Nutr Res Pract.  2024 Apr;18(2):194-209. 10.4162/nrp.2024.18.2.194.

Ellagic acid, a functional food component, ameliorates functionality of reverse cholesterol transport in murine model of atherosclerosis

• Park SH
• Kang MK
• Kim DY
• Lim SS
• Kang IJ
• Kang YH

• KMID: 2558498
• DOI: http://doi.org/10.4162/nrp.2024.18.2.194

Abstract

BACKGROUND/OBJECTIVES
High levels of plasma low-density lipoprotein (LDL) cholesterol are an important determinant of atherosclerotic lesion formation. The disruption of cholesterol efflux or reverse cholesterol transport (RCT) in peripheral tissues and macrophages may promote atherogenesis. The aim of the current study was to examine whether bioactive ellagic acid, a functional food component, improved RCT functionality and high-density lipoprotein (HDL) function in diet-induced atherogenesis of apolipoproteins E (apoE) knockout (KO) mice.
MATERIALS/METHODS
Wild type mice and apoE KO mice were fed a high-cholesterol Paigen diet for 10 weeks to induce hypercholesterolemia and atherosclerosis, and concomitantly received 10 mg/kg ellagic acid via gavage.
RESULTS
Supplying ellagic acid enhanced induction of apoE and ATP-binding cassette (ABC) transporter G1 in oxidized LDL-exposed macrophages, facilitating cholesterol efflux associated with RCT. Oral administration of ellagic acid to apoE KO mice fed on Paigen diet improved hypercholesterolemia with reduced atherogenic index. This compound enhanced the expression of ABC transporters in peritoneal macrophages isolated from apoE KO mice fed on Paigen diet, indicating increased cholesterol efflux. Plasma levels of cholesterol ester transport protein and phospholipid transport protein involved in RCT were elevated in mice lack of apoE gene, which was substantially reduced by supplementing ellagic acid to Paigen diet-fed mice. In addition, ellagic acid attenuated hepatic lipid accumulation in apoE KO mice, evidenced by staining of hematoxylin and eosin and oil red O. Furthermore, the supplementation of 10 mg/kg ellagic acid favorably influenced the transcriptional levels of hepatic LDL receptor and scavenger receptor-B1 in Paigen diet-fed apoE KO mice.
CONCLUSION
Ellagic acid may be an athero-protective dietary compound encumbering diet-induced atherogenesis though improving the RCT functionality.

Keyword

Apolipoproteins E; cholesterol; ellagic acid; high-density lipoprotein; reverse cholesterol transport

Figure

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  Fig. 1 Chemical structure of ellagic acid (A), HDL formation (B), and up-regulation of oxidized LDL-induced cellular ABCG1 expression and apoE secretion (C) by ellagic acid. J774A1 macrophages were treated with 1–10 μM ellagic acid for 18 h-stimulation with 50 μg/mL oxidized LDL. HDL formation was measured by using an enzyme-linked immunosorbent assay kit (B). For the measurement of ABCG1 expression and apoE secretion, total cell lysates and culture media were subjected to Western blot analysis with a primary antibody against ABCG1 or apoE (C). β-Actin was used as an internal control. The bar graphs represent quantitative results of blots obtained from a densitometer. Respective values (mean ± standard error of the mean, n = 3) in bar graphs not sharing a small letter are significantly different at P < 0.05.HDL, high-density lipoprotein; LDL, low-density lipoprotein; ABCG1, ATP-binding cassette transporter G1; apoE, apolipoproteins E.

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  Fig. 2 Schematic illustration of animal experimental design/timeline (A) and BW gain (B). WT mice (C57BL/6N) and apoE KO mice were fed with atherogenic Paigen diet for 10 weeks with and without oral administration of 10 mg/kg ellagic acid daily. BW was measured weekly for 10 weeks. Graph was shown the average BW of each group as a time-response of weeks. Statistical differences were shown as asterisks (*). Values (n = 16, each diet group) on line graphs indicate a significant difference at *P < 0.05, compared to WT.BW, body weight; WT, wild type; apoE KO, apolipoproteins E knockout.

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  Fig. 3 Effects of ellagic acid on transporter proteins responsible for peripheral cholesterol efflux. WT mice and apoE KO mice were fed atherogenic Paigen diet for 10 weeks with oral administration of 10 mg/kg ellagic acid daily. After the experimental period for 10 weeks, peritoneal macrophages were isolated from each mouse. Whole cell lysates were subject to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot with a specific antibody against ABCA1, ABCG1, and SR-B1 (A, B). β-Actin was used as an internal control. Respective values (mean ± standard error of the mean, n = 3) in bar graphs not sharing a small letter are significantly different at P < 0.05. Transcriptional levels of ABCA1, ABCG1, and SR-B1 were determined by using semi-quantitative reverse transcription polymerase chain reaction assay (C). GAPDH was used as a housekeeping gene for the co-amplification with ABCA1, ABCG1, and SR-B1.WT, wild type; apoE KO, apolipoproteins E knockout; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; SR-B1, scavenger receptor-B1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 4 Effects of ellagic acid on plasma levels of CETP, PLTP, LCAT, and PON1. WT mice and apoE KO mice were fed atherogenic Paigen diet for 10 weeks with and without oral administration of 10 mg/kg ellagic acid daily. Plasma levels of CETP, PLTP, LCAT, and PON1 were measured by using ELISA kits. All the ELISA procedures were followed according to the manufacturer’s instructions. Means (mean ± standard error of the mean, n = 3) in bar graphs without a common letter differ at P < 0.05.CETP, cholesterol ester transfer protein; PLTP, phospholipid transport protein; LCAT, lecithin-cholesterol acyltransferase; PON1, paraoxonase-1; WT, wild type; apoE KO, apolipoproteins E knockout; ELISA, enzyme-linked immunosorbent assay.

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  Fig. 5 Effects of ellagic acid on hepatic morphology (A) and lipid accumulation (B). WT mice and apoE KO mice were fed atherogenic Paigen diet for 10 weeks with oral administration of 10 mg/kg ellagic acid daily. Liver tissues were dissected and cut by 6 μm thickness with microtomes. For the hepatic morphology, the tissues were stained with H&E (A). Hepatic lipid accumulation was confirmed by staining with oil red O (B). Liver tissues were observed by microscopy with 200× magnification (scale bar = 50 μm).WT, wild type; apoE KO, apolipoproteins E knockout; H&E, hematoxylin and eosin.

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  Fig. 6 Effects of ellagic acid on expression of hepatic lipoprotein receptors. WT mice and apoE KO mice were fed atherogenic Paigen diet for 10 weeks with and without oral administration of 10 mg/kg ellagic acid daily. After the experimental period for 10 weeks, proteins and total mRNA of liver tissues were isolated. Western blot analysis showing protein levels of lectin-like oxidized LOX-1 and SR-B1 (A, B). Liver tissue extracts were subject to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot with a specific antibody against LOX-1 and SR-B1. β-Actin was used as an internal control. The bar graphs (mean ± standard error of the mean, n = 3) represent quantitative results of blots obtained from a densitometer. Transcription of SR-B1 and LDLR was determined by using real-time reverse transcription polymerase chain reaction with each primer. GAPDH was used as a housekeeping gene for the co-amplification with SR-B1 and LDLR. Respective values in bar graphs not sharing a small letter are significantly different at P < 0.05.WT, wild type; apoE KO, apolipoproteins E knockout; LOX-1, low-density lipoprotein receptor-1; SR-B1, scavenger receptor-B1; LDLR, low-density lipoprotein receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 7 Effects of ellagic acid on production and binding of apoA1. WT mice and apoE KO mice were fed atherogenic Paigen diet for 10 weeks with and without oral administration of 10 mg/kg ellagic acid daily. After the experimental period for 10 weeks, blood was collected. Proteins and total mRNA of liver tissues were collected. Plasma and liver tissue extracts were subject to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot with a specific antibody against apoA1. β-Actin was used as an internal control. The bar graphs (mean ± standard error of the mean, n = 3) represent quantitative results of blots obtained from a densitometer. Transcription of apoA1 and AIBP was determined by using real-time reverse transcription polymerase chain reaction with each primer. GAPDH was used as a housekeeping gene for the co-amplification with apoA1 and AIBP. Respective values in bar graphs not sharing a small letter are significantly different at P < 0.05.ApoA1, apolipoprotein A1; WT, wild type; apoE KO, apolipoproteins E knockout; AIBP, apoA1 binding protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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  Fig. 8 The effects of ellagic acid on hepatic induction of cholesterol transporters responsible for cholesterol efflux. WT mice and apoE KO mice were fed atherogenic Paigen diet for 10 weeks with and without oral administration of 10 mg/kg ellagic acid daily. After the experimental period for 10 weeks, proteins and total mRNA of liver tissues were isolated. Protein expressions of ABCA1 and ABCG1 were measured by Western blot analysis (A, B). Liver tissue extracts were subject to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot with a specific antibody against ABCA1 and ABCG1. β-Actin was used as an internal control. The bar graphs (mean ± standard error of the mean, n = 3) represent quantitative results of blots obtained from a densitometer. Transcription of ABCA1, ABCG1, and ABCG8 was determined by using real-time reverse transcription polymerase chain reaction with each primer (C-E). GAPDH was used as a housekeeping gene for the co-amplification with ABCA1, ABCG1, and ABCG8. Respective values in bar graphs not sharing a small letter are significantly different at P < 0.05.WT, wild type; apoE KO, apolipoproteins E knockout; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; ABCG8, ATP-binding cassette transporter G8; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

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