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Introduction
Curcumin is extracted from Curcumae
longae, and has been demonstrated to have a variety of pharmacological
effects, such as antitumoric, anti-inflammatory, as well as
anti-oxidative effects[1-3]. Some studies have revealed that
curcumin can decrease lipid peroxidation and cholesterol
levels of sera and tissues in mice and
human[4,5]. It is well known that atherosclerosis largely results from oxidative stress and
massive lipid deposition in the aorta.
Olszanecki et al[6] reported a preventive effect of curcumin on atherosclerotic
development in apoE/LDL-Receptor double-knockout mice.
A recent study showed that some genes, such as LDL-R,
sterol response element-binding protein (SREBP), and CD36,
involved in cholesterol homeostasis were influenced by
curcumin[7]. However, the detailed anti-atherosclerotic
mechanisms of curcumin are still to be elucidated.
Caveolin-1 is a 22-24 kDa constructive protein in
caveolae[8]. Evidence demonstrates that the caveolin-1 is able to
directly bind free cholesterol (FC) and form a cholesterol transport
complex with other elements[9-11]. In addition, the upregulation
of the caveolin-1 expression promotes intracellular
cholesterol efflux and an improved lipid-loaded
state[12]. SREBP-1 exists in the cytoplasm and nucleus, and is associated with
cholesterol catabolism[13]. Its active form can translocate
into the nucleus and regulate target gene transcription
through binding with the sterol regulatory element (SRE)
sequence within the promoter[14]. It was found that the SRE
sequence also exists in the caveolin-1 promoter that is
negatively regulated by SREBP-1[15]. A more recent report showed
that SREBP expression could be regulated by curcumin and
is associated with reverse cholesterol
transport[7].
The present paper addresses whether curcumin prevents
ox-LDL-induced cholesterol from accumulation in cultured
vascular smooth muscle cells (VSMC) through inhibiting the
nuclear translocation of SREBP-1 followed by raising the
caveolin-1 expression.
Materials and methods
Reagents Dulbecco's modified Eagle's medium (DMEM)
and fetal bovine serum were purchased from Gibco/BRL
(Grand Island, NY, USA). Curcumin
([E,E]-1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione, purity =99%),
N-acetyl-Leu-Leu-norleucinal (ALLN; purity =97%) and oil
red O powder were purchased from Sigma_Aldrich (St Louis,
MO, USA) and dissolved by DMSO (Sangon, Shanghai, China). All reagents were of analytical grade.
Animals and diets The apoE_/_
mice were obtained from the barrier unit at the Laboratory Animal Center of Chongqing
Medical University (Chongqing, China). The
apoE_/_ mice were randomized into 3 groups and had similar body weights
(n=7 in the apoE_/_ mice group;
n=8 in the apoE_/_ mice with curcumin group; and
n=8 in the apoE_/_ mice with lovastatin
group). C57BL/6J mice (n=7) were used as the control. Three
groups of apoE_/_ mice were given a high-fat diet (21% lard
and 0.15% cholesterol). The C57BL/6J mice were fed a
normal diet. The animals were housed in single pens under
controlled conditions (temperature between 18 °C and 22 °C,
relative air humidity between 30%_70%, with 4 air changes
per hour) and fed 3 times daily on a restricted schedule. All
mice received the same amount of food (4% body weight).
The dose of curcumin (purity =94%) administered (mixed with
diet) for the curcumin group was 20
mg·kg_1·d_1 per mouse (4
months). The dose of lovastatin administered for the
lovastatin group was 40
mg·kg_1·d_1.
The total study period was 5 months. Morphological changes of atherosclerotic
lesions in the aorta were measured by histological section
and hematoxylin-eosin (HE) staining.
Morphological examination of atherosclerotic lesions
At the end of the experimental period, the animals were
euthanized by phlebotomy under light anesthesia with
sodium pentobarbital (30 mg·kg_1 intravenously; Jilin Northern
Medicine, Jilin, China). The atherosclerotic lesions were
analyzed as described previously[16]. Briefly, the aorta was cut
into transversal sections of 4 µm and fixed in 10% formalin
for 24 h. The tissue slices were routinely processed and
embedded in paraffin. Lipid deposition in the aorta was
determined by the morphological assessment of the
percentage of lesion-covered aortas as visualized by HE staining
under a light microscope at 40× magnification.
Serum lipid and lipoprotein analysis Non-fasting mice
were anesthetized with sodium pentobarbital, and blood was
collected. Serum total cholesterol (TC), triglyceride (TG),
High Density Lipiprotein-Cholesterol (HDL-C) and Lower
Density Lipiprotein (LDL-C) were determined by commercial
enzymatic methods (test kits, Shanghai Rongsheng Biotechnology, Shanghai, China).
Cell culture Rat VSMC were maintained in DMEM
containing 10% fetal bovine serum in a humidified atmosphere
of 5% CO2 and 95% O2. The cells were pre-incubated with 50
mg·L_1 ox-LDL for 48 h and then treated with various
concentrations of curcumin (purity =94%; 12.5, 25, and 50
mmol·L_1) or ALLN (25
µmol·L_1) for 24 h. Untreated cells were used as
controls.
Oil Red O staining The culture was washed 3 times by
phosphate-buffered saline (PBS) to remove suspended cells.
The VSMC were fixed with 10% formalin for 10 min. After
washing with PBS, the cells were stained with oil red O
solution (solubilized in isopropanol: water, 3:2) for 30 min. Then
the cells were washed with 2-propanol for 10 s to remove
background staining. The cells were then stained with HE
for 5 min to stain the nuclei, and images were taken at 40×
magnification.
Intracellular lipid level analysis by HPLC
Cellular lipid (TC, FC, and cholesterol ester [CE]) contents were analyzed
by our method described previously[17]. Briefly, the VSMC
were scraped from the culture flasks into 0.9% NaCl (1 mL/50
cm2 flask) and homogenized on ice by sonication for 10 s.
After the protein concentration was determined using a BCA
kit (Pierce Biotechnology, Rockford, IL, USA), an equal
volume of freshly-prepared, cold (_20 °C) potassium hydroxide
in ethanol (150 g·L_1) was added to the cell lysates, and the
mixture was repeatedly vortexed until clear.
An equal volume of 3:2 hexane isopropanol
(v/v) was then added. The mixture was vortexed for 5 min, followed by centrifugation at
800×g (15 °C) for 5 min. The extraction procedure was
repeated twice. The combined organic phase was transferred
to clean tapered glass tubes and thoroughly dried under
nitrogen at 40 °C. The tubes were allowed to cool to room
temperature. One hundred microliters of
isopropanol-acetonitrile 20:80 (v/v) was added. The sample was solubilized in
an ultrasound water bath at room temperature for 5 min.
After centrifugation at 800×g for 5 min, the samples were
introduced into the HPLC device (Agilent 1100, Agilent
Technologies, Palo Alto, CA, USA). Cholesterol was eluted
with 1 mL·min_1 of eluent consisting of 20:80
isopropanol-acetonitrile (v/v), and detected by UV absorption at 206 nm.
Protein isolation and Western blotting The proteins
were isolated from flash-frozen apoE_/_
mice aortas as previously
described[18]. The total proteins (10-50
mg/lane) were electrophoresed, separated on 10% SDS-PAGE, and
transferred to a polyvinylidene difluoride membrane
(Millipore Corporation, Billerica, MA, USA), which was blocked in 5%
non-fat dry milk in Tris-buffered saline Tween (TBST; pH
7.6). The membrane was incubated overnight with a rabbit
polyclonal antibody to rat caveolin-1 or a monoclonal
antibody to mouse caveolin-1 and SREBP-1p68 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:1000
on a rotating platform at 4 °C. Subsequently, the membrane
was rinsed in TBST (pH 7.6) for 40 min and incubated with
horseradish peroxidase (HRP)-conjugated antirabbit
immunoglobulin G (IgG) antibodies (Boster, Wuhan, China)
diluted in TBST (1:4000) for 1 h on a rotating platform at 37 °C.
Bands were visualized using a HRP developer, and
background-subtracted signals were quantified on a laser
densitometer (Bio-Rad, Hercules, Califormia, USA). Blots were
probed with a mouse anti-â-actin monoclonal antibody (Boster,
China) to ensure equal protein loading. All protein levels were
assessed by densitometry, with b-actin used as a control.
Indirect immunofluorescence with double staining
For the immunofluorescence analysis,
2×105 cells were plated into each well of 6-well plate preplaced coverslips. The cells
were pretreated with ox-LDL for 48 h and treated with or
without curcumin. Then the cells were fixed with methanol
acetic acid (3:1) for 15 min at room temperature. The samples
were then permeabilized with 0.25% Triton X-100 in PBS
(Amresco, Solon, OH, USA) and 5% DMSO for 20 min at _20
°C, then washed twice with PBS containing 0.25% Triton X-100.
The cells were incubated with an anti-SREBP antibody (Santa
Cruz Biotechnology, USA) overnight at 4 °C in a humidified
chamber. After washing 3 times with PBS, the cells were
incubated with Cy3-labeled goat antirabbit IgG (Sigma, USA)
for 1 h and then incubated with
4',6-diamidino-2-phenylindole (DAPI)(from 0.1 to 1.0
mg·mL_1) for 10 min to display the cell
nucleus protected from light at room temperature. The cells
were then visualized using a fluorescence microscope
(Olympus, Tokyo, Japan), with red and blue representing the cytoplasm
and nucleus, respectively. Data were acquired with a Pixera
camera (Los Angeles, CA, USA).
Statistical analysis The results were expressed as
mean±SD. The two-tailed Student's t-test was used for
statistical comparisons. P<0.05 was considered to be
statistically significant. For
non-quantitative data, results are representative of at least 3 independent experiments.
Results
Curcumin improved atherosclerotic lesions and
increased plaque stability in apoE_/_ mice
To identify the anti-atherosclerotic role of curcumin, we first observed its effects
on plaque size and stability in apoE_/_ mice. HE staining
showed that the lesion area in apoE_/_mice seemed larger, the
aorta lumen became narrower, and the structure fibrous cap in
the lesions became brittle (Figure 1B). After administration of
curcumin (20
mg·kg_1·d_1) and lovastatin (40
mg·kg_1·d_1) for 4
months, the lesion area was reduced by 50%, and the plaque
size became more stable (Figure 1C, 1D).
Curcumin increased the caveolin-1 expression and
decreased the SREBP-1p68 expression of the aortic wall in
apoE_/_ mice Since caveolin-1 promotes intracellular
cholesterol efflux and SREBP-1 regulates caveolin-1 expression, we
were interested in examining whether the effect of
curcumin in reducing atherosclerotic lesions in
apoE_/_ mice was related to the SREBP-1/caveolin-1 pathway. The Western blotting
analysis showed an 80% lower caveolin-1 expression in
apoE_/_ mice as compared to C57BL/6J mice. However, the
caveolin-1 level in the curcumin group showed a 5-fold elevation as
compared with the models. In contrast,
apoE_/_ mice showed a 2-fold higher level of SREBP-1p68, an active form of
SREBP-1, compared to C57BL/6J mice. Curcumin significantly inhibited
high fat-induced the SREBP-1p68 expression in
apoE_/_ mice. The effects of curcumin were similar to lovastatin (Figure 2).
Curcumin improved plasma levels of lipids in
apoE_/_ mice TC, TG, and LDL-C were significantly increased in the
apoE_/_ mice fed a high-fat diet than those in the control group
(C57BL/6J mice). The administration of curcumin markedly decreased
plasma TC, TG, and LDL-C levels. Furthermore, a significant
increase in HDL cholesterol was observed in
curcumin-treated apoE_/_ mice. The regulating effect of curcumin on
blood lipids was similar to that of lovastatin (Table 1).
Curcumin inhibited cholesterol accumulation in rat
VSMC and origin lipid-loaded cells To further investigate
the mechanism of curcumin in inhibiting plaque size and
stabilizing plaque, we observed the effects of curcumin on
cholesterol accumulation in VSMC and origin lipid-loaded cells
induced by ox-LDL. Oil red O staining demonstrated that
many lipid droplets were full of lipid-loaded cells. Treatment
with curcumin (12.5, 25, and 50
µmol·L_1) for 24 h markedly reduced the number of lipid droplets (Figure 3) without
significant cellular toxicity (data not shown). The HPLC
analysis showed that curcumin decreased intracellular lipid levels
with a peak at 25 µmol·L_1 for 24 h; CE was reduced from
141±15 to 45±3.7 mg·g_1 protein (Table 2).
Curcumin facilitated caveolin-1 expression in rat VSMC
and origin lipid-loaded cells As already
stated, caveolin-1 plays a critical role in cholesterol
flux[10,11]. We tried to determine whether the intracellular caveolin-1 expression could
be influenced by curcumin during lipid-loaded state
improvement. Lipid-loaded cells were exposed to curcumin
at various concentrations (12.5, 25, and 50
µmol·L_1) for different durations (0, 6, 12, 24, and 48 h). Curcumin treatment
for 24 h significantly increased the caveolin-1 expression in
a concentration-dependent manner (Figure 4A) and a peak
at 25 µmol·L_1 (Figure 4B).
Curcumin inhibited the nuclear translocation of
SREBP-1 in rat VSMC and origin lipid-loaded cells
It has been reported that SREBP-1 regulates the caveolin-1
expression[22]. To examine the role of SREBP-1 under curcumin treatment,
we observed SREBP-1 translocation by immunofluorescence
with double staining. Compared with the controls, ox-LDL
enhanced the SREBP-1 expression and stimulated SREBP-1
translocation from the cytoplasm into the nucleus (Figure 5).
Yet there was a redistribution of SREBP-1 after the curcumin
treatment. The SREBP-1 expression decreased gradually,
and its substantial portion accumulated in the cytoplasm,
and not in the nucleus (Figure 5).
ALLN abolished the effect of curcumin on SREBP-1
translocation in rat VSMC and origin lipid-loaded cells
To further confirm the relationship between SREBP-1 and
curcumin, the lipid-loaded cells were treated with 25
µmol·L_1 curcumin for 24 h and then with or without ALLN, an
inhibitor of SREBP-1 metabolism (or a promoter of SREBP-1).
Compared with the controls, curcumin inhibited the SREBP-1
expression and its nuclear translocation. However, ALLN
abolished the effect of curcumin and raised the SREBP-1
expression, as shown in Figure 6, with an increased density
of red staining in the cell nuclei accompanying its nuclear
translocation augmentation.
ALLN attenuated the effect of curcumin on the
caveolin-1 expression in rat VSMC-derived lipid-loaded cells
In an effort to understand the role of the SREBP-1/caveolin-1
pathway in the anti-atherosclerosis of curcumin, we observed
the caveolin-1 expression in the presence or absence of
ALLN. The Western blotting analysis indicated that ALLN
could lower caveolin-1 expression in both conditions with or
without ox-LDL (Figure 7A). Further experiments revealed
that ALLN attenuated the curcumin-increased caveolin-1
expression in the presence of ox-LDL (Figure 7B), indicating
that curcumin enhanced caveolin-1 levels by inhibiting the
SREBP-1 expression.
ALLN attenuated the effect of curcumin
inhibiting cellular cholesterol accumulation in rat VSMC and origin
lipid-loaded cells Because ALLN influenced the effects of
curcumin on SREBP-1 translocation and caveolin-1 expression, whether intracellular lipid levels could be
eventually affected by ALLN requires further study. We noticed
that the pretreatment of ALLN for 24 h counteracted the
effects of curcumin on decreasing cellular lipid levels (Table
3), indicating that curcumin inhibits cellular cholesterol
accumulation through the regulation of the
SREBP-1/caveolin-1 signaling pathway.
Discussion
To clarify the pharmacological mechanism of curcumin
on anti-atherosclerosis, we observed its effects on
apoE_/_ mice and cultured rat VSMC and origin lipid-loaded cells. In
the present study, we reproduced the anti-arthrosclerosis of
curcumin at animal and cellular levels, and further
documented that these effects were related to the inhibition of
SREBP-1 nuclear translocation followed by the increment of
the caveolin-1 expression.
As a constituent of the spice turmeric, the antitumoric
and anti-inflammatory effects of curcumin have been
studied[1-3]. Although several lines of evidence strongly suggest
that curcumin could prevent atherosclerotic development
by regulating some elements in cholesterol
homeostasis[6,7], the potential mechanism was unclear. Our animal experiment
showed that the administration of curcumin inhibited plaque
formation and enhanced plaque stability in
apoE_/_ mice.
Caveolae, discovered approximately 50 years ago, was
mainly formed by the caveolin family and is known as an
important hub not only in signal transmission, but also in
lipid transport across the plasma
membrane[19,20]. Considering that plaque regression may be linked to intracellular lipid
efflux, and caveolin-1 may play a key role, we subsequently
studied the relationship between the caveolin-1 expression
and anti-atherosclerosis of curcumin in
apoE_/_ mice and cholesterol accumulation in cultured VSMC. Our results
demonstrated that curcumin promoted the caveolin-1 expression
in apoE_/_ mice. In rat VSMC and origin lipid-loaded cells
induced by ox-LDL, curcumin promoted the caveolin-1
expression and downregulated lipid levels in a
dose-dependent manner. The data demonstrated that curcumin
anti-atherosclerosis and this effect are perhaps linked to
caveolin-1 expression enhancement.
The SREBP-1 precursor is located on membrane of the
endoplasmic reticulum. When intracellular FC changes, its
active fragment (SREBP-1p68) transfers into the nucleus to
regulate caveolin-1
transcription[13,15], which implies that
SREBP-1 probably participates in the effects of curcumin on
the caveolin-1 expression and lipid levels. To identify this
possibility, we observed the expression and translocation of
SREBP-1 in cultured VSMC treated with ox-LDL or curcumin.
The results showed that ox-LDL induced the high
expression of SREBP-1, but curcumin counteracted this effect and
inhibited its nuclear translocation. Meanwhile, curcumin
upregulated the caveolin-1 expression and reduced the
intracellular lipid levels correspondingly. The relationship
between SREBP-1 and caveolin-1 in our experiment was
consistent with a previous study[15].
It has been reported that ALLN could enhance the
SREBP-1 level by inhibiting the catabolism of SREBP-1, and the latter
could negatively regulate the caveolin-1
expression[15,21]. However, we were interested in whether the effects of
curcumin on SREBP-1 translocation and subsequent caveolin-1
expression, as well as cellular lipid levels, could be decreased
by ALLN. Interestingly, in the present of ALLN at a
concentration of 25 µmol·L_1, when the lipid-loaded cells were
incubated with curcumin, the curcumin-induced blockage of
SREBP-1 translocation was obviously eliminated,
curcumin-induced caveolin-1 expression was reduced, and
intracellular lipid levels were upregulated again. These findings
strongly suggest the effects of curcumin on increasing the
caveolin-1 expression and lowering intracellular lipid levels,
which were mediated by the inhibition of the nuclear
translocation of SREBP-1.
In conclusion, our data demonstrate that curcumin
inhibits ox-LDL-induced cholesterol accumulation in cultured
VSMC by regulating the SREBP-1/caveolin-1 pathway. This
new mechanism may be beneficial for anti-atherosclerotic
research. Together with previous studies, our results
indicate the potential of curcumin or its chemically-modified
derivatives as ideal anti-atherosclerotic drugs.
Acknowledgements
The authors are indebted to Dr Xue-feng XIA (Department of Internal Medicine, University of Texas,
Houston Health Science Center, USA) for his valuable help in the
preparation of the manuscript.
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