Nrf2 and an antioxidant response and prevents hemin-induced toxicity in primary cultures of cerebellar granule neurons of rats. Oxid Med Cell Longev 2013; 2013: 14.

Yang D, Xiao CX, Su ZH, et al. (-)-7(S)-hydroxymatairesinol protects against tumor necrosis factor-α-mediated inflammation response in endothelial cells by blocking the MAPK/NF-κB and activating Nrf2/HO-1. Phytomedicine 2017; 32: 15-23.

Choi ES, Yoon JJ, Han BH, et al. Ligustilide attenuates vascular inflammation and activates Nrf2/HO-1 induction and, NO synthesis in HUVECs. Phytomedicine 2018; 38: 12-23.

Huang L, Guan T, Qian Y, et al. Anti-inflammatory effects of maslinic acid, a natural triterpene, in cultured cortical astrocytes via suppression of nuclear factor-kappa B. Eur J Pharmacol 2011; 672(1): 169-174.

Yap WH, Khoo KS, Ho ASH, et al. Maslinic acid induces HO-1 and NOQ1 expression via activation of Nrf2 transcription factor. Biomed Prev Nutr 2012; 2(1): 51-58.

Radding CM & Steinberg D. Studies on the synthesis and secretion of serum lipoproteins by rat liver slices. J Clin Invest 1960; 39(10) 1560-1569.

Yap WH, Phang SW, Ahmed N, et al. Differential effects of sPLA2-GV and GX on cellular proliferation and lipid accumulation in HT29 colon cancer cells. Mol Cell Biochem 2018; 447(1): 93-101.

Rudel LL, Greene DG & Shah R. Separation and characterization of plasma lipoproteins of rhesus monkeys (Macaca mulatta). J Lipid Res 1977; 18(6): 734-744.

Cox R & García-Palmieri M. Cholesterol, triglycerides, and associated lipoproteins. in Walker, H, Hall, W & Hurst, J (eds.), Clinical methods: the history, physical, and laboratory examinations. 1990; 3rd, Boston: pp. 153-160.

Shi H, Mao X, Zhong Y, et al. Lanatoside C promotes foam cell formation and atherosclerosis. Sci Rep 2016; 6: 20154.

Hossain E, Ota A, Karnan S, et al. Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages. Mol Cell Biochem 2015; 400(1): 29-40.

Luo Y, Duan H, Qian Y, et al. Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis. Cell Res 2107; 27: 352-372.

Chen CY, Shyue SK, Ching LC, et al. Wogonin promotes cholesterol efflux by increasing protein phosphatase 2B-dependent dephosphorylation at ATP-binding cassette transporter-A1 in macrophages. J Nutr Biochem 2011; 22(11): 1015-1021.

Meurs I, Out R, Van Berkel TJ, et al. Role of the ABC transporters ABCA1 and ABCG1 in foam cell formation and atherosclerosis. Future Lipidol 2008; 3(6): 675-687.

Yu XH, Fu YC, Zhang DW, et al. Foam cells in atherosclerosis. Clin Chim Acta 2013; 44: 245-252.

Schumacher T & Benndorf RA. ABC transport proteins in cardiovascular disease-a brief summary. Molecules 2017; 22(4): 589.


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