Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats
We examined expressions of E-selectin and myeloperoxidase (MPO) in cerebral tissues after cerebral ischemia-reperfusion (CIR) in rats and evaluated neuroprotective effects of atorvastatin under these conditions. Immunohistochemical methods were used to detect E-selectin and MPO expressioning tiss...
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Інститут фізіології ім. О.О. Богомольця НАН України
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Zhong, W. Wu, W. Cao, H. Tu, Q. Tang, X. 2019-02-17T20:10:49Z 2019-02-17T20:10:49Z 2014 Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats / H. Cao, W. Zhong, W. Wu, Q. Tu, X. Tang // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 368-374. — Бібліогр.: 29 назв. — англ. 0028-2561 https://nasplib.isofts.kiev.ua/handle/123456789/148306 519.876.2+612.018+612.015.1 We examined expressions of E-selectin and myeloperoxidase (MPO) in cerebral tissues after cerebral ischemia-reperfusion (CIR) in rats and evaluated neuroprotective effects of atorvastatin under these conditions. Immunohistochemical methods were used to detect E-selectin and MPO expressioning tissue. Small numbers of E-selectin- and MPO-positive cells were observed in the sham group within 4 to 24 h time intervals. In the operation group, numerous positive cells were found in the cortex and hippocampus after CIR. E-selectin expression occurred at 4 h, peaked at 12 h, and returned to nearly normal levels at 24 h. However, E-selectin expression in the intervention (CIR + atorvastatin) group was significantly lower than that in the operation group at each time (P < 0.05). Myeloperoxidase had nearly similar timing of changes in E-selectin expression, and MPO expressions at different time points in the intervention group were significantly lower than those in the operation group (P < 0.05). Therefore, expressions of E-selectin and MPO change dynamically after CIR. Atorvastatin, an agent having anti-inflammatory properties, demonstrates an obvious protective effect with respect to acute CIR-related damage. Ми досліджували експресію Е-селектину та мієлопероксидази (МПО) у тканинах мозку після церебральної ішемії– реперфузії (ЦІР) у щурів та оцінювали нейропротективний вплив аторвастатину в цих умовах. Для виявлення експресії Е-селектину та МПО були застосовані імуногістохімічні методики. У контрольній групі в проміжку 4–24 год спостерігалися невеликі кількості Е-селектин- та МПО-позитивних клітин. У групі, підданій ЦІР, у корі та гіпокампі відмічалася велика кількість позитивних клітин. Експресія Е-селектину проявлялася через 4 год, досягала максимуму через 12 год та поверталася до майже нормальних рівнів через 24 год. У той же час експресія Е-селектину в тварин, котрим уводили аторвастатин після ЦІР, у всіх часових проміжках була вірогідно нижчою, ніж у попередній групі (P < 0.05). Експресія МПО демонструвала часовий перебіг, приблизно подібний такому в експресії Е-селектину, і рівень експресії МПО в усіх часових інтервалах у групі, підданій дії аторвастатину, був істотно нижчим, ніж у групі без уведення цього агента (P < 0.05). Отже, експресія Е-селектину та МПО після ЦІР динамічно змінюється; аторвастатин – агент з антизапальною дією – продемонстрував очевидний протективний ефект щодо ушкоджень, індукованих гострою ЦІР. This work was supported by the Natural Science Foundation of Hunan Province, China (Grant No. 11JJ5081) and the Hunan Provincial Science and Technology Department, China (Grant No. 2012SK3226 and Grant No. 2011SK3236). en Інститут фізіології ім. О.О. Богомольця НАН України Нейрофизиология Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats Вплив аторвастатину на експресію Е-селектину та мієлопероксидази після ушкоджень мозку щурів, пов’язаних з ішемією–реперфузією Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| title |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats |
| spellingShingle |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats Zhong, W. Wu, W. Cao, H. Tu, Q. Tang, X. |
| title_short |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats |
| title_full |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats |
| title_fullStr |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats |
| title_full_unstemmed |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats |
| title_sort |
effects of atorvastatin on e-selectin and myeloperoxidase expressions after cerebral ischemia-reperfusion injury in rats |
| author |
Zhong, W. Wu, W. Cao, H. Tu, Q. Tang, X. |
| author_facet |
Zhong, W. Wu, W. Cao, H. Tu, Q. Tang, X. |
| publishDate |
2014 |
| language |
English |
| container_title |
Нейрофизиология |
| publisher |
Інститут фізіології ім. О.О. Богомольця НАН України |
| format |
Article |
| title_alt |
Вплив аторвастатину на експресію Е-селектину та мієлопероксидази після ушкоджень мозку щурів, пов’язаних з ішемією–реперфузією |
| description |
We examined expressions of E-selectin and myeloperoxidase (MPO) in cerebral tissues
after cerebral ischemia-reperfusion (CIR) in rats and evaluated neuroprotective effects of
atorvastatin under these conditions. Immunohistochemical methods were used to detect
E-selectin and MPO expressioning tissue. Small numbers of E-selectin- and MPO-positive
cells were observed in the sham group within 4 to 24 h time intervals. In the operation group,
numerous positive cells were found in the cortex and hippocampus after CIR. E-selectin
expression occurred at 4 h, peaked at 12 h, and returned to nearly normal levels at 24 h. However,
E-selectin expression in the intervention (CIR + atorvastatin) group was significantly lower
than that in the operation group at each time (P < 0.05). Myeloperoxidase had nearly similar
timing of changes in E-selectin expression, and MPO expressions at different time points in
the intervention group were significantly lower than those in the operation group (P < 0.05).
Therefore, expressions of E-selectin and MPO change dynamically after CIR. Atorvastatin,
an agent having anti-inflammatory properties, demonstrates an obvious protective effect with
respect to acute CIR-related damage.
Ми досліджували експресію Е-селектину та мієлопероксидази (МПО) у тканинах мозку після церебральної ішемії–
реперфузії (ЦІР) у щурів та оцінювали нейропротективний
вплив аторвастатину в цих умовах. Для виявлення експресії
Е-селектину та МПО були застосовані імуногістохімічні методики. У контрольній групі в проміжку 4–24 год спостерігалися невеликі кількості Е-селектин- та МПО-позитивних
клітин. У групі, підданій ЦІР, у корі та гіпокампі відмічалася
велика кількість позитивних клітин. Експресія Е-селектину проявлялася через 4 год, досягала максимуму через 12 год
та поверталася до майже нормальних рівнів через 24 год. У
той же час експресія Е-селектину в тварин, котрим уводили
аторвастатин після ЦІР, у всіх часових проміжках була вірогідно нижчою, ніж у попередній групі (P < 0.05). Експресія
МПО демонструвала часовий перебіг, приблизно подібний
такому в експресії Е-селектину, і рівень експресії МПО в
усіх часових інтервалах у групі, підданій дії аторвастатину,
був істотно нижчим, ніж у групі без уведення цього агента (P < 0.05). Отже, експресія Е-селектину та МПО після
ЦІР динамічно змінюється; аторвастатин – агент з антизапальною дією – продемонстрував очевидний протективний
ефект щодо ушкоджень, індукованих гострою ЦІР.
|
| issn |
0028-2561 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/148306 |
| citation_txt |
Effects of Atorvastatin on E-Selectin and Myeloperoxidase Expressions After Cerebral Ischemia-Reperfusion Injury in Rats / H. Cao, W. Zhong, W. Wu, Q. Tu, X. Tang // Нейрофизиология. — 2014. — Т. 46, № 4. — С. 368-374. — Бібліогр.: 29 назв. — англ. |
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Article
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4368
UDC 519.876.2+612.018+612.015.1
H. CAO, 1 W. ZHONG,1 W. WU,1 Q. TU,2 and X. TANG 1
EFFECTS OF ATORVASTATIN ON E-SELECTIN AND MYELOPEROXIDASE
EXPRESSIONS AFTER CEREBRAL ISCHEMIA-REPERFUSION INJURY IN RATS
Received 28.10.2013
We examined expressions of E-selectin and myeloperoxidase (MPO) in cerebral tissues
after cerebral ischemia-reperfusion (CIR) in rats and evaluated neuroprotective effects of
atorvastatin under these conditions. Immunohistochemical methods were used to detect
E-selectin and MPO expressioning tissue. Small numbers of E-selectin- and MPO-positive
cells were observed in the sham group within 4 to 24 h time intervals. In the operation group,
numerous positive cells were found in the cortex and hippocampus after CIR. E-selectin
expression occurred at 4 h, peaked at 12 h, and returned to nearly normal levels at 24 h. However,
E-selectin expression in the intervention (CIR + atorvastatin) group was significantly lower
than that in the operation group at each time (P < 0.05). Myeloperoxidase had nearly similar
timing of changes in E-selectin expression, and MPO expressions at different time points in
the intervention group were significantly lower than those in the operation group (P < 0.05).
Therefore, expressions of E-selectin and MPO change dynamically after CIR. Atorvastatin,
an agent having anti-inflammatory properties, demonstrates an obvious protective effect with
respect to acute CIR-related damage.
KEYWORDS: cerebral ischemia-reperfusion (CIR), E-selectin, myeloperoxidase,
atorvastatin, inflammation.
1Department of Neurology, Second Xiangya Hospital of the Central South
University, Changsha, China.
2 Departments of Geriatrics, Third Xiangya Hospital of the Central South
University, Changsha, China.
Correspondence should be addressed to X. Tang
(e-mail: txq6633@126.com).
INTRODUCTION
Atorvastatin is a lipid-lowering drug used in therapy of
cardio-cerebrovascular diseases where hyperlipidemia
is a significant independent risk factor. The following
questions seem to be logical: in addition to the lipid-
lowering effect in the above diseases, can atorvastatin
also play a role in some other way, and can patients
with normal blood lipid levels use statins?
Stroke is one of the three greatest threats to human
health, with high morbidity, disability, and mortality.
Cerebral ischemia-reperfusion is aggravated with
inflammation, resulting in secondary injury to
ischemic tissues [1, 2]. There is a series of reactions
developing after CIR, and inflammation is one of the
main negative factors inducing secondary injury to
ischemised tissues.
E-select in plays an important role in the
inflammatory cascade after CIR. This agent is an
inducible adhesion molecule that mediates adhesion
between activated endothelial cells and neutrophils
and is directly involved in aggregation and infiltration
of inflammatory cells. Within the period following
CIR injury, white blood cells travel through the blood
vessel wall, interact with endothelial cells, and release
a number of inflammatory factors. In brain ischemic
tissues, the levels of mRNA of adhesion molecules
are significantly increased, large amounts of adhesion
molecules are synthesized, and the level of E-selectin
correlates with the production of oxygen free radicals.
Blocking of expression of E-selectin and suppression
of adhesion processes and accumulation of neutrophils
in the ischemic area can reduce death of neurons
and limit the infarct volume. This may improve the
prognosis in stroke patients. In models of cerebral
ischemia, the survival rate of animals increased after
anti-E-selectin antibody treatment. Myeloperoxidase,
MPO, exists primarily in neutrophil azurophilic
granules, and its activity is a reliable index accessing
the infiltration of neutrophils in the tissues.
Statins function as both regulation and non-
regulation agents with respect to lipids. In a number
of studies, it was shown that statins can provide
protection against renal, pulmonary, and heart
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 369
EFFECTS OF ATORVASTATIN ON E-SELECTIN AND MYELOPEROXIDASE EXPRESSIONS
ischemia-reperfusion injury [3-5]. However, there is
no information about the effect of statins in the case of
CIR injury. Our experiments were aimed at modeling
of CIR, observation of the levels of expression
of E-selectin and MPO, and investigation of the
mechanism of possible anti-inflammatory actions of
atorvastatin.
METHODS
Animal Groups. Sprague-Dawley rats were provided
by the Laboratory animal center of the Xiangya
Medical School of the Central South University
(Changsha, China). A total of 96 rats weighing from
240 to 260 g were divided randomly into three groups,
sham-operated, experimental (CIR), and intervention
(CIR + atorvastatin) groups. In the latter group, each
rat received daily 6.5 mg/kg atorvastatin one week
before the operation; atorvastatin was dissolved in
0.9% normal saline (NS) and administered by gavage;
other groups were given a gavage of NS of the same
volume. Each group was further divided into sub-
groups surviving 4, 8, 12, and 24 h after CIR.
Rat CIR Model. Focal CIR was modeled in rats
according to the Longa suture method [6]. A silicone-
coated nylon monofilament was inserted across
bifurcation of the common carotid artery to the right
internal carotid artery and then penetrated into the
brain until a proximal occlusion of the right middle
cerebral artery (MCA) was achieved. After 2 hours of
occlusion, the filament was slowly withdrawn to allow
the MCA blood supply to recover, and reperfusion was
allowed to continue for 4, 8, 12 and 24 h. Rats were
anesthetized with isoflurane (4% for surgical induction
and 1.75% for a maintenance dose) for surgery. The
body temperature was maintained at 37°C. In the sham
group, rats received only anesthesia and vascular
separation.
Neurological Deficit Grading System. After
revival of the rats from anesthesia, we observed
neurological symptoms before selecting materials.
Neurological deficit was estimated according to the
Longa’s Score in the following mode. Score 0 was
given if the rat demonstrated no symptoms of neural
damage; score 1 was given if the rat couldn’t be fully
extend the forelimbs; score 2 was given if the rat
made circling movements to the opposite side; score
3 was given if the rat fell down to the opposite
side, and score 4 was used if the rat couldn’t walk
spontaneously, with the loss of consciousness. Rats
receiving scores 1 to 3 were selected as observational
objectives.
Tissue and Slice Processing. Celiac anesthesia was
achieved with 10% novochlorhydrate (350 mg/kg).
After the perfusion procedure, the entire brain tissue
was removed. We observed whether subarachnoid
hemorrhage occurred at the skull base, and knocked
out the ones that were bleeding. Then, the fixed brain
tissue was sliced in continuous 4-µm thick coronary
sections sliced between the optic chiasm and leading
edge of the pons, with conventional dehydration,
hyalinization, wax soaking and embedding.
Triphenyl Tetrazolium Chloride (TTC) Staining.
After anesthesia with 10% novochlorhydrate, brain
tissue was removed from the skull and refrigerated at
–20°C for 30 min. After that, five coronal slices were
made with a 2 mm interval behind the frontal pole (at
1, 3, 5, 7, 9, and 11 mm) and incubated in darkness
for 30 min with 2% TTC-PBS solution at a constant
temperature (37°C). Regions that were stained red
were considered normal, while regions of cerebral
infarction appeared white. After staining, sections
were fixed for 24 h with 10% formaldehyde solution,
and pictures were taken with a digital camera.
Immunohistochemical Staining. After dewaxation
and hydration, brain tissue paraffin sections were
washed with PBS buffer three times, each time for
5 min. Then, each section was blocked with 50 µl of
a peroxidase-blocking solution (to block endogenous
peroxidase activity) and incubated for 10 min at
room temperature. Washed with PBS buffer three
times, each section was incubated with 50 µl of
nonimmunologic animal serum for 10 min at room
temperature prior to overnight incubation at 4°C
with 50 µl monoclonal rabbit anti-mouse E-selectin
antibody. Washed in PBS buffer (5 min, 3 times)
each section was incubated with 50 µl biotin-labeled
sheep anti-rat IgG and 50 µl Streptomyces-avidin-
peroxidase solution for 10 min at room temperature.
Then each section was subjected to the action of 100
µl of freshly prepared AEC solution for 10 min and
observed under a microscope. Brown-color cites
were considered positive. Immunohistochemical
results were processed by the MIAS medical image
analysis system. Average grey values were selected
to stand for the levels of expression of E-selectin and
MPO. Polyclonal E-selectin antibodies (1:100) and
polyclonal MPO antibodies (1:100) were purchased
from Biosynthesis Biotechnology Co. (Beijing,
China). Immunohistochemical staining protocols
(SP method) and DAB coloration were used for
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4370
H. CAO, W. ZHONG, W. WU et al.
amplification and visualization.
Statistical Analysis. Numerical results are ex-
pressed below as means ± s.e.m. Differences between
the groups were estimated by ANOVA or the LSD-test,
and those with P < 0.05 were considered to be statisti-
cally significant.
RESULTS
TTC Staining. Brain sections in the sham group were
uniformly red (Fig. 1A). In the CIR group, blood supply
to the lateral regions was significantly reduced, as
was shown by the presence of infarcted white regions
(shown in Fig. 1B with black arrow). The contralateral
side remained red and, thus, well perfused. So, the
CIR model was found to be successful.
E-Selectin Expression. Positive expression looked
as was tan-yellow in the cell boundaries and cytoplasm.
In the sham group, immunohistochemical staining
showed that little E-selectin expression appeared
within the cortical areas supplied by the middle
cerebral artery (4 h, 8.44 ± 1.27; 8 h, 12.59 ± 0.80;
12 h, 13.89 ± 1.29, and 24 h, 9.85 ± 1.05; Figs. 2A;
4). In the operation (CIR) group, E-selectin expression
peaked at 12 h after CIR. There was intense tan-yellow
E-selectin-positive immune staining in the ischemized
cerebral cortex (4 h, 28.05 ± 1.28; 8 h, 69.33 ± 0.97;
12 h, 84.19 ± 1.06, and 24 h, 60.43 ± 1.33; in all cases,
P < 0.05; Figs. 2B; 4). Expression of E-selectin in the
intervention (+ atorvastatin) group peaked at 12 h
after CIR. The level of expression in each time point
in the intervention group was statistically lower than
that in the operation group, but higher than that in the
sham group (4 h, 22.32 ± 0.94; 8 h, 48.20 ± 1.09; 12 h,
62.93 ± 0.94, and 24 h, 41.47 ± 1.18; P < 0.05 in all
cases; Figs. 2C; 4).
MPO Expression. Positive cites were tan in their
color. In the sham group, immunohistochemical
staining showed that relatively low MPO expression
appeared within the cortical areas supplied by the
cerebral artery (4 h, 7.98 ± 1.05; 8 h, 7.79 ± 1.26; 12 h,
F i g. 1. Triphenyl tetrazolium
chloride staining of brain sections
in the sham (A) and operation
(cerebral ischemia-reperfusion)
group (B).
Р и с. 1. Забарвлення зрізів моз-
ку тріфенілтетразолієм у групі
контролю (A) та групі тварин
після церебральної ішемії–
реперфузії (B).
A
A
B
B C
F i g. 2. Expression of E-selectin in the sham (A), operation (B), and intervention (C) groups; 12 h after sham operation (A) and cerebral
ischemia-reperfusion (B and C); ×400.
Р и с. 2. Експресія Е-селектину в контрольних тварин (А), щурів після церебральної ішемії–реперфузії – ЦІР (В) та щурів після ЦІР
і введень аторвастатину (С).
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 371
EFFECTS OF ATORVASTATIN ON E-SELECTIN AND MYELOPEROXIDASE EXPRESSIONS
8.22 ± 0.89, and 24 h, 7.51 ± 1.17; Figs. 3A; 5).
In the operation group, tan MPO immunostaining
reached the maximum in the ischemic cerebral cortex
12 hours after CIR (4 h, 15.95 ± 2.00; 8 h, 20.45 ±
± 1.97; 12 h, 21.75 ± 2.61, and 24 h, 18.10 ± 0.49; P <
< 0.05 in comparisons with the respective sham values;
Figs. 3B; 5). In the intervention group, MPO expression
also peaked at 12 h after CIR. However, the levels of
MPO staining at all time points were significantly lower
than those in the operation group, but significantly
higher than those in the sham group (4 h, 12.55 ±
± 1.51; 8 h, 13.80 ± 0.44; 12 h, 15.14 ± 0.36, and 24 h,
11.84 ± 0.33; P < 0.05 in all cases; Figs. 3C; 5).
DISCUSSION
Recent researches indicated that excessive inflamma-
tion in cerebral tissues after CIR is one of the main
causes of the reperfusion injury [7]. Inflammation is
A B C
F i g. 3. Expression of myeloperoxidase in the sham, operation, and intervention groups (A-C, respectively); 12 h.
Р и с. 3. Експресія мієлопероксидази в трьох групах тварин (А–С відповідно).
0
10
20
30
40
50
60
70
80
90
4 8 12 24 h
0
5
10
15
20
25
4 8 12 24 h
F i g. 4. Dynamics of E-selectin expression in the sham, operation,
and intervention groups (1-3, respectively). Abscissa) Time, h;
ordinate) optic density according to the grey scale.
Р и с. 4. Динаміка експресії Е-селектину в трьох групах тварин
(1–3 відповідно).
F i g. 5. Dynamics of myeloperoxidase expression in the sham,
operation, and intervention groups (1-3, respectively). Designations
are similar to those in Fig. 4.
Р и с. 5. Динаміка експресії мієлопероксидази в трьох групах
тварин (1–3 відповідно).
1
1
3
3
2
2
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4372
H. CAO, W. ZHONG, W. WU et al.
characterized by infiltration by leukocytes, activation
of vascular endothelial cells, and increase in the level
of adhesion molecules on the surface of leukocytes,
which led to obstruction of microvessels by rolling
and adhering. This resulted in tissue damage because
of crossing the endothelium tissue and releasing of
such factors as reactive oxygen species, proteins, and
lipids [8, 9]. In some studies, it was demonstrated that
clearing from peripheral leukocytes and/or inhibiting
of the activity of leukocytes with certain drugs can
protect cerebral tissues from negative consequences of
ischemia-reperfusion [10, 11].
Selectin is one of the members of the adhesion
molecule family, and it began to undergo intense
research since it was cloned in 1989. The selectin
family includes E-selectin, L-selectin, and P-selectin.
E-selectin and L-selectin are, respectively, expressed
in endothelial cells and white blood cells, while
P-selectin has been mainly found in platelets and
endothelial cells. Normal platelets have no of small
amounts of P-selectin on their surface. After CIR,
P-selectin can migrate to the surface of endothelial
cells in a few minutes; it is involved in mediating
initial adhesion of leukocytes and endothelial cells.
Initial P-selectin-mediated adhesion is a short-lasting
and reversible process; further interactions between
white blood cells and endothelial cells are mediated
by E-selectin. This selectin plays an important
role in CIR-induced injury being involved in a late
inflammatory response. E-selectin does not occur on
the surface of endothelial cells and is not activated
until endothelial cells are stimulated by inflammation.
This is in accordance with our results that low levels of
E-selectin expression were observed in the sham group,
while such expression was dramatically higher in the
operation group. In a primate stroke model, treatment
with antibodies against adhesion molecular receptors
reduced a “non-circulating flow” phenomenon. In the
rat model with knockout of both CD18 and CD26E
genes, adhesion of leukocytes was suppressed by 95%.
The effects with respect to E-selectin are specific and
cannot be replaced by those with respect to any other
adhesion molecule [12]. Glycogen with E-selectin
antibody can crucially reduce adhesion and infiltration
of leukocytes, leading to intensification of blood flow
in the ischemized area and limitation of the cerebral
infarction area [13]. Selectin is, at present, widely
concerned as a new target for the drugs influencing
adhesion of leukocytes during inflammation [14].
Immunohistochemistry showed that E-selectin is
expressed in the ischemic lateral cortex, striatum,
and hippocampus. Little expression was observed in
the sham group, which confirmed what was found in
previous studies [15]. After CIR, E-selectin expression
demonstrated clear dynamics; it started at about 4 h,
peaked at 12 h, and remained obvious at 24 h but with
noticeable reduction. This timing of expression is
close to that described in other reports [16, 17].
Myeloperoxidase is a peroxidase enzyme that
in humans is encoded by the MPO gene. This
peroxidase is most abundantly expressed in neutrophil
granulocytes (a subtype of white blood cells) and
stored in azurophilic granules, which is the most
reliable assessment biomarker of infiltration of
neutrophils in the tissue. In our experiments, MPO
was expressed in the ischemic lateral cortex, striatum,
and hippocampus. There were few MPO-positive
cells after CIR at each time point in the sham group,
which agrees with previous reports. Compared with
the above (sham) group, there were much more MPO
positive cells in the cortex and hippocampus after
CIR. Expression of MPO also changed dynamically
(started at 4 h, peaked at 12 h, and remained at 24 h
with significant reduction). Such timing of expression
is similar to changes of E-selectin expression. The
degrees of changes correlated with the degrees of
neurological deficit. Thus, it can be speculated
that changes in E-selctin expression are positively
associated with the degree of inflammation.
Statins are defined as inhibitors of 3-hydroxy-3-
methylglutaryl-coenzyme A (HMG-CoA) reductase.
These agents function better in reducing levels of
low-density lipoproteins (LDLs) and other sorts of
cholesterol-lowering drugs; meanwhile they increase
the amount of high-density lipoproteins (HDLs). In
addition, statins function “outside” of regulation
of lipids, by improving the endothelial function,
influencing the structure and stability of plaques,
preventing thrombosis, and providing an anti-
inflammatory action. Mueck et al. [18, 19] found that
fluvastatin can decrease the level of ICAM-1, VCAM-1,
and E-selectin in a dose-dependent manner.
Our results showed that atorvastatin intervention
statistically significantly decreases expression of
E-selectin. The mechanism of cerebral protection by
reducing E-selectin expression at present remains
mostly unknown. However, according to some
references, it is probably related to both lipid-lowering
and non-lipid-lowering functions of atorvastatin.
Atorvastatin, when providing anti-inflammatory
effects, lowers the levels of triglycerydes, cytokines
(LDC-C and MCP-1) and increases the level of LDLs.
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4 373
EFFECTS OF ATORVASTATIN ON E-SELECTIN AND MYELOPEROXIDASE EXPRESSIONS
There is evidence that HDLs can inhibit E-selectin
expression. Increased E-selectin expression in the
umbilical vein in humans and L1 overexpressing in
pig aortic endothelial cells may be inhibited by HDLs
[20]. Thus, it can be speculated that atorvastatin may
raise the HDL amount to reduce E-selectin expression
and, in such a way, contribute to cerebral protection.
NF-κB is a transcription factor widely expressed
in eukaryotic cells, and it is proved to be activated
under conditions of ischemic damage. Activated
NF-κB regulates a series of gene expressions.
Atorvastatin and calcium can inhibit expression of
NF-κB p65 mRNA and NF protein in cerebral tissues
after ischemia-reperfusion, alleviating activation
of NF-κB. The reduction of expression of pro-
inflammatory molecules, in particular of adhesion
ones, can lead to alleviation of the adhesion processes
and congregation and infiltration of leukocytes in
the ischemia areas, contributing to the reduction
of CIR-related injury. Schneider et al. [6] utilized a
gene knockout technology to conclude that, after p65
knockout in Wistar rats, the infarct volume, water
content in cerebral tissues, and neurological deficit
were reduced more obviously than those in wild-type
rats. Thus, it was concluded that activation of NF-κB
can aggravate ischemic injury [6]. Similar experiments
were conducted by Liu et al. [21]. Zhang et al. [22]
showed that NF-κB p65 expression developed 2 h
after cerebral ischemia, peaked at 12 h, and attenuated
gradually at 24 h. The combination of TNF-β and
its receptors can activate NF-κB signal pathway,
leading to integration of NF-κB in the core [23]. A
general explanation is that activated JNK/p38 kinase
will phosphorylate c-JUN and bind promoters on the
corresponding sites to regulate E-selectin expression.
It is probable that NF-κB expression is the key
upstream event resulting in intensification of the action
of inflammatory factors and expression of adhesion
molecules. According to our research, it is likely that
atorvastatin impedes combination sites of E-selectin
promoters in activated NF-κB, inhibits NF-κB
signals, and reduces activated E-selectin expression.
Atorvastatin also inhibits the adhesion processes,
limits infiltration of leukocytes and vessel endothelial
cells, and in such a way contributes to cerebral
protection by reducing pro-inflammatory damage
after CIR. This statin is a promising neuroprotective
agent in the cases of such injury. At the same time,
the specific adjustment path is in this case not clear.
We suspect that may be there is a relationship with
NF-κB; so, further studies of the atorvastatin influence on
NF-κB are expedient, to make clearer of how
atorvastatin regulates inflammation to protect the
brain tissue from ischemic-reperfusion injury.
So, administrat ion of atorvastatin afforded
noticeable protection to nerve tissues against the
inflammation response and lipemic-oxidative injury
under conditions of CIR. Atrovastatin improved
lipid parameters and decreased the level of cerebral
lipid peroxidation and inflammation. We conclude
that atorvastatin is a prospective mean for treatment
of ischemic cerebrovascular disease. Except lipid-
lowering effects, statins can bring the benefits for
cerebrovascular disease patients with normal levels of
blood lipids. At the same time, it should be recognized
that only one dose was used in our experiments, and a
dose-effect relationship should be examined in future,
to make application of atorvastatin in clinics more
well-based.
Acknowledgment. This work was supported by the
Natural Science Foundation of Hunan Province, China (Grant
No. 11JJ5081) and the Hunan Provincial Science and
Technology Department, China (Grant No. 2012SK3226 and
Grant No. 2011SK3236).
All experiments on animals were carried out in agreement
with the international normative and approved by the Ethics
Committee of the Central South University, Changsha, China.
The authors, H. Cao, W. Zhong, W. Wu, Q. Tu, and X. Tang,
declare that they have no conflict of interest.
Х. Као1, В. Жонг1, В. Ву2, Кю. Ту2, Кс. Танг1
ВПЛИВ АТОРВАСТАТИНУ НА ЕКСПРЕСІЮ
Е-СЕЛЕКТИНУ ТА МІЄЛОПЕРОКСИДАЗИ ПІСЛЯ
УШКОДЖЕНЬ МОЗКУ ЩУРІВ, ПОВ’ЯЗАНИХ З
ІШЕМІЄЮ–РЕПЕРФУЗІЄЮ
1 Друга лікарня Ксіанг’я Центрального Південного
Університету, Чанша (Китай).
2 Третя лікарня Ксіанг’я Центрального Південного
Університету, Чанша (Китай).
Р е з ю м е
Ми досліджували експресію Е-селектину та мієлоперокси-
дази (МПО) у тканинах мозку після церебральної ішемії–
реперфузії (ЦІР) у щурів та оцінювали нейропротективний
вплив аторвастатину в цих умовах. Для виявлення експресії
Е-селектину та МПО були застосовані імуногістохімічні ме-
тодики. У контрольній групі в проміжку 4–24 год спостері-
галися невеликі кількості Е-селектин- та МПО-позитивних
клітин. У групі, підданій ЦІР, у корі та гіпокампі відмічалася
велика кількість позитивних клітин. Експресія Е-селектину
NEUROPHYSIOLOGY / НЕЙРОФИЗИОЛОГИЯ.—2014.—T. 46, № 4374
H. CAO, W. ZHONG, W. WU et al.
проявлялася через 4 год, досягала максимуму через 12 год
та поверталася до майже нормальних рівнів через 24 год. У
той же час експресія Е-селектину в тварин, котрим уводили
аторвастатин після ЦІР, у всіх часових проміжках була віро-
гідно нижчою, ніж у попередній групі (P < 0.05). Експресія
МПО демонструвала часовий перебіг, приблизно подібний
такому в експресії Е-селектину, і рівень експресії МПО в
усіх часових інтервалах у групі, підданій дії аторвастатину,
був істотно нижчим, ніж у групі без уведення цього аген-
та (P < 0.05). Отже, експресія Е-селектину та МПО після
ЦІР динамічно змінюється; аторвастатин – агент з антиза-
пальною дією – продемонстрував очевидний протективний
ефект щодо ушкоджень, індукованих гострою ЦІР.
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