Anti-Atherogenic Effect of Citrus Flavonoids, Naringin and Naringenin, Associated with Hepatic ACAT and Aortic VCAM-1 and MCP-1 in High Cholesterol-Fed Rabbits

Biochemical and Biophysical Research Communications 284, 681– 688 (2001) doi:10.1006/bbrc.2001.5001, available online at http://www.idealibrary.com on

Anti-Atherogenic Effect of Citrus Flavonoids, Naringin and Naringenin, Associated with Hepatic ACAT and Aortic VCAM-1 and MCP-1 in High Cholesterol-Fed Rabbits Chul-Ho Lee,* ,1 Tae-Sook Jeong,† ,1 Yang-Kyu Choi,* Byung-Hwa Hyun,* ,‡ Goo-Taeg Oh,* Eun-Hee Kim,* Ju-Ryoung Kim,† Jang-Il Han,† and Song-Hae Bok† ,‡ ,2 *Genetic Resources Center and †Cardiovascular Research Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong P.O. Box 115, Taejon 305-600, Korea; and ‡BioNutrigen Company, Ltd., #52 Eoun, Yusong, Taejon 305-333, Korea

Received May 2, 2001

The anti-atherogenic effects of the citrus flavonoids, naringin and naringenin, were evaluated in high cholesterol-fed rabbits. At 3 months of age, 30 male New Zealand White (NZW) rabbits were divided into three groups (n ⴝ 10 per group). The rabbits were fed a 1% cholesterol diet alone (control group) or a diet supplemented with either 0.1% naringin or 0.05% naringenin for 8 weeks. The plasma lipoprotein levels, total cholesterol, triglyceride, and high-density lipoprotein showed no significant differences in the control and experimental groups. Hepatic acyl-CoA: cholesterol acyltransferase (ACAT) activity was slightly low in naringin (5.0%)- and naringenin (15.0%)fed rabbits, compared to control group. The aortic fatty streak areas were significantly lower in both the naringin (19.2 ⴞ 5.6%)- and naringenin (18.1 ⴞ 6.5%)supplemented groups than in the control group (60.4 ⴞ 14.0%). The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), by semiquantitative RT-PCR analysis of the thoracic aorta, were significantly lower in the flavonoids supplemented groups than in the control group. These results suggest that the anti-atherogenic effect of the citrus flavonoids, naringin and naringenin, is involved with a decreased hepatic ACAT activity and with the downregulation of VCAM-1 and MCP-1 gene expression. © 2001 Academic Press Key Words: naringin; naringenin; fatty streak; ACAT; MCP-1; VCAM-1.

Atherosclerosis is characterized by intimal lesions by called atheromas or fibrofatty plaques that protrude into the vascular lumen, weaken the underlying media, 1

These authors contributed equally to this work. To whom correspondence should be addressed. Fax: ⫹82-42-8604592. E-mail: [email protected]. 2

and undergo a number of complications (1, 2). Atheromas are focal and sparsely distributed at first, but as the disease advances, they become more numerous, and sometimes cover the entire circumference of severely affected arteries. Consequently, in small arteries, atheromas are themselves occlusive, compromising blood flow to distal organs and causing ischemic injury (3, 4). Moreover, plaques can undergo disruption and precipitate thrombi that further obstruct blood flow (5). In large arteries, plaques are destructive, they encroach on the subjacent media and weaken the affected vessel wall, causing aneurysms or rupture, or favoring thrombosis (6). In addition, extensive atheromas are friable, often yielding emboli in the distal circulation supplied by both the descending and ascending aorta (7). In general, the concentration of plasma cholesterol in the body can be regulated by, the biosynthesis of cholesterol, the removal of cholesterol from the circulation, the absorption of dietary cholesterol, and the excretion of cholesterol via bile and feces. It is well known that two key enzymes, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA:cholesterol acyltransferase (ACAT) play a major role in the regulation of cholesterol metabolism. HMG-CoA reductase is a rate-limiting enzyme in the cholesterol biosynthetic pathway, whereas ACAT is involved in the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A, dietary cholesterol absorption, hepatic very low-density lipoprotein (VLDL)cholesterol secretion and the foam cell development in atherosclerosis (8). Accordingly, HMG-CoA reductase and ACAT has been a pharmaceutical target for developing cholesterol-lowering and/or antiatherosclerosis drugs (9, 10). Monocyte adhesion to the endothelium via adhesion molecules is one of the earliest events in atherogenesis. It has been suggested that vascular cell adhesion

681

0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1

PCR Primers and Conditions Used to Determine Gene Expression by RT-PCR Primers Target genes

Sense

Antisense

Size (bp)

AT (°C)

PCR cycles

VCAM-1 MCP-1 GAPDH

5⬘-GAACACTCTTACCTGTGCACAGC-3⬘ 5⬘-GTCTCTGCAACGCTTCTGTGCC-3⬘ 5⬘-GCGCCTGGTCACCAGGGCTGCTT-3⬘

5⬘-CCATCCTCATAGCAATTAAGGTGAG-3⬘ 5⬘-AGTCGTGTGTTCTTGGGTTGTGG-3⬘ 5⬘-TGCCGAAGTGGTCGTGGATGACCT-3⬘

567 327 465

63 63 63

30 30 27

Note. VCAM-1, vascular cell adhesion molecule 1; MCP-1, monocyte chemotactic protein 1; GAPDH, glyceraldehydes phosphate dehydrogenase; AT, annealing temperature.

molecule-1 (VCAM-1) plays a very important role in the recruitment of monocytes in atherosclerosis (11, 12). Thus the level of VCAM-1 may serve as a surrogate marker that reflects the cellular expression of VCAM-1. Oxidized LDL also induces local vascular cells to produce monocyte chemotactic protein-1 (MCP1), which causes monocyte recruitment and promotes the release of lipids and lysosomal enzymes into the extracellular space, thereby enhancing the progression of the atherosclerotic lesion (13). It has been demonstrated that MCP-1 expression occurs in the arterial wall in response to hypercholesterolemia in rabbits (14). Flavonoids, including apigenin, PD098063 (2-(3amino-phenyl)-8-methoxy-chromene-4-one), and quercetin, inhibit VCAM-1 or MCP-1 expression (15–17). A well-known antioxidant, probucol reduces the level of basal VCAM-1 expression in LDL receptor-deficient rabbits during early atherogenesis (18). Therefore, one possible mechanism for ameliorating atherosclerosis by naringin and naringenin is the down-regulation of proatherogenic molecules such as MCP-1 and VCAM-1. Some flavonoid compounds may possess significant antihepatotoxic, antiallergic, anti-inflammatory, antiosteoporotic, and even antitumor activities (19). Citrus fruits contain various flavonoids, and among these naturally occurring citrus flavonoids, naringenin, and naringin have been pharmacologically evaluated as a potential anticancer agent (20) and a hypolipidemic agent (21). Accordingly, this study was performed to examine

the effects of these citrus flavonoids on atherogenesis in rabbits on high cholesterol-fed diets. The present study was designed to examine the antiatherogenic effects of 0.1% (wt/wt diet) naringin and equivalent amount, 0.05% (wt/wt diet) of its aglycon, naringenin in high cholesterol-fed rabbits. We have

TABLE 2

Effect of Naringin and Naringenin Supplementation on the Changes of Body Weight in High Cholesterol-Fed Rabbits Body weight (kg) Groups

N

0 week

4 week

8 week

Control Naringin (0.1%, wt/wt diet) Naringenin (0.05%, wt/wt diet)

10 10

2.41 ⫾ 0.27 2.39 ⫾ 0.30

2.80 ⫾ 0.23 2.82 ⫾ 0.31

3.02 ⫾ 0.35 3.00 ⫾ 0.31

10

2.27 ⫾ 0.18

2.78 ⫾ 0.21

2.93 ⫾ 0.17

Note. All values are expressed as mean ⫾ SD.

FIG. 1. Effects of naringin and naringenin on the aortic fatty streak formations in rabbit model fed a high cholesterol diet for 8 weeks. (A) Gross photographs of oil red-O stained aorta between the second and seventh intercostals arteries (third portion). (B) A graph of artherosclerotic lesion size expressed as a percentage of the oil red-O positive area/measured internal surface in each group. Bars represent standard deviations. * are significantly different (P ⬍ 0.001) from control group.

682

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. Representative microscopic photographs of descending aorta (second portion) stained with hematoxylin and eosin (A, D, and G), and immunostained with macrophages (B, E, and H) and smooth muscle cell (C, F, and I) antibodies. Naringin (D, E, and F) and naringenin (G, H, and I) supplementations reduced the intimal thickness and decreased the amount of immunostained cells, compared with control group (A, B, and C). Bars represent 93 ␮m.

now examined the effects of naringin and naringenin on levels of plasma lipids, hepatic ACAT play a major role in the regulation of cholesterol metabolism, regression of atherosclerotic lesions in the aorta, and expression of endothelial VCAM-1 and MCP-1 gene. MATERIALS AND METHODS Animals and diets. New Zealand White (NZW) rabbits were purchased from Daehan Laboratory Animal Co. (Eum-sung, Korea). Thirty male NZW rabbits weighing between 2.2 and 2.5 kg at the age of 3 months were used in the experiment. The rabbits were divided into three groups (n ⫽ 10 per group), which were supplemented with a 1% cholesterol diet (RC4, Oriental Yeast Co. Ltd., Tokyo, Japan), or a 1% cholesterol diet containing either 0.1% naringin or 0.05% naringenin (Sigma Chemical Co., MO) for 8 weeks. All rabbits were individually caged and maintained in a controlled facility at 20 ⫾ 2°C, relative humidity (55 ⫾ 5%) and a strict 12-h light/dark cycle. Plasma lipids determination. Blood samples (3 ml), with ethylenediamine-tetraacetic acid (EDTA) as an anticoagulant, were obtained from the marginal vein of the ear, and centrifuged at 8,000g for 10 min. Collected plasma was analyzed in an automatic blood chemical analyzer (CIBA Corning, OH) and the plasma concentrations of total cholesterol, HDL-cholesterol and triglyceride obtained. Evaluation of aortic fatty streak lesions. Following blood collection, all rabbits were anesthetized with thiopental sodium (Choong-

wae Pharma Co., Seoul, Korea) and sacrificed by exsanguinations from the femoral artery. Immediately after opening the thoracic cavity, the aorta was excised, and adventitial tissue grossly adhering to the aorta removed. The aorta was then dissected longitudinally and separated into three portions. The first portion, a one-centimeter segment proximal to the outlet of the first intercostal artery was snap frozen in liquid nitrogen until further processing. The second portion, a segment between the first and the second intercostal artery, was routinely processed, paraffin-embedded and used for histological examination. The third portion, a segment between the second and the seventh intercostals arteries, was fixed in 10% neutral buffered formalin for 1 day. The aorta was then placed in absolute propylene glycol for 2 min and stained with oil red O for 4 h. After washing, the extent of the oil red O-positive area between the second and the seventh intercostal arteries was measured and expressed as a percentage of the internal surface using a computerassisted morphometry system (Image Pro Plus, MD). Measurement of hepatic ACAT activity. At necropsy, the liver tissues were obtained, and microsomes were prepared according to Erickson et al. (22) with a slight modification. The liver tissue was homogenized in a fourfold excess volume of a microsome buffer A (0.25 M sucrose, 1 mM EDTA, 10 mM Tris–HCl, pH 7.4). The homogenate was centrifuged at 14,000g for 15 min. The supernatant was then centrifuged at 100,000g for 1 h in the ultracentrifuge at 4°C. The pellet obtained was disrupted in a microsome buffer B (0.25 M sucrose, 10 mM Tris–HCl, pH 7.4) and recentrifuged at 100,000g for 1 h. The resulting microsomal pellets were dissolved in a micro-

683

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 3

Effects of Naringin and Naringenin Supplementation on the Plasma Lipids in High Cholesterol-Fed Rabbits Plasma lipids (mg/dl) Total cholesterol

HDL-cholesterol

Triglyceride

Groups

N

0 week

8 week

0 week

8 week

0 week

8 week

Control Naringin (0.1%, wt/wt diet) Naringenin (0.05%, wt/wt diet)

10 10 10

53 ⫾ 11 52 ⫾ 5 57 ⫾ 12

1696 ⫾ 383 1682 ⫾ 151 1676 ⫾ 297

23 ⫾ 6 21 ⫾ 7 24 ⫾ 7

78 ⫾ 8 81 ⫾ 26 89 ⫾ 22

66 ⫾ 32 69 ⫾ 27 63 ⫾ 23

114 ⫾ 60 123 ⫾ 24 107 ⫾ 23

Note. All values are expressed as mean ⫾ SD (mg/dl). No significant differences were observed among groups.

some buffer B and assayed for total protein by the method of Bradford (23). The ACAT activity was determined using freshly prepared hepatic microsomes according to the method developed by Brecher and Chan (24) with modification. The reaction mixture, containing 4 ␮l of microsomes (8 –10 mg/ml protein), 20 ␮l of 0.5 M potassiumphosphate buffer (pH 7.4, 10 mM dithiothreitol), 15 ␮l of bovine serum albumin (fatty acid free, 40 mg/ml), 2 ␮l of cholesterol in acetone (20 ␮g/ml, added last), and distilled water (up to 92 ␮l), was preincubated at 37°C for 15 min. The reaction was then initiated by adding 8 ␮l of [1- 14C]-oleoyl CoA solution (0.05 ␮Ci, final conc. 10 ␮M, Amersham Pharmacia Biotech Inc., Piscataway, NJ). After 15 min of incubation at 37°C, the reaction was stopped by adding 1.0 ml of an isopropanol-heptane (4:1, v/v) solution. A mixture of 0.6 ml of heptane and 0.4 ml of 0.1 M assay buffer (prepared by diluting 0.5 M assay buffer 1:5 in water) was then added to the terminated reaction mixture. The above solution was mixed and allowed to phase separation under gravity for 2 min. Cholesteryl oleate was recovered in the upper heptane phase. Finally, an aliquot (100 ␮l) of the supernatant was counted with a liquid scintillation counter (1450 Microbeta Trilux, Wallac Oy, Turku, Finland). ACAT activity was expressed as pmoles of cholesteryl oleate synthesized/min/mg microsomal protein. Immnunohistochemistry. The second portion, the paraffinembedded segment between the first and the second intercostal artery, was cut into 6 ␮m sections, and three serial sections were obtained every 240 ␮m. To observe the sections by light microscopy, the first section was stained with hematoxylin and eosin. To identify the intimal macrophages and smooth muscles, the second and third serial sections were immunostained with commercial mouse antibodies (DAKO Co., CA) to rabbit macrophages (1:500 dilution) and human smooth muscle cell actin (1:250 dilution) using an avidin/ biotin/horseradish peroxidase complex system (Novocastra Laboratories Ltd., Newcastle, UK), in accord with the manufacturer’s instructions. After immunostaining had been completed, the extent of the atherosclerotic lesions in each group was evaluated by semiquantitative method. A minimum of 20 sections immunostained with macrophage antibody in each rabbit were examined and the stage of each lesion was graded from 0 to 3 according to the relative content of intimal macrophage/foam cells (0 ⫽ normal intima without any subendothelial monocyte/macrophage; 1 ⫽ isolated monocytes/ macrophages in the subendothelial space; 2 ⫽ single monolayer of monocytes/macrophages underneath the endothelium; 3 ⫽ presence of multiple layers of monocytes/marophages as well as transitional and more advanced lesions). Then, the index of the atherosclerotic lesion in each group was obtained from the sum of the stage numbers divided by the number of sections examined. Determination of VCAM-1 and MCP-1 gene expressions. Total RNA was isolated from the first portion (one centimeter segment proximal to the outlet of the first intercostal artery) using TRIZOL Reagent (Life Technologies, Inc., MD). Equal amounts (250 ng) of

total RNA were reverse transcribed into cDNA in 50 ␮l reaction mixture using a recombinant M-MLV reverse transcriptase (Life Technologies, Inc., MD) and oligo dT 15 as primer. Reverse transcription was carried out for 60 min at 42°C followed by an inactivation step at 94°C for 10 min. PCR was performed on an Gene Cycler (Bio-Rad, Japan) in a total volume of 25 ␮l containing 2.0 ␮l of cDNA template, 0.24 ␮M primer, 100 ␮M dNTP, 2 mM MgCl 2, 100 ␮g/ml BSA, and 0.75 U Taq polymerase (Roche Molecular Biochemicals, Mannheim, Germany) in the PCR buffer provided with the enzyme. The PCR conditions of VCAM-1, MCP-1 and GAPDH genes are shown in Table 1, and all primers for each gene amplification were specifically designed as the same sets used in a previous report (18). VCAM-1 and MCP-1 gene expressions were determined by semiquantitative PCR, using GAPDH as an internal standard. To verify that the GAPDH expression in the aortic segment was constant and not affected by the supplementation of naringin and naringenin, GAPDH expression was determined by competitive PCR using a 168 bp-deleted competitor. All products obtained by PCR reactions were subjected to gel electrophoresis on a 2% agarose gel and stained with ethidium bromide. The intensity of each band was analyzed using gel documentation and analysis system (Viber Lourmat, France). VCAM-1 and MCP-1 expression levels were determined relative to the expression of GAPDH. Statistical analysis. Results were expressed as mean ⫾ SD. Student’s t-test was performed to compare means (the fatty streak lesions, body weights, plasma lipids, VCAM-1 and MCP-1 mRNA levels of animals) in different groups, and a P value of ⬍ 0.05 was considered to be statistically significant.

RESULTS Biochemical and Physiological Measurements In the control and experimental groups, the body weights of all rabbits increased progressively during the experimental period, but there were no significant differences within each group (Table 2). Changes in the plasma lipid levels are shown in Table 3. After 8 weeks, the plasma total cholesterol levels had significantly increased to almost 1700 mg/dl in all groups. The plasma total cholesterol levels of the different groups did not differ significantly during the experimental period. The triglyceride and HDL cholesterol levels of the naringin- or naringenin-supplemented groups were also not significantly different from those of the control group.

684

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Aortic Fatty Streak Lesions The fatty streak lesions of the ascending and descending thoracic aorta in each group were easily identified by staining with oil red O. Broad and fused fatty streak lesions were found in control rabbits supplemented with the 1% cholesterol diet alone, whereas only well-demarcated, 0.5 to 3 mm diameter small plaques were sparsely observed in the naringin- and naringenin-supplemented groups (Fig. 1A). The percentage area of occupied by atherosclerotic lesions on the inner surface between the second and seventh intercostal arteries (the third portion) significantly reduced in the 0.1% naringin (19.2 ⫾ 5.6%)- and the 0.05% naringenin (18.1 ⫾ 6.5%)-supplemented groups compared to control group (60.4 ⫾ 14.0%, P ⬍ 0.001) (Fig. 1B). Histological and Immunohistochemical Findings Histologically, the second portion, the aortic segment between the first and second intercostal arteries was stained with hematoxylin and eosin, and revealed intimal thickening mainly due to an accumulation of foam cells, and an infiltration and proliferation of smooth muscle cells in the intima and a deposition of extracellular matrix substances. The intimal thickening in the naringin (Fig. 2D)- and naringenin (Fig. 2G)-supplemented groups were observed as low levels, compared to the control group (Fig. 2A). By immunohistochemistry using antibodies against macrophage and smooth muscle actin, largely macrophages and partly smooth muscle cells were observed in the atherosclerotic intima of the control group (Figs. 2B and 2C). However, small numbers of positive cells for macrophage antibody and relatively few cells against smooth muscle actin were observed in the naringin (Figs. 2E and 2F)- and naringenin (Figs. 2H and 2I)treated groups. Semiquantitative analysis of the intimal thickening of each group, according to subendothelial macrophages content, showed significantly lower scores in the naringin- and in the naringeninsupplemented group than in the control group (P ⬍ 0.001) (Fig. 3). Hepatic ACAT Activities The hepatic ACAT activities of the naringin- and naringenin- fed groups were slightly lowered 5.0% (247 ⫾ 27 pmol 䡠 min ⫺1 䡠 mg protein ⫺1) and 15.0% (221 ⫾ 16 pmol 䡠 min ⫺1 䡠 mg protein ⫺1), compared to that of the control group (260 ⫾ 30 pmol 䡠 min ⫺1 䡠 mg protein ⫺1, P ⬍ 0.05), respectively. VCAM-1 and MCP-1 Expressions Gene expressions of VCAM-1 and MCP-1 at the first portion (one centimeter segment proximal to the outlet of the first intercostal artery) of the thoracic aorta were

FIG. 3. Effects of naringin and naringenin on the levels of intimal thickening. The index scores were obtained from the semiquantitative method as described in materials and methods. Bars represent standard deviations. * are significantly different (P ⬍ 0.001) from control group.

determined by quantitative PCR using GAPDH as an internal standard. Results were expressed as ratios between VCAM-1 or MCP-1 and GAPDH. To verify that GAPDH constitutes a suitable internal standard, its expression in thoracic aorta was examined quantitatively by competitive PCR using a shortened cDNA competitor. As expected, the gels showed only two bands, which represented the 465-bp PCR product of the aortic GAPDH and the truncated product of the competitor (Fig. 4A). In addition, the expression levels of VCAM-1 and MCP-1 were significantly lower in the naringin- and naringenin-supplemented animals than in the control group (P ⬍ 0.01) (Figs. 4B, 4C, and 4D). DISCUSSION This study was undertaken to evaluate whether or not the citrus flavonoids, naringin and naringenin can inhibit the progression of atherosclerosis in NZW rabbit on a high cholesterol diet. The flavanone glycoside, naringin are deglycosylated by bacteria in the gastrointestinal tract to naringenin prior to absorption (25). Thus, this aglycon flavonoid could act directly in the liver. The present study investigated the antiatherogenic effects of 0.1% (wt/wt diet) naringin and equivalent amount, 0.05% (wt/wt diet) of its aglycon, naringenin in high cholesterol-fed rabbits. We found that supplementing the diet with naringin or naringenin efficiently inhibited the experimentally induced atherosclerosis. Slight suppression of hepatic ACAT activity, the low expression of endothelial VCAM-1 and MCP-1 genes, and the inhibition of monocyte/macrophage adhesion accompanied this anti-atherogenic effect to the vascular wall. However, the treatment with

685

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 4. Effects of naringin and naringenin on the expressions of aortic (first portion) VCAM-1 and MCP-1 genes determined by semiquantitative RT-PCR as described under Materials and Methods. (A) Competitive expressions of aortic GAPDH determined by competitive PCR using as competitor a 168 bp-deleted cDNA fragment of the PCR product. (B) Ethidium bromide stained agarose gels showing RT-PCR products amplified with VCAM-1 and MCP-1 primers in different groups. Decreased expressions of VCAM-1 (C) and MCP-1 (D) determined relative to the expression of GAPDH were observed in naringin- and naringenin-supplemented groups. Bars represent standard deviations. * are significantly different (P ⬍ 0.01) from control group.

naringin or naringenin did not affect the levels of plasma cholesterol, triglyceride and HDL-cholesterol. In previous studies, hypocholesterolemic effects of dietary naringin and naringenin associated HMG-CoA reductase and ACAT in high cholesterol fed rats were reported (26, 27). It was also reported that the level of dietary cholesterol could affect plasma cholesterol levels even though hypocholesterolemic agents such as the antioxidant (probucol) and the enzyme inhibitor (lovastatin) were supplemented (28, 29). That is, when the same amount of anti-atherogenic agent was supplemented, a reduction in plasma cholesterol level was observed in rabbits fed relatively low concentration of dietary cholesterol, but this did not occur in rabbits on high cholesterol diets (30, 31). In the present study, the plasma cholesterol level had increased to almost 1700 mg/dl in all groups, and so hypocholesterolemic effects of dietary naringin and naringenin associated HMGCoA reductase could not observed in 1% high cholesterol-fed rabbits. However, the hepatic ACAT activities were slightly lower in naringin (5.0%)- and narin-

genin (15.0%)-supplemented rabbits than in the control group. Naringin and naringenin exhibited a slightly inhibitory activity on hepatic ACAT by 30.6 and 36.2% at 500 ␮M, respectively, when measured in vitro. Although more studies are needed, with various animal models, to explain the inhibitory activities of naringin and naringenin on ACAT in vivo, naringin and naringenin may inhibit foam cell development via reducing apolipoprotein B (apoB) secretion and cholesterol ester synthesis. These results are supported by recent studies by Wilcox et al. (32) and Borradaile et al. (33) that showed that naringenin substantially reduced apoB secretion and cholesterol esterification by inhibiting ACAT activity in HepG2 cells. In preliminary experiment, naringin had strong antiatherogenic action that may not be associated with its very mild lipid lowering action in high cholesterol-fed rabbits (34). From these results, we assumed that the anti-atherogenic effect such as apparent reduction of aortic fatty streak formation might be related with another factor in cholesterol-fed rabbits supplemented

686

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with naringin or naringenin. The accumulation of foam cells in the vascular wall is one of the characteristic features of atherosclerotic lesions. Studies in experimental animals and humans have indicated that most of the foam cells, especially in early atherosclerotic lesions, are monocyte-derived macrophage (35, 36). One of the early visible changes in the atherosclerosis of experimental animals involves the increased adherence of monocytes to arterial endothelium, followed by transendothelial migration into subendothelial space and differentiation into macrophages (37, 38). VCAM-1 and MCP-1 are assumed to play an important role in the adhesion of monocytes to the endothelium. In this study, the expression level of VCAM-1 and MCP-1 genes was investigated in the aortic segment, and this was found significantly decreased in naringin- and naringenin-supplemented rabbits, compared to the control group. Also, using an immunohistochemical method, it was found that the foam cell number and the thickness of the subendothelial layer were significantly reduced in the naringin- and naringenin-treated groups. These histological findings support that the anti-atherogenic effects of naringin and naringenin result from the inhibition of VACM-1 and MCP-1 expressions, followed by the prevention of monocyte adhesion and macrophage infiltration in aortic endothelium. This is the first study to demonstrate that naningin and naringenin inhibit VACM-1 and MCP-1 expressions. However, it seems that the signaling of VCAM-1 and MCP-1, lipoprotein oxidation and other factors must be considered to clarify the detailed mechanistic roles of VCAM-1 and MCP-1. In summary, our studies found that the citrus flavonoids, naringin and naringenin significantly inhibited aortic fatty streak formation in rabbit on high cholesterol diets. This anti-atherogenic effect seems to be closely involved with a decreased hepatic ACAT activity, and the down-regulation of VCAM-1 and MCP-1 genes. These results suggest that the antiatherogenic effect of naringin and naringenin in high cholesterol-fed rabbits was not due to the regulation of plasma lipid profile. However, the exact mechanism behind the anti-atherogenic effects of naringin and naringenin was not fully elaborated by this study, and further investigative work is underway.

3.

4.

5.

6.

7.

8.

9.

10.

11. 12.

13.

14. 15.

16.

ACKNOWEDGMENTS This work was supported by grants from the Ministry of Science and Technology (BSKG1420-1999053-2 and 00-J-BP-01-B-66), the Ministry of Health and Welfare (HMP-97-D-4-0015), and the Ministry of Agriculture and Forestry (BSAG760M-1000050-3), Korea.

REFERENCES

17.

18.

1. Brown, A. J. (2000) Atherosclerosis: Cell biology and lipoproteins. Curr. Opin. Lipidol. 11, 667– 669. 2. Beisiegel, U., and St. Clair, R. W. (1996) An emerging under687

standing of the interactions of plasma lipoproteins with the arterial wall that leads to the development of atherosclerosis. Curr. Opin. Lipidol. 7, 265–268. Brown, B. G., and Maher, V. M. (1994) Reversal of coronary heart disease by lipid-lowering therapy. Observations and pathological mechanisms. Circulation 89, 2928 –2933. Guazzi, M. D., Bussotti, M., Grancini, L., De Cesare, N., Guazzi, M., Pera, I. L., and Loaldi, A. (1997) Evidence of multifocal activity of coronary disease in patients with acute myocardial infarction. Circulation 96, 1145–1151. Nishimura, S., and Katoh, K. (2000) Development of acute coronary syndrome and progression of coronary artery disease: A serial clinical-angiographic analysis. Intern. Med. 39, 331–333. Rekhter, M. D., Hicks, G. W., Brammer, D. W., Work, C. W., Kim, J. S., Gordon, D., Keiser, J. A., and Ryan, M. J. (1998) Animal model that mimics atherosclerotic plaque rupture. Circ. Res. 83, 705–713. Eggebrecht, H., Oldenburg, O., Dirsch, O., Haude, M., Baumgart, D., Welge, D., Herrmann, J., Arnold, G., Werner, S. K., and Erbel, R. (2000) Potential embolization by atherosclerotic debris dislodged from aortic wall during cardiac catheterization: Histological and clinical findings in 7,621 patients. Catheter Cardiovasc. Interv. 49, 389 –394. Chang, T. Y., Chang, C. C. Y., and Cheng, D. (1997) Acylcoenzyme A: Cholesterol acyltransterase. Annu. Rev. Biochem. 66, 613– 638. Hilleman, D. E., Wurdeman, R. L., and Lenz, T. L. (2001) Therapeutic change of HMG-CoA reductase inhibitors in patients with coronary artery disease. Pharmacotherapy 21, 410 – 415. Krause, R. B., and Bocan, T. M. A. (1995) Experimemtal Animals (Ruffolo, R. R., Jr., and Hollinger, M. A., Eds.), pp. 173–198, CRC Press, Boca Raton, FL. Ross, R. (1995) Cell biology of atherosclerosis. Annu. Rev. Physiol. 57, 791– 804. Li, H., Cybulsky, M. I., Gimbrone, M. A., Jr., and Libby, P. (1993) An atherogenic diet rapidly induces VCAM-1, a cytokineregulatable mononuclear leukocyte adhesion molecule, in rabbit aortic endothelium. Arterioscler. Thromb. 13, 197–204. Parhami, F., Fang, Z. T., Fogelman, A. M., Andalibi, A., Territo, M. C., and Berliner, J. A. (1993) Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J. Clin. Invest. 92, 471– 478. Clinton, S. K., and Libby, P. (1992) Cytokines and growth factors in atherogenesis. Arch. Pathol. Lab. Med. 116, 1292–1300. Gerristsen, M. E., Carley, W. W., Ranges, G. E., Shen, C. P., Phan, S. A., Ligon, G. F., and Perry, C. A. (1995) Flavonoids inhibit cytokine-induced endotherial cell adhesion protein gene expression. Am. J. Pathol. 147, 235–237. Wo¨lle, J., Hill, R. R., Ferguson, E., Devall, L. J., Trivedi, B. K., Newton, R. S., and Saxena, U. (1996) Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid: Lack of effect on transcription factor NF-␬B. Aeterioscler. Thromb. Vasc. Biol. 16, 1501– 1508. Ishikawa, Y., Sugiyama, H., Stylianou, E., and Kitamura, M. (1999) Biofalvonoid quercetin inhibits interleukin-1-induced transcriptional expression of monocyte chemoattractant protein-1 in glomerular cells via suppression of nuclear factorkappaB. J. Am. Soc. Nephrol. 10, 2290 –2296. Fruebis, J., Gonzalez, V., Silvestre, M., and Palinski, W. (1997) Effect of probucol treatment on gene expression of VCAM-1, MCP-1, and M-CSF in the aortic wall of LDL receptor-deficient rabbits during early atherogenesis. Arteroscler. Thromb. Vasc. Biol. 17, 1289 –1302.

Vol. 284, No. 3, 2001

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

19. Carlo, G. D., Mascolo, N., Izzo, A. A., and Capasso, F. (1999) Flavonoids: Old and new aspects of a class of natural therapeutic drugs. Life Sci. 65, 337–353. 20. So, F. V., Guthrie, N., Chambers, A. F., Moussa, M., and Carroll, K. K. (1996) Inhibition of human breast cancer cell proliferation and delay of mammary tumorigenesis by flavonoids and citrus juices. Nutr. Cancer 26, 167– 81. 21. Bok, S. H., Shin, Y. W., Bae, K. H., Jeong, T. S., Kwon, Y. K., Park, Y. B., and Choi, M. S. (2000) Effects of naringin and lovastatin on plasma and hepatic lipids in high-fat and highcholesterol fed rats. Nutr. Res. 20, 1007–1015. 22. Erickson, S. K., Shrewsbury, M. A., Brooks, C., and Meyer, D. J. (1980) Rat liver acyl coemzyme A:cholesterol acyltransferase: its regulation in vivo and some of its properties in vitro. J. Lipid Res. 21, 930 –941. 23. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254. 24. Brecher, P., and Chan, C. (1980) Properties of acyl-CoA:cholesterol acyltransferase in aortic microsomes from atherosclerotic rabbit. Biochim. Biophys. Acta 617, 458 – 471. 25. Ameer, B., Weintraub, R. A., Johnson, J. V., and Yost, R. A. (1996) Flavonone absorption after naringin, hesperedin, and citrus administration. Clin. Pharmacol. Ther. 60, 34 – 40. 26. Bok, S. H., Lee, S. H., Park, Y. B., Bae, K. H., Son, K. H., Jeong, T. S., and Choi, M. S. (1999) Plasma and hepatic cholesterol and hepatic activities of 3-hydroxy-3-methyl-glutaryl-CoA reductase and acyl-CoA: cholesterol acyltransferase are lower in rats fed citrus peel extract or a mixture of citrus bioflavonoids. J. Nutr. 129, 1182–1185. 27. Lee, S. H., Park, Y. B., Bea, K. H., Bok, S. H., Kwon, Y. K., Lee, E. S., and Choi, M. S. (1999) Cholesterol-lowering activity of naringenin via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase in rats. Ann. Nutr. Metab. 43, 173–180. 28. Carew, T. E., Schwenke, D. C., and Steinberg, D. (1987) Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: Evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc. Natl. Acad. Sci. USA 84, 7725–7729.

29. Bocan, T. M., Mazur, M. J., Mueller, S. B., Brown, E. Q., Sliskovic, D. R., O’Brien, P. M., Creswell, M. W., Lee, H., Uhlendorf, P. D., and Roth, B. D. (1994) Antiatherosclerotic activity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase in cholesterol-fed rabbits: a biochemical and morphological evaluation. Atherosclerosis 111, 127–142. 30. Fujioka, T., Nara, F., Tsujita, Y., Fukushige, J., Fukami, M., and Kuroda, M. (1995) The mechanism of lack of hypocholesterolemic effects of pravastatin sodium, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, in rats. Biochem. Biophys. Acta 1254, 7–12. 31. Bocan, T. M., Mueller, S. B., Brown, E. Q., Uhlendorf, P. D., Mazur, M. J., and Newton, R. S. (1992) Antiatherosclerotic effects of antioxidants are lesion-specific when evaluated in hypercholesterolemic New Zealand white rabbits. Exp. Mol. Pathol. 57, 70 – 83. 32. Wilcox, L. J., Borradaile, N. M., Kurowska, E. M., Telford, D. E., and Huff, M. W. (1998) Naringenin, a citrus flavonoid, markedly decreases apoB secretion in HepG2 cells and inhibits acyl-CoA: cholesterol acyltransferase. Circulation 98, I-537 (Abstract). 33. Borradaile, N. M., Carroll, K. K., and Kurowska, E. M. (1999) Regulation of HepG2 cell apolipoprotein B metabolism by the citrus flavanones hesperetin and naringenin. Lipids 34, 591– 598. 34. Choi, S. C., Kim, H. S., Jeong, T. S., Bok, S. H., and Park, Y. B. (1998) Antiatherogenic effect of naringin independent of lipidlowering action in hypercholesterolemic rabbits. Kor. Circ. J. 28, 1873–1881. 35. Gerrity, R. G. (1981) The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am. J. Pathol. 103, 181–190. 36. Watanabe, T., Hirata, M., Yoshigawa, Y., Nagafuchi, Y., Toyoshima, H., and Watanabe, T. (1985) Role of macrophages in atherosclerosis. Sequential observations of cholesterol-induced rabbit aortic lesion by the immunoperoxidase technique using monoclonal anti-macrophage antibody. Lab. Invest. 53, 80 –90. 37. Joris, I., Zand, T., Nunnari, J. J., Krolikowski, F. J., and Mauno, G. (1983) Studies on the pathogenesis of atherosclerosis: I. Adhesion and emigration of mononuclear cells in the aorta of hypercholesterolemic rat. Am. J. Pathol. 113, 341–358. 38. Faggiotto, A., Ross, R., and Harker, L. (1984) Studies of hypercholesterolemia in the nonhuman primate. I. Changes that lead to fatty streak formation. Atherosclerosis 4, 323–340.

688