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CARDIOVASCULAR JOURNAL OF AFRICA • Volume 28, No 5, September/October 2017
AFRICA
299
Recent studies suggest that JNK induces apoptosis by directly
phosphorylating BA, Bim
EL
, and Bim
L
.
8-13
In addition, JNK also
phosphorylates and thus inactivates the anti-apoptotic Bcl-x
L
and Bcl-2.
14-16
In contrast to augmented studies on the regulation
of Bcl-2 family members by JNK, there is no proof that p38
regulates apoptosis through direct activation/inactivation of
Bcl-2 family proteins.
6,17
Moreover, Bcl-2 protein is a major
regulator of the intrinsic apoptosis signalling pathway. In recent
years it has been elucidated that it modulates the apoptotic
events in vascular cells.
7,18
Activation of caspase-3 plays a unique role in apoptosis
and is considered the final step prior to DNA fragmentation.
Caspase-3 triggers apoptotic DNA fragmentation by cleaving
DFF45 (DNA fragmentation factor 45) or ICAD (inhibitor of
caspase-activated DNase), which is changed to active DFF40/
CAD (caspase-activated DNase).
Statins, as classic inhibitors of 3-hydroxy-3-methylglutaryl-
coenzyme A (HMG-CoA) reductase, have been shown to
potentiate decreased plasma levels of cholesterol and the ratio of
oxLDL to native LDL, leading to attenuation of the development
of atherosclerosis.
19
Recently, the non-cholesterol-lowering effect
of statins, including their effect on platelet adhesion,
20
cytokine
release,
14
and anti-inflammatory effects
21
have been explored.
Atorvastatin (AV) is a lipophilic member of the statin
family and is mainly recommended for the treatment of
hypercholesterolaemia. It has been shown to have anti-
inflammatory benefits in the coronary arteries,
3,19,22
but the
effect of AV on the carotid arteries is seldom investigated.
Since inflammation and apoptosis are common events
in atherosclerosis, we speculated that AV may attenuate
cholesterol-induced injuries in carotid tissue via its influence
on inflammation and apoptosis in the carotid arteries. We
therefore evaluated the effects of AV on the MAPK signalling
pathway and apoptosis in this tissue.
Methods
In this applied, experimental study, 40 male Wistar rats (250
±
25 g) were obtained from the breeding colony of the Pasteur
Institute, Karaj, Iran. The experiment was carried out in 2015 in
the laboratories of the Neurosciences Research Centre (NSRC)
located at Tabriz University of Medical Sciences, Tabriz, Iran.
The study was approved by the ethics committee of Tabriz
University of Medical Sciences (approval number: A125345) and
conformed to the
Guidelines of the National Institute of Health
for the Care and Use of Laboratory Animals
(NIH Publications
No. 80-23).
The animals were kept under controlled conditions at 22
±
1°C
with a 12-hour light:dark cycle and 50–55% relative humidity.
They had free access to standard rodent chow and water, and
were housed in individual cages for 96 hours before use.
Atorvastatin (purity
≥
98%) (Lipitor
®
; Pfizer Inc, New York,
NY, USA), cholesterol (purity
>
99.9%), and the protease
inhibitor cocktail were purchased from Sigma-Aldrich (St
Louis, MO, USA). Rabbit anti-Bcl-2, anti-phospho-p38 (anti-
P-p38), anti-p38, HRP-conjugated goat anti-rabbit, anti-cleaved
caspase-3 and anti-B-actin polyclonal antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All
biochemical kits for colorimetric assays of plasma lipid profiles
were purchased from Zist Chimi Inc (Tehran, Iran).
We used simple randomisation by coin to divide the rats into
four dietary groups: normal diet (ND;
n
=
10), high-cholesterol
diet (HD;
n
=
10), high-cholesterol diet plus AV (HD
+
AV;
n
=
10) and the AV control group (AV;
n
=
10). HD rats received
the normal chow diet plus 2% cholesterol (Sigma-Aldrich,
No: C8667) whereas the ND group was fed only the normal
chow diet. Rats in the AV and HD
+
AV groups were given AV
(20 mg/kg) dissolved in 2 ml warm water before intra-gastric
administration.
23
All animals had access to food and water
ad
libitum
daily during the experiment.
Aftereightweeksof feedingtheHDdietwiththeadministration
of AV, the rats were intraperitoneally anesthetised using xylazine
(4 mg/kg; Sigma-Aldrich) and ketamine hydrochloride (10%, 40
mg/kg; Sigma-Aldrich). Following ligation of the left and right
common carotid arteries (CCAs), blood samples were withdrawn
directly from the heart of the rats and collected in a serum
separator tube. The blood was allowed to coagulate for two
hours at room temperature and centrifuged (Beckman model L
centrifuge) at 3 000 ×
g
for 20 minutes. The serum was saved for
biochemical analyses. The CCAs were removed, put into liquid
nitrogen and kept at –70°C for immunoblotting analysis.
Biochemical measurement of plasma levels of triglycerides
(TG), total cholesterol (TC) and high-density lipoprotein
cholesterol (HDL-C) were determined photometrically in a
Vitros 5600 autoanalyser (Ortho-Clinical Diagnostics Inc, USA)
in the endpoint manner using Ziest Chimi kits (Tehran, Iran).
To calculate LDL-C levels, Friedewald’s formula
24
was applied
as follows:
LDL-C (mg/dl)
=
TC (mg/dl) – TG (mg/dl)
____________________
5 – HDL-C (mg/dl)
.
25
Western blotting technique was used to evaluate the expression
of bcl-2, cleaved caspase-3 and phospho-p38 proteins, based
on the Santa Cruz online protocol. A 10% carotid tissue
homogenate in RIPA lysis buffer (Sigma) containing protease
inhibitor cocktail (Sigma-Aldrich) was prepared after being
centrifuged (SW14R, Froilabo, France) at 4°C and 13 000 ×
g
for 15 minutes.
Protein concentration was measured using the Bradford assay
(Bio-Rad Laboratories, CA, USA); 10 µg protein was loaded
into each well of 4–10% SDS polyacrylamide gel. Following
electrophoresis, proteins were blotted onto the membrane
(polyvinylidene fluoride, Bio-Rad) and blocked in 3% bovine
serum albumin (BSA) in phosphate-buffered saline (PBS) and
0.1% Tween 20 (PBST). Membranes were blotted overnight
at 4°C with the following primary antibodies diluted in PBST
containing 0.1% Tween: anti-Bcl-2 (N-19) (1:500; catalogue
number sc-492), anti-cleaved caspase-3 p11 (h176)-R (1:500;
catalogue number sc-22171-R) or anti-P-p38 antibody (Tyr 182)
(1:500; catalogue number sc-101759) and anti-p38 antibody
(1:500; catalogue number sc-535). After a rinsing step with
PBST, the membrane was incubated with a secondary antibody
(HRP-conjugated goat anti-rabbit) (1:5000; catalogue number
sc-2004).The membranes were then rinsed in PBST containing
0.05% Tween.
The immune complex was detected with a chemiluminescence
method using ECL-plus kits (GE Healthcare, USA). B-actin
protein expression was used as the loading control. The intensity
of the bands was determined and analysed with the Spectrum
multispectral imaging system by the Vision Work LS image
acquisition and analysis software (UVP, Germany).