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Int J Angiol Vol 17 No 4 Winter 2008186
Attenuation of the development of
hypercholesterolemic atherosclerosis by
thymoquinone
Ahmed Ragheb1, Fawzy Elbarbry2, Kailash Prasad3, Adel Mohamed4, Mohamed S Ahmed5, Ahmed Shoker1
1Department of Medicine, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan; 2School of Pharmacy, Pacific
University, Hillsboro, Oregon, USA; 3Department of Physiology; 4Department of Anatomy and Cell Biology; 5Department of Pathology,
College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan
Correspondence: Dr Ahmed Shoker, Department of Medicine, Division of Nephrology, University of Saskatchewan, 103 Hospital Drive,
Saskatoon, Saskatchewan S7N 0W8. Telephone 306-966-2630, fax 306-966-7996, e-mail ass787@mail.usask.ca
The role of reactive oxygen species (ROS) in hypercholes-
terolemia-induced atherosclerosis has been previously
reported (1,2). Hypercholesterolemia stimulates polymorpho-
nuclear leukocytes and activates vascular endothelial cells,
which results in excess ROS release (3,4). In addition, hyper-
cholesterolemia increases platelet-activating factor concentra-
tions, which in turn increases interleukin (IL)-1b and tumour
necrosis factor-alpha (TNF-a) synthesis and release (5). IL-1b,
IL-8 and IL-6, TNF-a and interferon-gamma are known to
activate granulocytes that generate ROS (6). TNF-a also indu-
ces NADPH-oxidase enzyme to liberate ROS in the vascular
endothelial cells (7).
Thymoquinone (TQ) (2-isopropyl-5-methyl-1,4-benzo-
quinone) is the bioactive constituent of the volatile oil of
Nigella sativa seeds (8). The oil and seed constituents, in par-
ticular TQ, have shown potential medicinal properties in trad-
itional medicine. N sativa seeds have long been used in folk
medicine for a wide range of illnesses including bronchial
asthma, headache, dysentery, infections, obesity, back pain,
hypertension and gastrointestinal problems (9). TQ has anti-
oxidant and anti-inflammatory activities (10), as well as lipid-
lowering effects (11).
Because of these pharmacological properties, it is hypoth-
esized that TQ may attenuate hypercholesterolemia and the
development of hypercholesterolemic atherosclerosis. The
main objectives of the present study were to investigate
whether TQ attenuates the development of atherosclerosis,
whether this effect is associated with reduction in serum lipids and
oxidative stress, and whether the effects are dose-dependent.
METHODS
Drugs
TQ (Sigma-Aldrich, USA) was dissolved in pure corn oil
(Sigma-Aldrich, USA) to give a concentration of 75 mg/mL.
Animals and experimental design
Twenty-four female New Zealand white rabbits, weighing
1.8 kg to 2 kg and aged six to eight weeks, were assigned to four
groups. After one week of adaptation, the rabbits were fed a
regular diet (group I), a high-cholesterol diet alone (group II), or
a high-cholesterol diet plus TQ 10 mg/kg/day or 20 mg/kg/day
(groups III and IV) for eight weeks. The diet was prepared by
Purina (USA) and did not contain any antioxidants. TQ was
given through nasogastric tubes. Water was given ad libitum.
The rabbits were housed in individual cages at room temper-
ature (22°C to 24°C) and a relative humidity of 40% to 60%
under a 12 h light/12 h dark cycle. The experimental protocols
were approved by the Ethics Committee of the University of
ORIGINAL ARTICLE
©2008 Pulsus Group Inc. All rights reserved
A Ragheb, F Elbarbry, K Prasad, A Mohamed, MS Ahmed,
A Shoker. Attenuation of the development of
hypercholesterolemic atherosclerosis by thymoquinone. Int J
Angiol 2008;17(4):186-192.
Thymoquinone (TQ), derived from Nigella sativa seed, is an antioxidant.
The present study investigated whether TQ attenuates the development of
atherosclerosis, and/or reduces the serum lipid levels and oxidative stress in
rabbits. New Zealand white female rabbits were assigned to four groups of
six animals each: group I, control; group II, 1% cholesterol diet; group
III, 1% cholesterol plus TQ (10 mg/kg/day; through a nasogastric tube)
diet; and group IV, 1% cholesterol plus TQ (20 mg/kg/day; through a
nasogastric tube) diet. Blood samples were collected at baseline and
after four and eight weeks on the experimental diets for measurement
of serum lipids, total cholesterol (TC), triglycerides (TG), low-den-
sity lipoprotein cholesterol (LDL-C), high-density lipoprotein cho-
lesterol (HDL-C), TC/HDL-C ratio and oxidative stress biomarkers
(malondialdehyde [MDA] and protein carbonyls). At the end of the
eight weeks, the aorta was removed for the assessment of atheroscle-
rotic changes, MDA and protein carbonyls. Group II animals devel-
oped atherosclerosis (45%±11% of the intimal surface of aorta was
covered with atherosclerotic plaques), which was associated with an
increase in the serum TC, TG, LDL-C, HDL-C, TC/HDL-C, MDA
and protein carbonyls. In group III, TQ decreased serum TC, LDL-C,
MDA and protein carbonyls by 26%, 29%, 85% and 62%, respectively,
and aortic MDA by 73%, which was associated with a 40% reduction
of the development of aortic atherosclerosis. The higher dose of TQ in
group IV had effects similar to the lower dose (group III), except that
this dose further decreased serum TG. It is concluded that TQ attenu-
ates hypercholesterolemic atherosclerosis and this effect is associated
with a decrease in serum lipids and oxidative stress.
Key Words: Atherosclerosis; Hypercholesterolemia; Reactive oxygen spe-
cies; Thymoquinone
Thymoquinone and atherosclerosis
Int J Angiol Vol 17 No 4 Winter 2008 187
Saskatchewan, Saskatoon, Saskatchewan, and the animal care
was according to the approved standards for Laboratory Animal
Care. Following 18 h of fasting, blood samples (from the ear
marginal vein) were collected before (zero week), and after
four and eight weeks on the respective diets for measurement of
total cholesterol (TC), triglycerides (TG), low-density lipopro-
tein cholesterol (LDL-C), high-density lipoprotein cholesterol
(HDL-C), TC/HDL-C and serum malondialdehyde (MDA).
Serum protein carbonyls was measured only at week 8. At the
end of the study (eight weeks), rabbits were anesthetized with
Euthanyl (sodium pentobarbital; Bimeda-MTC Animal Health
Inc, Canada) (50 mg/kg intravenously) in the marginal ear
vein. Aortas were removed for the assessment of atheroscler-
otic changes, and measurement of aortic MDA and protein
carbonyls.
Serum lipids
TC, TG and HDL-C were measured on an automated Synchron
LX20 Clinical System Analyzer (Beckman Coulter Inc, USA).
LDL-C was calculated. Risk ratio was estimated using the quo-
tient TC/HDL-C.
Preparation of the aortic tissue for measurement of MDA
and protein carbonyls
The aortic rings were excised at the roots of the aortic arches
and kept in 10% buffered formalin for histological examina-
tion. Aortas between the origin and bifurcation to the iliac
arteries were removed, cleansed of gross adventitial tissue and
cut longitudinally into two halves. One half was used for assess-
ment of atherosclerotic changes, and the other half was kept on
ice for preparation of the supernatants, by a previously
described method (12), for measurement of the aortic tissue
MDA and protein carbonyls.
Serum and aortic MDA (thiobarbituric acid-reactive
substances)
MDA levels in the aortic tissue supernatant and serum were
measured as thiobarbituric acid-reactive substances by a previ-
ously described method (12,13). Thiobarbituric acid-reactive
substances were extracted in a mixture of butanol and pyridine
(15:1) that was separated by centrifugation. The fluorescence
intensity of the butanol-pyridine solution was measured at
553 nm with excitation at 513 nm. The MDA content of the
tissue was expressed as nmol/mg proteins and that of serum as
nmol/mL.
Serum and aortic protein carbonyls
The protein carbonyl levels in the aortic tissue supernatant and
serum were measured using a Cayman Chemical Carbonyl
Protein Assay Kit (Cayman Chemical Company, USA). The
absorbance was measured at wavelengths between 360 nm and
386 nm using an ELx 808TM Absorbance Microplate Reader
(BioTek Instruments Inc, USA). The protein carbonyl content
of the tissue was expressed as nmol/mg proteins and that of
serum as nmol/mL.
Tissue protein measurement
Protein content was determined using a Modified Lowry Protein
Assay Kit (Pierce Biotechnology, USA). The absorbance was
measured at a wavelength of 750 nm using an ELx 808TM
Absorbance Microplate Reader.
Assessment of atherosclerotic changes in the aorta
Assessment of atherosclerotic changes in the aorta was per-
formed by using Herxheimer’s solution containing Sudan IV
for lipid staining, as described previously (3,14). Photographs
of the stained intimal surface of the aorta were taken with a
digital camera. The total and atherosclerotic areas of the
intimal surface of the aorta were measured using Scion Image
for Windows (Scion Corporation, USA) image analysis soft-
ware. The extent of atherosclerosis was expressed as a percent-
age of total intimal surface area.
Histological examination
The aortic ring specimens were crosswise and embedded in
paraffin. Paraffin sections of 4 µm thickness were cut and
stained with hematoxylin and eosin (H&E) and examined by
light microscopy. A Sigma-Aldrich Accustain Elastic Stain Kit
(Sigma-Aldrich Canada Ltd) was used to visualize elastic lam-
inae and examine the changes in the elastic fibres by light
microscopy.
Quantification and histological assessment of atherosclerotic
lesions in aortic rings
Sections stained with H&E were used to compare the total
cross-sectional intimal and medial areas and the intima/media
thickness (I/M) ratio between the groups. Using Scion Image
for Windows image analysis software, the total cross-sectional
intimal area was measured between the endothelial cell mono-
layer and the internal elastic lamina (IEL), and the total cross-
sectional medial area was measured between the external
elastic lamina and the IEL. The intimal and medial surface
areas, as well as the I/M ratio, were taken as measures of the
severity of atherosclerosis (15).
Statistical analysis
Results were expressed as mean ± SD. Repeated-measures
ANOVA was used for statistical analysis. A value of P<0.05
was considered to be significant.
RESULTS
Body weight
Sequential changes of the body weight of the rabbits in the four
groups at zero, four and eight weeks were documented in kilo-
grams. Compared with baseline measurements, there was a
significant increase in the body weight in all groups at eight
weeks. At zero, four and eight weeks, the mean weights in
group I were 1.74±015 kg, 2.45±0.08 kg and 2.92±0.12 kg,
respectively. In group II, the weights were 1.8±0.07 kg,
2.5±0.13 kg and 2.95±0.09 kg, respectively, and in group III,
the respective weights were 1.78±0.09 kg, 2.56±0.14 kg and
2.94±0.22 kg. In group IV, the weights were 1.75±0.1 kg,
2.45±0.22 kg and 2.93±0.20 kg, respectively. No significant
differences in weights were observed among the groups through-
out the study period.
Serum lipids
The baseline levels of serum TC in groups I, II, III and IV were
1.38±0.2 mmol/L, 1.26±0.3 mmol/L, 1.5±0.7 mmol/L and
1.5±0.3 mmol/L, respectively. The baseline levels were not
significantly different among the groups. The sequential chan-
ges in serum TC in the four groups are shown in Figure 1.
Serum levels of TC remained unchanged in group I with
Ragheb et al
Int J Angiol Vol 17 No 4 Winter 2008188
respect to zero time. Serum levels of TC were elevated in
groups II, III and IV compared with their basal levels and these
levels were higher compared with those in group I at weeks 4
and 8. The levels at week 8 were not different from those at
week 4 in all groups, and these levels were lower in groups III
and IV compared with group II.
The baseline levels of serum TG in groups I, II, III and IV
were 0.79±0.2 mmol/L, 1.02±0.3 mmol/L, 0.62±0.1 mmol/L
and 0.45±0.1 mmol/L, respectively. The baseline levels were
not significantly different among the groups. The sequential
changes in serum TG in the four groups are shown in Figure 1.
Serum levels of TG remained unchanged in group I with respect
to zero time. Serum levels of TG increased in groups II, III and
IV compared with the initial levels and these levels were higher
compared with those in group I at weeks 4 and 8. The levels at
week 8 were not different from those at week 4 in all groups and
these levels were lower in group IV compared with group III.
The baseline levels of serum LDL-C in groups I, II, III and IV
were 0.4±0.2 mmol/L, 0.35±0.2 mmol/L, 0.72±0.7 mmol/L and
0.59±0.3 mmol/L, respectively, and were not significantly differ-
ent from each other. The sequential changes in LDL-C in the
four groups are shown in Figure 2. Serum levels of LDL-C
remained unchanged in group I with respect to zero time. Serum
LDL-C levels increased in groups II, III and IV compared with
the baseline levels and these levels were higher compared with
those in group I at weeks 4 and 8. LDL-C levels at week 8 were
not different from those at week 4 in all groups and these levels
were lower in groups III and IV compared with group II.
Figure 1) Sequential changes in serum total cholesterol (TC) and triglycerides (TG) in the four groups. Results are expressed as mean ± SD.
Group I: normal diet; group II: 1% cholesterol diet; group III: 1% cholesterol diet plus thymoquinone 10 mg/kg/day; group IV: 1% cholesterol
diet plus thymoquinone 20 mg/kg/day. aP<0.05, zero time versus four and eight weeks in the respective groups; bP<0.05, group I versus other
groups; cP<0.05, group II versus groups III or IV; dP<0.05, group III versus group IV
Figure 2) Sequential changes in serum low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in the
four experimental groups. Results are expressed as mean ± SD. Group I: normal diet; group II: 1% cholesterol diet; group III: 1% cholesterol
diet plus thymoquinone 10 mg/kg/day; group IV: 1% cholesterol diet plus thymoquinone 20 mg/kg/day. aP<0.05, zero time versus four and
eight weeks in the respective groups; bP<0.05, group I versus other groups; cP<0.05, group II versus groups III or IV
Thymoquinone and atherosclerosis
Int J Angiol Vol 17 No 4 Winter 2008 189
The initial levels of serum HDL-C in groups I, II, III and IV
were 0.62±0.2 mmol/L, 0.44±0.2 mmol/L, 0.49±0.2 mmol/L
and 0.7±0.1 mmol/L, respectively, and were not significantly
different among the groups. The sequential changes in HDL-C
in the four groups are shown in Figure 2. Serum levels of
HDL-C remained unchanged in group I with respect to zero
time. Serum HDL-C levels increased in groups II, III and IV
compared with their initial levels and these levels were higher
compared with those in group I at weeks 4 and 8. HDL-C levels
at week 8 were not different from those at week 4 in all groups
and these levels were lower in groups III and IV compared with
group II at weeks 4 and 8.
The baseline values of the risk ratio TC/HDL-C in groups I,
II, III and IV were 2.31±0.4, 3.04±1, 3.03±1 and 2.15±0.3,
respectively. The basal values were not significantly different
among the groups. The sequential changes in the risk ratio in
the four groups are shown in Figure 3. The risk ratio remained
unchanged in group I and increased in groups II, III and IV
with respect to zero time. The risk ratio values were increased
to a similar extent in groups II, III and IV compared with those
in group I at weeks 4 and 8. There were no significant differ-
ences among groups II, III and IV at weeks 4 and 8.
MDA levels
Serum MDA: The basal values of serum MDA in groups I, II,
III and IV were 0.93±0.4 mmol/L, 0.84±0.3 mmol/L,
0.86±0.2 mmol/L and 0.32±0.1 mmol/L, respectively, and were
not significantly different among the groups except in group IV,
which had a lower value than the other groups. The sequential
changes in serum MDA in the four groups are shown in Figure 3.
The values remained unchanged in group I but increased in
group II at weeks 4 and 8 and in groups III and IV at week 4
only with respect to zero time. The increases in groups III and
IV were similar but lesser than those in group II at weeks 4 and
8. Serum MDA values in group II were higher than those in
groups I, III and IV at four and eight weeks.
Aortic tissue MDA: The MDA contents of the aortic tissue in
the four groups are shown in Table 1. The mean aortic MDA
content from group I was 0.25±0.06 nmol/mg protein. The
value in group II was 2.12-fold higher than that in group I. The
values in groups III and IV were 73% and 75%, respectively,
lower than in group II. The 1% cholesterol diet increased the
levels of aortic tissue MDA while TQ at doses of 10 mg/kg/day
and 20 mg/kg/day reduced these levels by 73% and 75%,
respectively.
Protein carbonyl levels
Serum protein carbonyls: Serum protein carbonyl levels in the
four groups at eight weeks are shown in Table 1. The mean
serum protein carbonyl level in group I was 1.34±0.25 nmol/mL
at the end of the eight weeks. The 1% cholesterol diet in group
II increased the levels of serum protein carbonyls to a mean of
5.34±1.36 nmol/mL. The values in groups III and IV were
62% and 57%, respectively, lower than in group II. The 1%
TABLE 1
Serum protein carbonyls and aortic malondialdehyde
(MDA) and protein carbonyl levels in the four groups at
eight weeks
Group I Group II Group III Group IV
Serum protein
carbonyl content,
nmol/mL
1.34±0.25 5.34±1.36* 2.01±0.97** 2.27±0.4**
Aortic MDA,
nmol/mg
0.25±0.06 0.53±0.32* 0.14±0.07** 0.13±0.06**
Aortic protein
carbonyl content,
nmol/mg
0.40±0.42 0.48±0.38 0.42±0.42 0.36±0.11
Results are expressed as mean ± SD. *P<0.05, group I versus other groups;
**P<0.05, group II versus groups III and IV. Group I: normal diet; group II: 1%
cholesterol diet; group III: 1% cholesterol diet plus thymoquinone 10 mg/kg/day;
group IV: 1% cholesterol diet plus thymoquinone 20 mg/kg/day
Figure 3) Sequential changes in total cholesterol (TC)/high-density lipoprotein cholesterol (HDL-C) ratio and serum malondialdehyde (MDA)
in the four experimental groups. Results are expressed as mean ± SD. Group I: normal diet; group II: 1% cholesterol diet; group III: 1%
cholesterol diet plus thymoquinone 10 mg/kg/day; group IV: 1% cholesterol diet plus thymoquinone 20 mg/kg/day. aP<0.05, zero time versus
four and eight weeks in the respective groups; bP<0.05, group I versus other groups; cP<0.05, group II versus groups III or IV; eP<0.05, group
IV versus other groups
Ragheb et al
Int J Angiol Vol 17 No 4 Winter 2008190
cholesterol diet increased the levels of serum protein carbonyls
by 3.89-fold compared with the regular diet (group I), while
TQ in groups III and IV produced inhibition of serum protein
carbonyl levels by approximately 1.5-fold compared with the
control group.
Aortic tissue protein carbonyls: Aortic tissue protein carbonyl
levels of the four groups are summarized in Table 1. The mean
protein carbonyl content of the aortic tissue from group I was
0.40±0.42 nmol/mg protein. The values in all groups were
similar.
Histological effects
Extent of atherosclerotic plaque formation: Representative
photographs of the atherosclerotic changes in the intimal sur-
face of the aortas from each group are shown in Figure 4 and
the results are summarized in Table 2. There were no athero-
sclerotic changes in the intimal surface of the aortas from
group I. Significant areas of the intimal surfaces of aortas from
groups II (45.08%±11.65%), III (27.81%±4.99%) and IV
(20.67%±4.38%) were covered with atherosclerotic plaques.
Administration of TQ at doses of 10 mg/kg/day and 20 mg/kg/
day reduced the development of atherosclerosis by 40% and
53%, respectively. No significant difference was found between
the effects of the two doses of TQ.
Microscopic changes in the aortic segments: Representative
photographs of the histological sections of the aorta stained
with H&E and the Accustain Elastic Stain Kit from the four
groups are shown in Figures 5 and 6, respectively.
In Figure 5, the control group (group I) specimens showed
normal arterial wall structure with no atherosclerotic chan-
ges. Group II showed moderate to severe atherosclerotic
lesions. Administration of TQ at doses of 10 mg/kg/day and
20 mg/kg/day with the 1% cholesterol diet, in groups III and
IV, showed only mild localized areas of atherosclerosis.
In Figure 6, group I specimens were intact throughout the
entire wall of the artery. Group II showed diffuse damage in the
IEL at the sites of the atherosclerotic changes. Administration
of TQ at doses of 10 mg/kg/day and 20 mg/kg/day with the 1%
cholesterol diet, in groups III and IV, showed only focal damage
in the IEL while most of the wall showed intact IEL.
Changes in intimal and medial thicknesses in the aortic seg-
ments: The intimal and medial surface areas and ratios are
summarized in Table 2. The intimal and medial cross-sectional
areas and the I/M ratio values in group I were 7.6±1.5 ×103 µm²,
52.3±9.7 ×103 µm² and 0.15±0.01 ×103 µm², respectively. Both
the intimal cross-sectional area and I/M ratio values were
increased by 4.3- and 3.5-fold, respectively, in group II while
the medial cross-sectional area remained unchanged.
Administration of TQ reduced the intimal cross-sectional area
and I/M ratio by 52% and 45%, respectively, in group III, and by
60% and 50%, respectively, in group IV. No significant differ-
ences were found between the effects of the two doses of TQ.
TABLE 2
Atherosclerotic changes in the four groups
Group I Group II Group III Group IV
Atherosclerosis
(% of total
intimal
surface)
0 45.08±11.65* 27.81±4.99*†20.67±4.38*†
Intimal area 7.58±1.52 32.38±7.13* 15.54±6.86*†13.31±4.23*†
Medial area 52.31±9.71 64.56±10.88 52.49±17.13 50.46±13.65
I/M ratio 0.15±0.01 0.52±0.06* 0.29±0.04*†0.26±0.02*†
Results are expressed as mean ± SD. Intimal (I) and medial (M) surface areas
are expressed as ×103 μm2. *P<0.05, group I versus other groups; †P<0.05,
group II versus groups III and IV. Group I: normal diet; group II: 1% cholesterol
diet; group III: 1% cholesterol diet plus thymoquinone 10 mg/kg/day; group IV:
1% cholesterol diet plus thymoquinone 20 mg/kg/day
Figure 4) Representative photographs of the intimal surfaces of
aortas from the four experimental groups showing Sudan IV-stained
lipid deposits (dark staining in groups II, III and IV). Note the dif-
ference between the extent of atherosclerosis between group II and
groups III and IV. Group I: normal diet; group II: 1% cholesterol
diet; group III: 1% cholesterol diet plus thymoquinone 10 mg/kg/day;
group IV: 1% cholesterol diet plus thymoquinone 20 mg/kg/day
Figure 5) Representative photographs of the microscopic changes from
the four groups stained with hematoxylin and eosin. Note the normal
arterial wall structure in group I (original magnification ×10), the
increased intimal thickness in group II that involves most of the aortic
circumference and the lesser thickness that involves much smaller por-
tions of the aortic circumference in groups III and IV (original magni-
fication ×4). Group I: normal diet; group II: 1% cholesterol diet;
group III: 1% cholesterol diet plus thymoquinone 10 mg/kg/day; group
IV: 1% cholesterol diet plus thymoquinone 20 mg/kg/day
Thymoquinone and atherosclerosis
Int J Angiol Vol 17 No 4 Winter 2008 191
DISCUSSION
TQ doses used in the present study were similar to those used
by other investigators. Based on that, there was no significant
difference in the body weight gain among the studied groups,
indicating that the various interventions did not affect the
body weight. The high-cholesterol diet induced a significant
increase in the serum levels of TC, TG, LDL-C, HDL-C and
the TC/HDL-C ratio. Similar changes in the lipid profile were
reported in several studies (4,16). Administration of TQ at a
dose of 10 mg/kg/day reduced the levels of the TC, LDL-C and
HDL-C. However, the TC/HDL-C ratio did not show any
change due to the concomitant decreases in both TC and the
HDL-C levels. The TG levels were not affected by TQ admin-
istration. No previous data are available on the effects of TQ
on lipid profile in rabbits. However, Zaoui et al (11) reported
that N sativa seed oil has a lipid-lowering effect in rats. TQ was
also reported to produce significant reductions in the levels of
TC, TG, LDL-C and HDL-C in albino rats, but with no linear
dose- or time-dependent effect (17). The mechanism of these
hypolipidemic effects is unknown.
Consistent with previous reports (2,4,16), the present study
demonstrated the coexistence of severe hyperlipidemia, enhanced
ROS activity and atherosclerosis in this rabbit model. The study
showed for the first time, to our knowledge, the increased levels
of serum protein carbonyls in rabbits fed a high-cholesterol diet.
Protein carbonyls are a marker of irreversible damage due to
enhanced ROS activity (18), which validates this model for the
study of ROS injury. The extent of atherosclerosis in the present
study was 45% in the 1% cholesterol-fed group of rabbits. The
cholesterol diet also increased the intimal cross-sectional area
and I/M ratio 4.3 and 3.5 times, respectively, compared with
the control. The intimal cross-sectional area and I/M ratio
were considered previously as markers for the severity of ath-
erosclerosis (15). Finally, the cholesterol diet induced marked
destruction to the IEL. In the current study, the hypercholes-
terolemic atherosclerosis was associated with increases in the
serum and aortic MDA and protein carbonyls, suggesting an
increase in the levels of ROS. Several mechanisms were postu-
lated to explain the role of ROS in the pathogenesis of athero-
sclerosis. One of these mechanisms is the oxidation of LDL
molecules to highly immunogenic oxidized LDL molecules in
the presence of excess ROS (19). Oxidized LDL molecules are
found in the subendothelial layers and help activate monocytes
that are transformed into macrophages, upregulating their
scavenger receptors and toll-like receptors that then phagocyt-
ose them. With progressive accumulation of oxidized LDL,
macrophages modulate their phenotype, turning them into
foam cells. Foam cells are the principal component of the fatty
streaks, the first step in atheromatous plaque formation (20).
ROS may also induce atherosclerosis by producing endothelial
cell injury (21) and modulation of adhesion molecules (22).
The antiatherogenic effect of TQ may be due to its anti-
oxidant and/or anti-inflammatory effects. Previous in vitro and
in vivo studies reported that TQ is a potent superoxide anion
scavenger (10). TQ was also found to inhibit the cyclooxygen-
ase and lipoxygenase enzymes (23). Cyclooxygenase enzymes
generate prostaglandins and thromboxane from arachidonic
acid, while lipoxygenase enzymes catalyze the formation of
leukotrienes (24). Oxygen radicals are produced during the
synthesis of prostaglandins and leukotrienes (25). In the
present study, the use of TQ decreased the levels of the serum
and aortic MDA and the serum protein carbonyls, suggesting a
decrease in the levels of the ROS. The higher dose of TQ did
not further lower the levels of the serum or aortic MDA, or the
serum protein carbonyls. Results from previous experiments
also showed that increasing TQ dose over 10 mg/kg/day was
not more beneficial in protecting rats against cisplatin-induced
nephrotoxicity (26). The present study is the first to demon-
strate the effects of TQ on aortic MDA and protein carbonyls
in hypercholesterolemic atherosclerotic rabbits. In other mod-
els, TQ administration significantly reduced the brain tissue
MDA in ischemia-reperfusion injury (15), the hepatic MDA in
carbon tetrachloride-induced hepatotoxicity (27) and the
renal MDA in gentamicin-induced nephrotoxicity (28). The
decrease in the levels of serum and aortic ROS induced by TQ
is probably due to antioxidant properties of TQ (10). In the cur-
rent study, the antiatherosclerotic effect of TQ (10 mg/kg/day)
was manifested in group III by a 40% decrease in the extent of
atherosclerosis, a 52% decrease in the intimal surface area and
a 45% decrease in the I/M ratio compared with group II. It also
minimized hypercholesterolemia-induced damage in the IEL of
the arteries. The 20 mg/kg/day dose showed 53%, 60% and
50% decreases in the atheromatous and intimal areas and I/M
ratio, respectively, in group IV.
CONCLUSION
TQ reduced the development of atherosclerosis and this was
associated with reductions in serum lipids and oxidative stress.
Increasing the dose of TQ from 10 mg/kg/day to 20 mg/kg/day
was not associated with a further improvement. Further testing
in humans is suggested.
Limitations of the study
Caution should be exercised to interpret our results.
Although our data indicate that reduction of oxidative
Figure 6) Representative photographs of the microscopic changes from
the four groups stained with elastic fibre stain. Note the black-stained
elastic fibres, the relatively well-defined internal elastic lamina (IEL)
and the poorly defined external elastic lamina (EEL) (original magni-
fication ×4). Group I: normal diet; group II: 1% cholesterol diet;
group III: 1% cholesterol diet plus thymoquinone 10 mg/kg/day;
group IV: 1% cholesterol diet plus thymoquinone 20 mg/kg/day
Ragheb et al
Int J Angiol Vol 17 No 4 Winter 2008192
stress and/or hyperlipidemia is associated with attenuation
of atherosclerotic plaque development, it is plausible that TQ
may have other functions independent of its effect on the ROS
system. Further work is necessary to identify the cause of
reduction in lipid parameters by TQ. Of further interest, it
would be important to study the macrophage content of ath-
erosclerotic lesions in rabbits with and without TQ treatment.
Our data also do not address the potential toxicity of TQ.
ACKNOWLEDGEMENT: The authors thank Mr Mabood Qureshi
and Mrs Heather Neufeld for their support in performing the bio-
chemical and histological assays of the study.
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