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Introduction
Myocardial infarction is one of the major causes of death in many countries. Although restoration of blood flow is the
only way to save the myocardium from necrosis, reperfusion-induced injuries with the help of thrombolytic therapy are at the
background of a high mortality rate[1]. Extensive studies show that myocardial ischemia-reperfusion (MI/R) injury is
associated with increased generation of reactive oxygen species (ROS). These oxygen free radicals may result in depressions in
contractile function, arrhythmias, depletion of endogenous antioxidant network, and membrane permeability
changes[2].
In recent years, accumulating experiments have shown that metallothionein (MT), a low molecular non-enzyme protein, is
a strong endogenous cytoprotective agent against cardiovascular injury. It plays an important role in detoxifying against
harmful heavy metals, scavenging oxygen radicals, and stabilizing
biomembranes[3]. The antioxidant function of MT was first
suggested in the early 1980s. Studies in vitro
have revealed that MT reacts directly with reactive oxygen species, including
superoxide, hydroxyl, and hydrogen peroxide. In particular, the antioxidant function of MT in the heart has been explored
extensively. The data gathered from recent studies using a cardiac-specific, MT-overexpressing transgenic mouse have
provided direct evidence to support this physiological role of
MT[4]. Under acute and chronic oxidative stress conditions
such as treatment with doxorubicin, ischemia-reperfusion, and dietary copper-induced restriction, MT-overexpressing transgenic
mouse hearts displayed a marked resistance to the injurious consequences, including biochemical, pathological, and
functional alterations. This protective action of
MT correlates with its inhibition of reactive oxygen species-induced lipid
peroxidation[5]. Exogenous administration with MT or induction of endogenous MT production may be used as a new
strategy in prevention and therapy of cardiovascular
diseases[6,7].
The janus kinase/signal transducer and activator of transduction (JAK/STAT) pathway is a newly discovered
intracellular signal-transducing pathway that is activated by
oxygen radicals, various cytokines, and growth factors in
ischemic heart diseases. Recent studies have demonstrated
that MI/R induce rapid activation of this
pathway[4]. Although the functional consequences of this event remains to be elucidated, there is emerging evidence that JAK/STAT signaling plays
an important role in the development of the cardioprotected phenotype associated with ischemic precon-ditioning.
Some studies in vivo also indicated that activation of STAT 3 protects myocardium from ischemia-reperfusion
injury at least partially through metallothionein, including
MT-1 and MT-2[8]. Specifically, brief episodes of MI/R activated JAK 1 and
JAK 2, followed by recruitment of STAT 1 and STAT 3, resulting in transcriptional upregulation of
MT[9,10].
Flavonoids are ubiquitous compounds found in a wide variety of edible plants, fruits, vegetables, grains and red wine.
They are an integral part of the human diet, and are important and effective constituents of some medicines, especially of
Chinese herbal medicines[11]. Epidemiological studies have indicated an association between the consumption of flavonoids
and a reduced incidence of diseases such as
stroke[12], coronary heart
diseases[13] and
cancer[14]. Glycyrrhiza glabra
(family: Leguminosae), the licorice plant, is widely used in Chinese medicine and food and has a history of consumption for the past
6000 years. Isoliquiritigenin (ISL), 2',4',4'-three hydroxy chalcone, one of the components in the root of
Glycyrrhiza glabra, is a member of the flavonoids, which have shown various biochemical activities, such as vasorelaxant effect, antioxidant,
anti-platelet, anti-tumor, anti-allergic, antiviral activities and estrogenic
properties[15]. However, the effects of ISL on MI/R
injury have not been studied. Therefore, the protective effects of ISL and its molecular mechanism were investigated by
using coronary artery occlusion_reperfusion rat model
in vivo, which is considered to more closely mimic the clinical
situation.
Materials and methods
Reagents The roots of Glycyrrhiza
glabra used in this study were collected in Yili, Xinjiang Province, China and
authenticated by Dr Ming-xi JIANG, Wuhan Institute of Botany, Chinese Academy of Sciences. The extraction and
identification of ISL are identical with our description in a previous
study[16]. ISL was finally suspended in a 0.5%
carboxymenthylcellulose (CMC)-saline solution. Creatinine phosphokinase (CPK) and lactate dehydrogenase (LDH) assay
kits were from Jian Cheng Biology Research Institute (Nanjing, China).
The antibodies for MT-1, MT-2, rabbit anti-JAK 2,
anti-STAT 1, anti-STAT 3, anti-ERK 1, anti-ERK 2, and anti-Akt
polyclonal antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Phospho-JAK 2,
STAT 1, STAT 3, ERK 1, ERK 2, and Akt were from New England
Biolabs (Beverly, MA, USA). RNA polymerase chain reaction (PCR) kit (AMV) was purchased from TaKaRa Biotechnology
(Dalian, China). All other chemicals were reagents of molecular biology grade obtained from standard commercial sources.
AG490 was purchased from Calbiochem-Novabiochem (La Jolla, CA, USA).
Animals and treatment Male Sprague-Dawley rats (Grade II,
Certificate No 2004-0004) weighing 250_300 g were purchased
from the Experimental Animal Center of Wuhan University. The rats were maintained under standard laboratory conditions
at 25±2oC, relative humidity 50%±15% and normal photo period (12-h dark/12-h light). The animals were fed normal diet and
water ad libitum. All study protocols were approved by internationally accepted principles and the Guidelines for the Care
and Use of Wuhan University, Wuhan, China. Rats were randomly divided into six groups, each consisting of ten animals,
(i), sham group; (ii), vehicle group (0.5% sodium CMC+MI/R); (iii), ISL-administration groups (MI/R+ISL), in which the rats
were subdivided into 5, 10, and 20 mg/kg groups; and (iv), AG490 group (MI/R+ISL 20 mg/kg+AG490 1 mg/kg). On d 7, 1 h
after the above treatments, the rats were subjected to the following evaluation tests.
Surgical preparation Rats of all experimental groups were anaesthetized intraperitoneally with pentobarbitone sodium
(60 mg/kg). The neck was opened with a ventral midline incision, and a tracheotomy was performed, the rats were ventilated
with room air from a positive pressure ventilator (Crompton Parkinson, Doncaster, Yorkshire, UK). The right carotid artery
was cannulated with a polyethylene tube to obtain the blood and extract the serum used for assaying the activities of LDH
and CPK at the end of surgery protocol. A left thoracotomy was performed at the fifth intercostals space and the pericardium
was opened to expose the heart. The left arterial descending coronary artery (LAD) was
ligated 2 mm from its origin by a 5_0 silk suture with a traumatic needle and ends of this ligature were passed through a small
vinyl tube to form a snare. The thoracic cavity was covered with saline-soaked gauze to prevent the heart from drying. The
animals were allowed to stabilize for 10 min before LAD ligation. Myocardial ischemia was induced by one stage occlusion
of the LAD by pressing the polyethylene tubing against the ventricular wall and then fixing it in place by clamping the vinyl
tube with a hemostat. Electrocardiographic leads were attached subcutaneous electrodes to monitor limb lead II. The animals
then underwent 15 min of ischemia, confirmed visually
in situ by the appearance of regional epicardial cyanosis and
ST-segment elevation. The myocardium was reperfused by releasing the snare gently for a period of 2
h[17]. A lead II electrocardiogram was monitored throughout the study. At the end of reperfusion period, rats were killed for biochemical studies and
other analysis.
Evaluation of myocardial infarct size
The frozen heart tissue samples were cut into 2-mm transverse slices. The slices
were incubated in 1% triphenyl tetrazolium chloride (TTC) in pH 7.4 buffer at 37oC for 20 min. TTC stains living tissue a deep
red color while necrotic tissue is TTC negative and is tan in color. The infarct and risk zone considered to be the area lacking
fluorescence under UV light was traced.
Determination of arrhythmias
Before and during ischemia and reperfusion periods, ECG changes were recorded. The
incidences and duration of ventricular premature beats (VPB), ventricular tachyarrhythmia (VT), and ventricular fibrillation
(VF) in surviving animals were determined.
Measurement of plasma LDH and CPK activities
The blood samples drawn from the carotid artery at the end of MI/R
period were collected in heparinized tubes. These samples were kept at 4oC until they were centrifuged at
2000×g for 15 min. The plasma was recovered and aliquots were used for determination of LDH and CPK activities with commercial kits.
Reverse transcription PCR analysis
Total RNA and then cDNA were prepared from rats myocardial tissues using TRIzol
RNA extraction and RT-PCR kits. cDNA synthesis, semi-quantitative RT-PCR for MT-1, MT-2, cyclooxygenase-2 (COX-2),
iNOS and GAPDH mRNA, and the analysis of the results obtained were performed as described in a previous
study[18]. The sense and anti-sense primer sequences used were: MT-1: 5'-GGTCTTCTCTGTTGGGGACA-3', 5'-GCTG
G-GTTGGGTTGGTCCGATACTA-3'; MT-2: 5'-TAGATGGA-TCCTGCTCCTGC-3', 5'-CACTTGTAGGAAGCCCTCTT-3'; COX-2:
5'-CCATGTCAAAACCGTGGTGAATG-3', 5'-AGTG-GAGTTGGGCAGTCATCAG-3'; iNOS:
5'-GTGAGGATCAA-AACTGGGG-3', 5'-ACCTGCAGGTTGGACCACTG-3'; GAPDH: 5'-CCTCTATGCCAACACAGT-3',5'-AGCCACC-AATCCACACAG-3'. These
primers set yielded PCR products of 145, 53, 374, and 200 bp for MT-1, MT-2, and GAPDH, respectively. PCR (26 cycles for
MT-1, MT-2 cDNA, 35 cycles for COX-2 cDNA, 32 cycles for iNOS cDNA, and 23 cycles for GAPDH cDNA) was performed
using the GeneAmp PCR System 2400 (Perkin Elmer Life and Analytical Sciences, Boston, MA, USA). The PCR products
were electrophoresed through a 1.5% agarose gel and visualized by ethidium bromide staining and UV irradiation.
Western blot analysis Left ventricles from the hearts were homogenized in a buffer containing 25 mmol/L Tris-HCl, 25
mmol/L NaCl, 1mmol/L orthovanadate, 10 mmol/L pyrophosphate, 10 mmol/L okadaic acid, 0.5 mmol/L EGTA , and 1
mmol/L PMSF. Protein (100 µg) of each heart homogenate was incubated with 1
mg of antibody against MT-1, MT-2, JAK 2, STAT 1, STAT 3,
ERK 1, ERK 2, and Akt for 1 h at
4oC. The immune complexes were precipitated with protein A Sepharose, immunoprecipitates separated by SDS-PAGE and
immobilized on polyvinylidene difluoride membrane. The membrane was immunoblotted with PY20 to evaluate the
phosphorylation of JAK 2, STAT 1, STAT 3, ERK 1, ERK 2, and
Akt. The membrane was stripped and reblotted with specific antibodies
against MT-1, MT-2, JAK 2, STAT 1, STAT 3, ERK 1, ERK 2, and Akt.
The resulting blots were digitized and subjected to
densitometric scanning using a standard NIH image program.
Statistic analysis Results of all the above estimations have been indicated in terms of mean±SD. The difference between
means was analyzed by ANOVA test. Minimum level of significance was fixed at
P<0.05.
Results
Effects of ISL on myocardial infarction
The myocardial infarction induced by ischemia-reperfusion was measured by
TTC staining. As compared with the vehicle group, the percentage of myocardial infarct size was obviously decreased in the
ISL 20 mg/kg group from 50.91%±1.90% to 20.45%±
1.03% (P<0.01) (Table 1). Pretreatment with tyrphostin AG490 significantly blocked the anti-infarct effect of ISL.
Effects of ISL on ischemia-reperfusion arrhythmias
There was no significant difference between vehicles and rats
treated with ISL with respect to heart rate (HR) immediately prior to coronary occlusion. These were 435±6 and 423±8
beats/min. After coronary artery occlusion, all animals exhibited cardiac arrhythmias, which occurred as VPB, VT, and VF. In some
animals, atrioventricular (A-V) block also occurred. On subsequent reperfusion, arrhythmia severity was much less marked
in those rats given ISL (Table 2). VPB developed in 100% of vehicles versus 62.5%,
42.8%, and 25% (P<0.05) of ISL 5, 10, and
20 mg/kg treated animals, respectively. The incidences of VF that occurred in 83% of the vehicle group were decreased to 0
in the ISL 20 mg/kg group. Also, ISL markedly shortened the duration of ventricular arrhythmias.
Effects of ISL on plasma LDH and CPK activities
The biochemical indicators of myocardial damage were evalu
ated in the MI/R period. When plasma LDH and CPK activities in rats treated with ISL, the results shown in Table 3 were
obtained. Compared to the vehicle group, pretreatment of rats with ISL (5, 10, and 20 mg/kg, ig) markedly decreased the LDH
and CPK activities by 22%, 34%, 38% and 35%, 52%, 58%. The decreases of LDH and CPK releases partly disappeared with
the inhibition of AG490.
Effects of ISL on MT-1, MT-2 mRNA and protein expressions
This study investigated the effect of ISL on the regulation
of MT expressions in MI/R rat model. RT-PCR demonstrated specific amplifications of MT, COX-2, and iNOS cDNA. MT-1
and MT-2 mRNA were upregulated by ISL in a dose-dependent manner. There were no significant differences of COX-2 or
iNOS mRNA levels among ISL-treated groups when compared to the vehicle group (Figure
1). With the dose of ISL increased, the protein expressions of MT-1, MT-2 were obviously increased. This result indicated that MT expressions were upregulated
by ISL in myocardium through transcriptional activation. In contrast, the increased MT expression in the ISL 20 mg/kg group
was blocked by tyrphostin AG490.
Effects of ISL on JAK /STAT phosphorylations
To determine the underlying molecular mechanism of the JAK 2
inhibitor-induced decrease of MT expression , the phosphorylation of JAK 2, STAT 1, STAT 3, ERKs, and Akt were examined by
Western blot analysis. The results suggested that pretreatment with AG490 30 min before the coronary artery ligation
combined with ISL 20 mg/kg significantly inhibited the phosphorylation of STAT 3. STAT 1 was weakly phosphorylated in
MI/R rats, however, it was not obviously affected by AG490 treatment
(Figure 2). Other signal transducing molecules downstream of gp130, such as ERK and Akt were also analysed with Western blotting by using phosphor-specific antibodies.
As shown in Figure 2, neither Akt nor ERK was activated in MI/R rats of ISL-treated groups, indicating that JAK 2/STAT 3
may be exclusively activated.
Discussion
In the present study, we demonstrated that ISL induced the upregulation of MT expression. As expected, ISL also
resulted in cardioprotection as evidenced by attenuated
ventricular arrhythmias, reduced infarct size, and decreased the releases of LDH and CPK.
The ISL treated MI/R rats
revealed increased phosphorylations of JAK2/STAT3 but not of ERKs and Akt
pathway. Inhibition of JAK2 kinase with tyrphostin, AG490 resulted in the inhibition of STAT3 phosphorylation, decreased the expressions of MT-1 and MT-2, and
abrogated the cardioprotection effects of ISL.
Myocardial reperfusion through thrombolysis, percutaneous transluminal coronary angioplasty, or coronary artery
bypass grafting is standard treatment in acute myocardial infarction. However, these therapies initiate a second phase of
myocardial injury either by acceleration of detrimental processes initiated during ischemia or by inducing
additional pathological processes following
reperfusion[19,20]. Our results indicated that in MI/R model group, serious
myocardial infarction and ventricular arrhythmias were observed. Besides this, the activities of LDH and CPK in the plasma
were much higher than those in the sham group.
Increasing clinical and experimental data have provided evidence that ischemic cell injury is mediated by
ROS[21]. The role of oxygen free radicals in the pathophysiology of ischemia-reperfusion injury is supported by the increased formation of lipid
peroxides and other toxic products following such as
injury[22]. The interaction of ROS with cell membrane lipids and
essential proteins contributes to myocardial cell damage, leading to inflammatory reactions, irreversible tissue injury, and
consequently, to impaired cardiac
function[23].
MT, a low molecular protein rich in
L-cysteine have been shown to be associated with resistance to oxidative stress in
cardiac protection against ischemia-reperfusion
damage[24]. In this study, we found that both the mRNA and protein
expressions of MT were upregulated by ISL. These results are in accordance with previous studies on the antioxidative role of ISL.
Originally described as the regulator for cytokine
signaling, JAK/STAT pathway is now recognized as an important
membrane-to-nucleus signaling pathway for a variety of stress responses including ischemia, hypoxia, and oxidative stress.
In response to ischemic stress, phosphorylation of JAK occurs followed by tyrosine phosphorylation and dimerization of
cytosolic STAT monomers. The STAT dimers readily translocate to the nucleus, bind to the promoter regions of the DNA and
regulate transcription of genes[25]. The JAK/STAT pathway, especially the JAK2/STAT3-dependent pathway, may have a
cytoprotective effect on stretched myocardium in MI/R
hearts[4]. Treatment with JAK2 inhibitor reduced the
phosphorylation of STAT1, 3 in MI
hearts[26]. STAT3 also showed a transducer protective signal against oxidative stress and
doxorubicin-induced cardiomyopathy[27]. The current study showed that ISL increased JAK2/STAT3 phosphorylation levels and led to
upregulation of MT levels in MI/R rat. These results are consistent with previously published reports that state that specific
activation of STAT3 transducers antioxidative signals through MT and confer resistance against ROS stress in the heart.
It has been reported that COX-2 and iNOS are the downstream targets of the JAK/STAT pathway and play an important
role in ischemia-reperfusion
injury[4,28]. Thus, we examined the mRNA expression of COX-2 and iNOS by RT-PCR and found
no significant difference of iNOS and COX-2 mRNA levels in ISL-treated groups when compared to the vehicle group. These
findings suggest that COX-2 and iNOS is may not be involved in ISL-induced cardioprotection.
In summary, we demonstrated that ISL, a flavonoid, strongly reduced myocardial infarct size and prevented
reperfusion-induced arrhythmias in the model of MI/R rats
in vivo. It also appears that activation of JAK2/STAT3 and subsequent
upregulation of MT is instrumental, at least in part, in ISL-mediated cardioprotection.
Acknowledgment
We would like to thank the Center for Drug Nonclinical Safety Studies of Wuhan University for technical assistance
during this work.
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