228 Asian Pacific Journal of Tropical Medicine (2013)228-231

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Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

Document heading doi:

Effect of rosiglitazone on rabbit model of myocardial

ischemiareperfusion injury
Xia-Qing Gao1, Hua-Wei Li1, Xue Ling2*, Ya-Hui Qiu2, Yue Gao2, Yang Zhang1
1
Liaoning Medical University, Jinzhou 121000, China
23
rd Section, Department of Cardiology, 3rd Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, China

ARTICLE INFO ABSTRACT

Article history: Objective: To explore mechanism and protective effect of rosiglitazone on myocardial ischemia Methods: A total of 48 male
Japanese white big-ear rabbits were
Received 10 October 2012
reperfusion (I/R) injury.

Received in revised form 15 November 2012Accepted 15 January 2013dose of rosiglitazone group randomly divided into control group (D).
Plasma concentration of and also reduced the concentration of (A), I/R group(B), low dose of rosiglitazone group (C), high

Available online 20 March 2013

plasma serum creatine kinase (CK), CK-MB, high-sensitivity C-reactive protein (hsCRP), ultra-

Keywords:Myocardial infarctionRosiglitazoneIschemia reperfusion injury superoxide dismutase (MDAmyocardial infarction were tested.

hours after GSH and -PX)ET, nitric oxide I were decreased in /R the hearts were harvested for

pathological and ultrastructural analysis. ((SODNO) and endothelin), malondialdehyde C, D

group compared with group Results:(ET Plasma concentration of ) were measured (MDA),

lactic acid glutathione skin peroxidase B. 1 h later after Plasma concentration of CK, CK-

IMB/R. , hsTwenty-four CRPATrea of -, SODNO, and GSH-Px were increased significantly in

C, D group compared with group B. Compared with group B, pathological and ultrastructural

changes in C and D group were slightly. There was significant difference in myocardial

P
infarction area between group C, D and group B ( <0.05). MConclusions:yocardial infarction

area and arrhythmia rate were lower in group Rosiglitazone may protect myocardium from

I/R injury by enhancing C, D compare with group T-SOD and B.

GSH-Px concentration, inhibit inflammatory reaction, and improve endothelial function.

1. Introduction
 Xia-Qing Gao et al./Asian Pacific Journal of Tropical Medicine (2013)228-231
Myocardial ischemia-reperfusion injury often occurs
after acute myocardial infarction thrombolysis, PTCA
and coronary artery bypass grafting or other treatments.
The mechanism of the injury is a patho-physiology
phenomena caused by the expression of cytokines,
especially the surface adhesion molecules [1], which can
start and induce the aggregation and adhesion of
inflammatory cells and cause immune injury of tissue
cell. It seriously affects the treatment efficacy and
prognosis of patients with myocardial infarction.
Peroxisome proliferators-activated receptor (PPAR) is a
member of nuclear receptor superfamily.
*Corresponding author: Ling Xue, Professor, 3rd Section, Department of
Cardiology, 3rd Affiliated Hospital of Liaoning Medical University, Jinzhou 121000,
China.
Tel: +86 13841690961
E-mail: Lysyxueling@163.com
Foundation project: Financially supported by Planning Program of Department of
Science and Technology of Liaoning Province (Grant No. 2011225015).
Retinoic acid, degenerate prime, thyroid hormone,
vitamin D, and orphan receptors are also members of the
毭 is mainly
nuclear receptor superfamily[2]. PPAR
expressed in
adipose tissues and the immune system,
which closely related to the adipocyte differentiation,
insulin resistance and body immune[3].
can regulate the production and transportation of the
Through activating nuclear receptors PPAR 毭 ,
rosiglitazone glucose, and also regulate the transcription
of the insulin response genes. And rosiglitazone can
relieve ischemiareperfusion myocardial injury by some
physiological effects such as inhibiting the
inflammation, improving the endothelial function and
reducing the oxidative stress[4,5]. In this study we
investigated the protective effect of rosiglitazone on
myocardial ischemia-reperfusion injury and its
mechanism. We expect to provide new ideas for the
clinical treatment of myocardial ischemia-reperfusion,
thus more patients can be benefit from it.
2. Materials and methods
2.1. Animal
Male Japanese long-ear white rabbits, (2.50 依 0.25) kg,
provided by medical experimental animal center of
Liaoning Medical College. Permit number of
experimental animals was SYXK (Liao) 20030019.
2.2. Medicines and reagent
Rosiglitazone hydrochloride tablets were from the
Shanghai First Biochemical Pharmaceutical Co., Ltd.,
batch number
2.3. Instruments
HITACHI 7170A automatic biochemical analyzer; 721
visible spectrophotometer (Shanghai Precision &
Scientific Instrument Co., Ltd.); Ultraviolet visible
spectrophotometer type-UV300; K15-C low temperature
ultracentrifuge (Beijing medical centrifuge factory); SHH.
7121.6OO electric constant temperature tris-use water
tank (Beijing YongGuangming medical equipment
factory); Shimadzu LC-9A liquid chromatograph;
Fluorescent detector type-RF535; IX70 inverted
fluorescence microscope; The OLYMPUS optical
microscope type-CH20; OLYMPUS microscope
photography type-C35A; Image analyzer CIAS-1000;
Electronic balance; Enzyme mark instrument MC-
ASCENT; Electrophoresis apparatus (DYY-6C); Low
temperature refrigerator and ultra low temperature
refrigerator (MDFU40865); Animal ventilator (HX-200).
2.4. Establishment of myocardial ischemia-reperfusion
injury models in rabbits
Healthy male Japanese white big-ear rabbits were under
anesthesia with urethane (20%, 1 g/kg). Animals were
then bound to a plate with the back side down and chest
shearing. A skin incision was made from the
sternoclavicular joint line to the point above the xiphoid
process along the sternum midline. The 2nd, 3rd, 4th ribs
were cut in the left sternal margin, stretched with a
thoracic incision, then the pericardium was opened to
expose the heart. A small round looper stab 2 mm below
the left bundel of coronary artery was hold by needle
holder with the 2/T line for ligation.
229
2.5. Grouping and administration
A total of 48 rabbits were randomly divided into four
exposed but not ligated. groups: (1) Sham operation
group: Rabbits were fed with common Coronary artery
was only pellet feedstuff for 3 days; (2) Ischemia-
reperfusion group: ligated for 30 min, under reperfusion
for 60 min. The rabbits were feed with common pellet
feedstuff for 3 days; (3) Low dose rosiglitazone group:
ligated for 30 min, under reperfusion for 60 min. Rabbits
were fed with common pellet feedstuff for 3 days and
rosglitazone 0.5 mg/kg/day; (4) High dose rosiglitazone
group: ligated for 30 min, under reperfusion for 60 min.
Rabbits were fed with common pellet feedstuff for 3
days and rosglitazone 3 mg/kg/day. Successful ligation
was confirmed by significantly ascending
ST-segment of ECG in the ischemia-reperfusion group,
low dose rosiglitazone group and high dose rosiglitazone
group after operation. After reperfusion for 60 min,
common carotid artery blood was added with heparin
anticoagulant tube for biochemical testing.
2.6. Indexes measurement
 Xia-Qing Gao et al./Asian Pacific Journal of Tropical Medicine (2013)228-231
Plasmic level of myocardial enzymes plasma serum The results showed that compared with the sham
(CT n 栺, group,the
CK-MB), high-sensitivity C-reactive protein
CTn 栺 , CK-MB, and hsCRP levels in plasma were
(hsCRP) were measured in carotid arterial blood under significantly increased in the ischemia-reperfusion
anticoagulation. The blood was centrifuged at 2 500 rpm group. And compared with the ischemia-reperfusion
for 10mins, and then the supernatant was collected to group,the CTn 栺 , CK-MB, hsCRP levels in plasma in the
determine plasma SOD, high dose rosiglitazone group and the low-dose
MDA, GSH-PX, NO and ET. rosiglitazone group were decreased significantly (Table
The hearts were harvested, then were rinsed with PBS 1).
buffer. The ischemic area of the heart were cut into or
approximately 0.25% NBT phosphate buffer 1 mm in
℃
parallel, and were placed in (pH=7.4), 37 ,under water
Table 1
TTC Econtent ffect on cardiac enzymes (mean 依 SD). (CTn 栺, CK-MB)
1% bath for 30 min. The dyed and undyed areas were
content and hsCRP
isolated under dissecting microscope. The infarcted
myocardium was pale and the none-infarcted
myocardium was blue. The BI2000 image analysis system Groups CTn 栺 (毺 g/mL) CK-MB (毺 g/mL) hsCRP (U/L)
was used to calculate the percentage of the infarcted Group A 110.35 依 25.96 △ 134.75 依 35.12△ 47.10 依 2.82△
myocardium area accounted for the total ventricular area. Group B 182.37 依 30.46★ 214.52 依 43.78★ 77.35 依 1.32★
Isolated hearts were washed with stationary liquid. The Group C 136.42 依 28.37★ 157.21 依 45.91★ 53.38 依 1.56★
left ventricular anterior myocardial tissues were cut into
strips, and then placed in stationary liquid for 2 h,
followed by rinsed with 0.09 mmol KH2PO4 for 15 min,
and stored in this solution. It was dehydrated in
ascending series of ethanol, cleared in xylene, embedded
in paraffin, and stained by HE staining. Then the
Group D 120.53 依 31.03 142★.36 依 40.13 50.24 依 1.47
structural c myocardial pathological change was
observed under light microscope. P
group, Note: C△ompared with group P <0.01. B, <0.01; compared
with the sham
2.7. Statistical analysis

The experimental data were analyzed with SPSS17.0

3.2. Effect on NO, ET and GSH-Px content in plasma
statistical software. All measurement data were
expressed with mean 依 SD. And single-factor analysis the The results showed that compared with the sham

of variance q-test was used to compare between two

group,
groups. ) was applied in the comparison among many
groups, P <0.05
(ANOVA

indicated statistical significance. Comparatively

describing 230 method was used to analyze the group, the rosiglitazone group and the low-dose
pathomorphological data. rosiglitazone group levels increased. decreased in the
ischemia-reperfusion group, while significantly. were
increased significantly, while NO, GSHNOT, -he content
3. Results of ETPx levels in plasma were significantly ,
CGSHompared with the ischemia-reperfusion -Px levels
in plasma in the high dose NO, ET, GSHET- levels
hsCRP content in plasma 3.1. Effect on cardiac enzymes decreased Px in plasma had ET
(CTn 栺, CK-MB) content and
 Xia-Qing Gao et al./Asian Pacific Journal of Tropical Medicine (2013)228-231
no significant difference between rosiglitazone high 3.4. Myocardial infarct size
dose group and rosiglitazone low dose group (Table 2).

Table 2
Effect on NO, ET, GSH-Px content in plasma after the myocardial
ischemia and reperfusion injury (mean 依 SD).
and the high-dose rosiglitazone group [significantly
Groups NO(毺 g/mL) ET (pg/mL) GSH-Px (U0
Group A 71.28 依 4.32 40.23 依 6.35 218.23 依 21.75△ Group
decreased myocardial infarct size of the ischemia-
△ △
reperfusion group with the ischemic reperfusion group,
B 32.51 依 5.25★ ★ 297.57 依 7.58★ 112.42 依 31.58★ ★ Group C the myocardial infarct [size of the low-dose rosiglitazone
57.32 依 6.34★ 143.56 依 6.54★ 176.56 依 33.81★ ★
group [(41As compared with sham-operated group [.3 依
8.5)%] was significantly increased (P<0.05). Compared
with the sham (P( <23(019..405. 依 (226)6
依. .5C6..5)ompared 5%依%)]7 were ]., the 1)%]

Group D 65.84 依 5.05 121.28 依 8.82 187★ ★ .34 依 27.29 ★

N<0ote: compared with ischemia-reperfusion group, .01; compared

P P
with the sham group, △ P <0.01. <0.05,

3.3. Effect on T-SOD and MDA content in plasma group, the rosiglitazone low-dose group and the
rosiglitazone high-dose group showed no significant
difference (P> 0. 05).

3.5. Pathological structures under light microscopy

the in the ischemia-reperfusion group, and the
significantly decreased. The results showed that
compared with the sham group, MDA levels in plasma
were significantly increased Compared with the
ischemia-T-SOD was group were decreased significantly, of swelling and disorder myocardial fibers, inflammatory
while the dose rosiglitazone group and the low-dose
rosiglitazone increased significantly reperfusion group,
under the light microscope, without inflammatory cell
the (TMDAable levels in plasma in the high 3). T-
SOD was
Myocardial fibers arranged in neat rows in the sham

group Group I/R were infiltration of the myocardial

interstitial.
Table 3
Effect on the T-SOD and MDA content in plasma (mean 依 SD).

Groups T-SOD(U/L) MDA(nmol/mL)

Group A 521.38 依 34.42△ 7.14 依 0.42 △ cells and even focal necrosis in the myocardial
Group B 390.54 依 33.86★ ★ 12.84 依 0.79★
Group C 454.58 依 25.43★ ★ 8.10 依 0.54★

groups were in uniform size, myocardial fibers

interstitial. M

★
Group D 489.27 依 15.62 7.46 依 0.37

C
N★ ★ote: P <ompared with ischemia-reperfusion group were mild
P yocardial cell nuclei in high and low dose
0.01; compared with the sham group, △ P <0 . 05 <.0.05,
 Xia-Qing Gao et al./Asian Pacific Journal of Tropical Medicine (2013)228-231
rosiglitazone infiltration at different degrees.swelled, and 毭
transcriptional effects which Wsignal ith the presence

there were fibroblasts and inflammatory cell

of TRANSDUCERLPS, active protein and activator of

4. Discussion

PPAR- 毭 , 15 d- PGJ 2 at very low

transcription 21 , NF- 毷 B, and

the 1[8]. With

monocytes, mast cells, and macrophages all play

important also involved in the reperfusion
injuryprocess which was participated by a variety of
inflammatory roles in this process, while cell nucleus
inflammatory cytokines. threatening the human life and
health at present, while myocardial ischemia -
reperfusion can cause irreversible cells, thus aggravate
myocardial damage and increase mortalitydamage,death
and apoptosis to the damaged myocardial Acute
myocardial infarction is the leading disease [6].
Myocardial ischemia reperfusion injury is a TNF- 毩 ,
[7]IL.-1, ILN-eutrophils, 6, IL-PPAR8 were -

PPAR ligand -毭 15 is expressed in many immune cells,

PGJ2
and d- can regulate many immune responses. is

mediated by concentrations can inhibit the

 crosstalk between RAGE and nitrative thioredoxin inactivation

Am J during diabetic myocardial ischemia-reperfusion injury.

Xia-Qing Gao et al./Asian Pacific Journal of Tropical Medicine (2013)228-231
231
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 Xia-Qing Gao et al./Asian Pacific Journal of Tropical Medicine (2013)228-231
induce the production of -[11,12]PX-S6Ttudies have shown that as , which can inhibit monocyte inflammatory
lymphocytes and shows anti inflammatory , and produce the anti-inflammatory effect. NOCK, and also reduced the
concentration HT-he activation of owever, the specific mechanism is MBIL c-TNI2ILT- lymphocyte is to control , hs2
gene expression of the CRPPPAR, PPARMDA-R 毭-osiglitazone and can inhibit 毭 PPARF receptor rom the ET-. 毭 It

inhibit activated protein-

content of our further study.inhibit the damage of myocardial ischemia and improve the by

inhibiting agonist, rosiglitazone can affect the formation and activation and inducible oxide synthase by regulating the

expression ischemia-reperfusion injury by inhibiting inflammation, function of heart. improving endothelial function

and reducing the generation of of inflammatory cells and inflammatory cytokines, inhibit of oxygen free radicals.

PPAR-毭 [13,14]Jun . IMCPNn addition, R-osiglitazone can improve myocardial terminal kinase phosphorylation and - 1,

intercellular adhesion molecule 1T DNAhe specific mechanism is the Khandoudi binding activity, et al[15] found that

TZDs can the expression of

Conflict of interest statement

We declare that we have no conflict of interest.

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