Niimi et al. Lipids in Health and Disease 2013, 12:166
http://www.lipidworld.com/content/12/1/166

RESEARCH

Open Access

Probucol inhibits the initiation of atherosclerosis
in cholesterol-fed rabbits
Manabu Niimi1, Yuka Keyamura2, Masanori Nozako2, Takashi Koyama2, Masayuki Kohashi2, Reiko Yasufuku2,
Tomohiro Yoshikawa2 and Jianglin Fan1*

Abstract
Background: Probucol and statin are often prescribed for treating atherosclerosis. These two drugs exhibit different
mechanisms but it is unknown whether they have the same anti-atherogenic properties. In the current study, we
examined whether these two drugs at optimal doses could inhibit the initiation of atherosclerosis in cholesterol-fed
rabbits in the same way.
Methods: New Zealand White rabbits were fed a cholesterol-rich diet for 5 weeks to produce the early-stage lesionsof atherosclerosis. Drug-treated rabbits were administered either probucol or atorvastatin and serum lipids andaortic atherosclerotic lesions were compared with those in a control group.
Results: Atorvastatin treatment significantly reduced serum total cholesterol levels while probucol treatment led
to significant reduction of high-density lipoprotein cholesterol levels without changing total cholesterol levels
compared with those in the control group. Compared with the control, probucol treatment led to 65% (p < 0.01)
reduction while atorvastatin treatment led to 23% (p = 0.426) reduction of the aortic lesion area. Histological andimmunohistochemical analyses revealed that the lesions of the probucol-treated group were characterized byremarkable reduction of monocyte adherence to endothelial cells and macrophage accumulation in the intima
compared with those of both atorvastatin and control groups. Furthermore, low-density lipoprotein (LDL) isolated
from the probucol group exhibited prominent anti-oxidative reaction, which was not present in LDL isolated fromeither the atorvastatin-treated or the control group.
Conclusions: This study suggests that probucol inhibits the initiation of atherosclerosis by reducing monocyte
adherence and infiltration into the subintima. Anti-oxidization of LDL by probucol protects more effectively against
early-stage lesion formation than statin-mediated lipid-lowering effects.
Keywords: Atherosclerosis, Monocyte, Probucol, Atorvastatin, Hypercholesterolemia

Background
Atherosclerosis is a chronic inflammatory disease that is
initiated by monocyte adhesion to arterial intimal surfaces followed by migration into the subintimal space.
Intimal monocytes are further differentiated into macrophages and then transformed into foam cells afteringesting deposited lipids. High levels of plasma lowdensity lipoprotein (LDL), especially oxidized LDL, arethe major atherogenic factor for the recruitment of
monocyte adhesion and induction of foam cell formation* Correspondence: jianglin@yamanashi.ac.jp
1
Department of Molecular Pathology, Interdisciplinary Graduate School of
Medicine and Engineering, University of Yamanashi, Yamanashi, Japan
Full list of author information is available at the end of the article[1,2]. Therefore, reducing plasma LDL levels and inhibition of LDL oxidization are the primary targets for theprevention and treatment of atherosclerotic disease.
Probucol is not only a lipid-lowering drug but also has
anti-oxidant activity [3]. For this reason, it is often prescribed for the treatment of hypercholesterolemia andxanthoma. Probucol reduces plasma LDL-cholesterol
through increasing LDL catabolism and bile synthesis in
the liver [4,5]. However, in addition to LDL-cholesterol
lowering effects, probucol also simultaneously reduces
plasma high-density lipoprotein (HDL) levels. In thepast, low levels of plasma HDL-cholesterol (HDL-C) induced by probucol were considered as a notorious side
effect, but now it is clear that the functions of HDL© 2013 Niimi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication
waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise
stated.

Niimi et al. Lipids in Health and Disease 2013, 12:166
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Results
Serum lipids and lipoprotein profile

As shown in Figure 1, the average total cholesterol (TC)level at the baseline (1 week) was 700 mg/dL after cholesterol diet feeding for 1 week. Serum TC level of thevehicle group was increased to 1500 mg/dL at 5 weeks.
Atorvastatin treatment led to a significant reduction ofserum TC level at 3 and 5 weeks but did not change
HDL-C and triglyceride (TG) levels. Probucol treatment
did not affect TC and TG levels but significantly reduced
HDL-C level at 3 and 5 weeks. Analysis of lipoprotein
profiles by high performance liquid chromatography(HPLC) revealed that the increased serum TC level wasessentially caused by elevated serum very-low-density
lipoprotein and LDL fractions in cholesterol-fed rabbits
and these fractions were markedly reduced in theatorvastatin-treated group while HDL fractions were reduced in the probucol-treated group (Figure 2).
Aortic atherosclerosis

Analysis of en face aortic lesion area revealed that, although both drug treatments inhibited the lesion size

1600

VehicleAtorvastatin
ProbucolTC (mg/dL)

1200
800

*

*
drug treatment

400

HDL-C (mg/dL)

0
50
40
30
20

**

10

***

0
80
60
TG (mg/dL)

particles such as reverse cholesterol transport [6-8] andscavenger receptor class B type I activity are actually upregulated [9,10]. Previous studies showed that probucol
treatment protects against atherosclerosis in WHHL andcholesterol-fed rabbits [11,12]. Clinically, probucol has
been widely used in Asia, such as Japan, China, andKorea, for the treatment of hyperlipidemia and atherosclerosis. Recently, a clinical trial demonstrated that
long-term probucol treatment reduced the risk of cardiovascular events in familial hypercholesterolemia [13].
In spite of this, the lipid-lowering effect of probucol israther moderate compared with that of statins, hydroxymethylglutaryl (HMG)-CoA reductase inhibitors, themost popular lipid-lowering drugs for the treatment ofhyperlipidemia. Statins reduce cholesterol biosynthesis
through inhibiting HMG-CoA reductase activity and increase hepatic LDL receptor activity [14]. Furthermore,statins also have anti-inflammatory effects designated aspleiotropic effects. Nevertheless, treatment with statin
therapy alone can lead to only 30% reduction of the incidence of coronary events [15,16]. Therefore, other nonstatin therapies are required for many patients who are
not responsive to statins [2,17].
In the current study, we compared these two drugs interms of atheroprotective effects in cholesterol-fed rabbits.
We were particularly interested in the initiation of atherosclerosis and made a side-to-side comparison of these two
drugs. Our results suggested that the anti-oxidative effects
exerted by probucol may be more beneficial for the inhibition of monocyte adhesion and infiltration during atherogenesis than statin-mediated cholesterol lowering effects.

Page 2 of 8

*
40
20
0

0

1

2
3
(weeks)4

5

Figure 1 Serum concentrations of total cholesterol (TC),HDL-cholesterol (HDL-C), and triglycerides (TG). Data areexpressed as mean ± SEM (n = 8, each group). *P < 0.05,**P < 0.01, ***P < 0.001 vs. vehicle.

(65%↓ in the probucol group and 23%↓ in the atorvastatin group compared with that in the vehicle group),
statistical significance was only found in probucoltreated group (Figure 3, left). Histological examinations
showed that the aortic lesions were mainly composed ofinfiltrating macrophages (Figure 3, right). Comparedwith the vehicle group, intimal lesion area was remarkably reduced in the probucol group (92%↓ in aortic archand 95%↓ in thoracic aorta). The atorvastatin group also
had less intimal lesions, but the difference was notNiimi et al. Lipids in Health and Disease 2013, 12:166
http://www.lipidworld.com/content/12/1/166

150

significant (51%↓ in aortic arch and 16%↓ in thoracic
aorta, p > 0.05). Immunohistochemical staining showedthat the reduced lesion areas found in both drug-treatedgroups were mainly caused by reduced macrophage infiltration: 96%↓ in aortic arch and 94%↓ in thoracic aorta
in the probucol group, and 51%↓ in aortic arch and15%↓ in thoracic aorta in the atorvastatin group compared with those in the control group, while a significant
difference was only found in the probucol group.

100

Scanning electron microscopic observation

Chow-fed
rabbit serum
20

Page 3 of 8

Cholesterol diet-fed
rabbit serum
300

VehicleAtorvastatin
Probucol

LDL

VLDL
250
15
TC (mV)

HDL
10

LDL

200

5
HDL

50
0
20

25

0
30
35
20
25
Retention time (min)30

35Figure 2 Analysis of serum lipoprotein profile by HPLC gel
filtration. Serum lipoproteins were fractionated using a gel filtration
column with on-line post-column cholesterol detection. Serum
lipoprotein profiles of normal rabbits fed a chow diet and cholesterolenriched for 5 weeks after drug treatment for 4 weeks. Data
are expressed as mean value of 8 rabbits from each group.

VehicleAtorvastatin

ProbucolWe also examined the aortic surface changes by scanningelectron microscopy and observed monocyte-endothelialinteractions. Under the scanning electron microscopy, thelesions of the aorta were visible even at low magnification.
While there was variation in lesion features from place toplace in the three groups, the number of monocytes adhered to endothelial cells was generally less in the probucol group than in the atorvastatin or control group(Figure 4), which was consistent with the observations inlight microscopic histological study described above. At
high magnification, there were diverse features of intimallesions (Figure 5): some areas appeared as raised ridges or
became bulged or protuberant with or without monocytes
VehicleHE

Atorvastatin

Probucol

EVG

30

20

10

0

M

Microscopic
lesion area (mm2)Gross lesion area (%)

P=0.426

Macrophage area
(mm2)

**

40

VehicleAtorvastatin
Probucol

0.2

0.1

0
0.12

**

*

Aortic arch Thoracic aorta

0.08
0.04
0

*

*

Aortic arch Thoracic aorta

Figure 3 Analysis of aortic atherosclerosis. Representative photographs of pinned-out aortic arch from each group (left, top). Scatter plot of
the aortic atherosclerotic lesions on the surface was quantified using an image analysis system (left, bottom). Representative micrographs of theaortic arch from each group (right, top). Serial frozen sections of aortas were stained with hematoxylin-eosin (HE), elastica van Gieson (EVG), or
immunohistochemically stained with mAb against macrophages (Mϕ). Intimal microscopic lesions on EVG-stained sections (right, middle) andMϕ-positive area (right, bottom) were quantified with an image analysis system. The values are expressed as mean ± SEM (n = 8, each group).
*P < 0.05, **P < 0.01 vs. vehicle.

Niimi et al. Lipids in Health and Disease 2013, 12:166
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Page 4 of 8

VehicleVehicle

250 m

50 m

Atorvastatin

Atorvastatin

250 m

50 m

Probucol

Probucol

250 m

50 m

Figure 4 Representative micrographs of the aortic arch from each group observed under a scanning electron microscope. At low
magnification, monocytes that adhered to the intimal surface of the aorta in the probucol group are small in number compared with those in
the atorvastatin and control groups (left). Many monocytes are clearly visible in the atorvastatin and control groups at high magnification (right).

attached on the surface. Many monocytes adhered to theendothelial surface either singly or in clusters. The bulged
or protuberant areas contained many monocytes beneathendothelial cells, which can be observed in the areas
where endothelial cells were detached or split. These lesions were also fewer in the probucol-treated group thanin the other two groups.
LDL oxidizability

We compared the suppressive capacity of probucol andatorvastatin on plasma LDL oxidization, a major insult
and trigger for atherogenesis. As shown in Figure 6, LDL
isolated from probucol-treated rabbits but not atorvastatin-treated rabbits showed significant suppressive
ability against copper-induced oxidization expressed as aprolonged lag-time, and decreased maximal oxidation
speed and maximal diene concentrations.

Discussion
In the current study, we compared the atheroprotective
effects of probucol and atorvastatin, two hypolipidemic
drugs for the treatment of hyperlipidemia. It is generally
believed that probucol reduces plasma cholesterol
through increasing LDL catabolism and bile excretion,
whereas atorvastatin inhibits HMG-CoA reductase

activity and increases hepatic LDL receptor activity [14].
Here, we demonstrated that, at the optimal doses ofeach drug, probucol exerts a greater inhibitory effect on
the initiation of cholesterol-diet-induced atherosclerosis
in rabbits than atorvastatin. It has been reported previously that probucol at high dose (1%) and for a long
period (2 ~ 6 months) protects against atherosclerosis inboth WHHL and cholesterol-fed rabbits [11,12]. Here,we further demonstrated that probucol even at lower
dose (0.3%) for a short period (4 weeks) was still able toreduce the lesions of atherosclerosis in cholesterol-fed
rabbits and the lesions were characterized by reduced intimal macrophage accumulation. It is interesting to note
that, unlike atorvastatin, probucol administration for ashort period did not lead to a significant cholesterollowering effect, while HDL-C was significantly low.
Therefore, several possible mechanisms may operate independently upon the lipid-lowering effect regarding
probucol’s anti-atherogenic effects. First, probucol hasvery strong anti-oxidative effects, whereas atorvastatin
does not. In this situation, one may consider that theanti-atherogenic effects induced by anti-oxidation by
probucol are stronger than the lipid-lowering effects of
atorvastatin. It may also suggest that oxidation in the arterial wall may be more important than merely elevation

Niimi et al. Lipids in Health and Disease 2013, 12:166
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Page 5 of 8

A

E

50 m

B

50 m

F

50 m

50 m

C

G

50 m

50 m

D

H

10 m

10 m

Figure 5 Different intimal changes of the aorta often observed under a scanning electron microscope. (A) Normal-appearance intimalendothelial cells. (B) Intimal surface becomes bulged without monocyte adherence (right area). (C-D) Monocytes are found under split
endothelial cells, possibly due to artifacts of the fixation and drying processes. (E-H) Monocyte adherence to the bulged or protruded intima.

of plasma cholesterol in the initiation of atherosclerosis.
The second possible mechanism may be attributed to HDL
modulation induced by probucol. It has been reported thatHDL functions such as reverse cholesterol transport [6-8]and anti-oxidative activity [18] are markedly up-regulated
in probucol-treated patients, while HDL-C levels are low.
If these functions occur in the arterial wall, accumulationof lipids would be diminished. Nevertheless, these results
may raise an important question: Do we need to treat
hyperlipidemic patients with atorvastatin combined withprobucol? In Japan, probucol is used for hyperlipidemic
patients who are resistant to statins [19,20]. In China,
however, probucol is widely used for the treatment ofhyperlipidemia and given the same priority as statins. Socalled “PAS” therapy (probucol, aspirin, and statin) in
China is often prescribed for hyperlipidemic patients forboth primary and secondary prevention of coronary arterydiseases [21]. Although this question still awaits more clinical investigation, apparently, the anti-oxidative capacity issuperior to the lipid-lowering effect for protection against
atherosclerosis initiation.

Conclusions
Our studies provide evidence that probucol inhibited theinitiation of atherosclerosis without improving serum
TC levels, despite a lowering of serum HDL-C levels.
These results suggest that the anti-oxidative effects
exerted by probucol are more efficient and effective forthe inhibition of atherosclerosis than the cholesterollowering effects.
Methods
Chemicals

Probucol was synthesized at Otsuka PharmaceuticalCo., Ltd. (Tokushima, Japan). Atorvastatin calcium
was purchased from Sequoia Research Products Ltd.
(Pangbourne, UK).

Niimi et al. Lipids in Health and Disease 2013, 12:166
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Page 6 of 8

Analysis of lipoproteins using HPLC

Absorbance at 234 nm

1.0

0.8
0.6
0.4
0.2

0
0
800

200

***

400
Time (min)
0.020

600

800

1.2

VehicleAtorvastatin
Probucol

200

0.015

OD max (A234)

400

V max (ΔA234/min)

Lag time (min)

1.0
600

Analysis of aortic atherosclerosis

0.8
0.6

0.010

0.005

***

***

0.4
0.2

0

0

Serum lipoproteins were analyzed using HPLC (Tosoh
Co., Tokyo, Japan) according to the procedure describedby Usui et al. [22] with slight modification. In brief, sera
were separated on 2 tandem connected gel filtration
columns TSKgel LipopropakXL (Tosoh Co., Tokyo,
Japan) with phosphate-buffered saline containing 0.005%
Brij-35 and 0.05% sodium azide. The column effluent
was mixed with on-line connected cholesterol colorimetric reagent (Wako Pure Chemical Industries Ltd., Osaka,
Japan) and the absorbance at 600 nm was monitored
after the enzymatic reaction in a reaction coil at 37°C.
The lipoprotein fractions of very-low-density lipoprotein,
LDL, and HDL were defined based on retention time.

0

Figure 6 Kinetic evaluation of LDL oxidation induced bycopper. Representative kinetic graph is shown in the top panel.
Lag-time, maximal oxidization speed (V max), and maximal dieneconcentration (OD max) were calculated. The values are expressed
as mean ± SEM (n = 8-9, each group). ***P < 0.001 vs. vehicle.

Experimental protocol

Male New Zealand White rabbits (1.0-1.5 kg at age of
6-7 weeks old) provided by Kitayama Labes Co., Ltd.
(Nagano, Japan) were fed an atherogenic diet containing
0.5% cholesterol for 1 week. Using the TC levels (around
700 mg/dl), we selected those rabbits that showed a reasonable response to cholesterol diet feeding for the following experiments. These rabbits were further fed witheither the same atherogenic diet (vehicle group) or supplemented with either 0.3% (w/w) probucol or 0.005%(w/w) atorvastatin for another 4 weeks. All diets wereprepared by Oriental Yeast Co., Ltd. (Tokyo, Japan).
Blood samples were obtained from the auricular veinbiweekly for biochemical analysis. At the end of theexperiment, all rabbits were sacrificed for evaluation ofaortic atherosclerosis. All animal experiments were
performed with the approval of the Animal Care Committee of Otsuka Pharmaceutical Company and the
University of Yamanashi.
Analysis of serum biochemistry

Serum levels of TC, TG, and HDL-C were measured using
commercial assay kits (Wako Pure Chemical Industries
Ltd., Osaka, Japan) and automated clinical analyzer
BIOLIS 24i (Tokyo Boeki Machinery, Ltd., Tokyo, Japan).

At the end of the experiment, all rabbits were euthanized by injection of an overdose of sodium pentobarbital solution. Aortas were rapidly dissected free fromthe arch to the iliac artery bifurcation. The aortas wereanalyzed for en face gross lesions and microscopic quantification of intimal lesion areas. En face lesion area wasquantitated as described previously [23]. Aortas wereopened longitudinally and mounted on a black rubberboard, and then were photographed using a digital camera. Using Photoshop software (Adobe Systems Inc., San
Jose, CA), atherosclerotic lesions were visualized first
and then quantitatively measured using the image analysis system WinRoof (Mitani Co., Tokyo, Japan). For
microscopic lesion analysis, two segments (aortic archand thoracic aorta) fixed in 10% formalin were embedded in OCT compound (Sakura Finetek Japan Co., Ltd.,Tokyo, Japan). Frozen sections were stained withhematoxylin-eosin (HE) and elastica van Gieson (EVG).
The intimal lesion size was quantitatively measured
using the image analysis system WinRoof as described
previously [24]. For microscopic evaluation of the cellular components of the lesions, serial frozen sections of
the aorta were immunohistochemically stained with
RAM11 (Dako Japan Inc., Tokyo, Japan) monoclonal
antibody against rabbit macrophage using Histofine Simple Stain MAX-PO(M) kits (Nichirei Biosciences Inc.,Tokyo, Japan). Macrophages in the lesions were measured by quantifying the positively stained area using theimage analysis system.
Scanning electron microscopic observation

To observe intimal surface changes of aorta aftercholesterol-rich diet feeding, we perfused three rabbits
from each group at the end of the experiment using
phosphate-buffered saline followed by fixative solution
containing 2.5% glutaraldehyde and 2% paraformaldehyde (pH 7.4) for 20 min. After that, aortas were dissected free from the aortic root to the common carotid

Niimi et al. Lipids in Health and Disease 2013, 12:166
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artery bifurcation. Aorta was cut into 13 pieces as specimens (approximately 3 mm square). These specimens
were further fixed in the same solution at 4°C overnight.
Fixed specimens were post-fixed with 1% osmium tetroxide (pH 7.4) and dehydrated with graded ethanol
followed by isoamyl acetate. Aortic segments were driedin CO2 using critical point dryer HCP-2 (Hitachi Koki Co.
Ltd., Tokyo, Japan) and were sputter-coated with a thin gold
layer in an ion coater IB-3 (EIKO Co., Tokyo, Japan). The
aortic surface was examined under a scanning electron
microscope JSM-6510 (JEOL Ltd., Tokyo, Japan).
Evaluation of LDL oxidizability

Plasma LDLs (d = 1.006-1.063) were isolated by ultracentrifugation [25] from rabbits at 4 weeks after cholesterol-rich-diet feeding. The susceptibility of LDL tocopper-induced oxidation was assessed by determining
the formation of conjugated dienes as described previously [26]. In brief, LDL (50 μg protein/mL) was incubated with 5 μM CuSO4, and the formation of
conjugated dienes was determined by monitoring thechange of absorbance at 234 nm at 37°C with a microplate reader SpectraMax 190 (Molecular Devices,
Sunnyvale, CA). Lag-time, maximal oxidization speed,
and maximal diene concentrations were calculated forthe estimation of oxidization sensitivity.
Statistical analysis

All data were expressed as mean ± SEM. Statistical significance was determined by one-way ANOVA (Dunnett’s test) using SAS software (SAS Institute Japan Ltd.,Tokyo, Japan). P values less than 0.05 were consideredsignificant or otherwise specified.

Page 7 of 8

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.
15.

16.

Competing interests
The authors declare no conflict of interest.
Authors’ contributions
MN, TY and JF designed the study. MN, YK, MN, TK, MK and RY performed
the experiments and MN analyzed the data. MN and JF wrote themanuscript. All authors read and approved the final manuscript.

17.
18.

Author details
1
Department of Molecular Pathology, Interdisciplinary Graduate School of
Medicine and Engineering, University of Yamanashi, Yamanashi, Japan. 2Free
Radical Research Project, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.

19.

Received: 12 September 2013 Accepted: 29 October 2013
Published: 4 November 201320.

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Cite this article as: Niimi et al.: Probucol inhibits the initiation ofatherosclerosis in cholesterol-fed rabbits. Lipids in Health and Disease2013 12:166.

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