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Introduction
Cardiac hypertrophy is a pathological response of the
heart to chronic pressure or volume overload.
Epidemiological studies revealed that cardiac hypertrophy was an
independent risk factor for ischemic heart disease, arrhythmia,
and sudden death. Moreover, the hypertrophic heart often
leads to dilated cardiomyopathy, and eventually causes
congestive heart failure after sustained overload. Therefore, it
is important to determine the mechanism of the development
of cardiac hypertrophy and prevent or treat
it[1]. Recent evidence shows that cardiac apoptosis, although at a low
level, is present in overload cardiac
hypertrophy[2,3]. It was proposed that chronic, low level of cardiac myocyte
apopto-sis is a causal component in the pathogenesis of heart failure.
Cardiac apoptosis might be a critical factor during the
transition from compensatory hypertrophy to heart
failure[3,4]. The mechanisms of cardiac apoptosis include extrinsic factors
and some cellular signal pathways. Bcl-2 expression has
been proposed as an important marker of myocardial cell
survival probability[5]. Tumor suppressor protein p53, a
transcriptional modulator of the bcl-2 genes, can produce cell
cycle arrest and facilitate
apoptosis[5]. Condorelli et
al[6] reported cardiomyocyte apoptosis contributed to the
transition from pressure overload-induced cardiac hypertrophy to
heart failure with dramatic downregulation of bcl-2,
predisposing cardiomyocytes to apoptosis. Leri
et al[7] reported the upregulation of local renin-angiotensin system in
stretched cardiomyocytes increased susceptibility of
myocytes to undergo apoptosis, coupled with the
activation of p53 and bcl-2 decrease. In addition, in pressure
overload-induced cardiac hypertrophy, excessive reactive
oxygen species (ROS) may be generated in cardiac tissue.
In vitro and in vivo studies have demonstrated that ROS may
activate necrosis, apoptosis, even hypertrophy in
cardio-myocytes[8,9].
Recently, more and more antioxidants have shown
inhibitory effects on cardiac
hypertrophy[10,11]. Epigallocate-chin gallate (EGCG), the major component of polyphenols in
green tea, has recently attracted considerable attention for
antioxidative, anti-inflammatory, antitumorigenic and
antisenescent properties[12]. Recent research has shown that
EGCG and green tea exerts protective effects against
cardiovascular diseases[13_15]. Townsend
et al[15] reported that green tea extracts and EGCG protected cardiomyocytes against
ischemia/reperfusion-induced apoptotic cell death both
in vivo and in vitro. More recently, results form both our
laboratory[16] and from Li et
al[17] showed that EGCG effectively
inhibited cardiac hypertrophy in mice and rats. However, to date,
little is known about the effects of EGCG on cardiac apoptosis
in pressure overload-induced cardiac hypertrophy. It is
known that EGCG may have an exceptional antioxidant
capacity, even far exceeding than that of vitamin E and
vitamin C[18]. We hypothesized that the inhibitory effects of
EGCG on cardiac hypertrophy might be related to its
antioxidant effects by scavenging ROS and inhibiting cardiac
apoptosis in the hypertrophic myocardium.
To test this hypothesis, an abdominal aortic constriction
model was established to detect the effects of EGCG on
pressure overload-induced oxidative stress and cardiac apoptosis
in cardiac hypertrophy with an analysis of p53 and bcl-2
protein expression in the hypertrophic myocardium.
Mean-while, cultured newborn rat cardiomyocytes were exposed
to exogenous hydrogen peroxide to determine the effects of
EGCG on oxidative stress-induced cardiomyocyte injury and
apoptosis in vitro.
Materials and methods
Materials EGCG used in animal experiments was
purchased from Sichuan Leshan Yujia Tea Science and
Technology Development Co Ltd (Leshan, Sichuan, China, purity
>95%), while EGCG used in the cell experiments was
purchased from Sigma (St Louis, USA). Captopril (Cap) was
purchased from Jiangsu Huanghe Pharmaceutical Co Ltd
(Jiangsu, China). Assay kits of malondialdehyde (MDA),
superoxide dismutase (SOD), glutathione peroxidases
(GSH-Px) and lactate dehydrogenase (LDH) were purchased from
Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
The terminal transferase-mediated dUTP-biotin nick
end-labeling (TUNEL) assay kit was purchased from Fujian Maixin
Biotechnology Co Ltd (Fuzhou, Fujian, China). Mouse
anti-p53 monoclonal antibody (sc-100) and rabbit anti-bcl-2
polyclonal (sc-492) antibody were purchased from Santa Cruz
Biotechnology (Santa Cruz, California, USA). Mouse
anti-á-sarcomeric actin monoclonal antibody was purchased from
Sigma (St Louis, USA). Dulbecco's modified Eagle's
medium (DMEM) was purchased from Gibco (Carlsbad, California, USA).
Cardiac hypertrophy model induced by abdominal aortic
constriction Male Sprague-Dawley rats (187.4±9.7 g) were
obtained from the Experimental Animal Center of Soochow
University (Suzhou, China; Certificate No 20020008, Grade
II). The NIH Guidelines for the Care and Use of Laboratory
Animals were followed in all animal procedures. The rats
were anesthetized by sodium pentobarbital 35 mg/kg ip. A
mid-abdominal incision was made to expose the abdominal
aorta. The aorta above the left renal artery was dissected
and constricted at the suprarenal level using a 7 gauge needle
(outside diameter 0.5 mm), which was ligated with aorta and
withdrawn afterwards. In the age and body weight-matched
sham-operated rat, the abdominal aorta was isolated without
ligation. After surgery, the rats were administered with
penicillin 200 kU·kg-1·d-1
intramuscularly (im) for 1 week to prevent
infection[19,20]. One week after surgery, the model rats
were randomly divided into 5 groups: the aortic constriction
(AC) group, EGCG 3 dose groups, and the Cap group, which
were intragastrically (ig) infused with nomal saline (NS), EGCG
25, 50, and 100 mg/kg, and Cap 50 mg/kg for 6 weeks,
respectively. The rats of the sham-operated group were
infused with NS for 6 weeks.
Determination of systolic blood pressure and heart
weight indices Before the operation and each week after
operation, systolic blood pressure (SBP) was measured
between 10:00 and 12:00 AM in each rat by use of the tail cuff
method after the rats were warmed at 37 °C for 30
min[17]. Six weeks after administration (7 weeks after AC), blood samples
were collected from aorta for the detection of MDA, SOD,
and GSH-Px; the rats were then sacrificed and the hearts
were excised. After rinsing in phosphate buffered saline
(PBS), the atria and right ventricular free wall were carefully
dissected from the left ventricle. The wet weights of the
whole heart and left ventricle were measured. The degree of
cardiac hypertrophy was estimated by calculating the heart
weight index (HWI£½HW/body weight (BW)) and left
ventricular weight index
(LVWI=LVW/BW)[19,20].
Detection of MDA content, and SOD and GSH-Px
activities in serum and cardiac homogenates The blood samples
were centrifuged at 1700×g for 10 min to obtain serum. The
left ventricle was homogenized in 9 (v/v) volumes of ice-cold
PBS, and the homogenates were centrifuged at
3000×g 4 °C for 15 min to obtain the supernate. MDA content, and SOD
and GSH-Px activities in serum and cardiac homogenates
were measured by assay kits, respectively, according to the
manufacturer's instructions[21].
Histological analysis The left ventricles were fixed in
10% formalin. The fixed hearts were embedded in paraffin,
and sectioned at 4-µm thickness, then stained with
hematoxylin and eosin or by van Gieson method.
TUNEL assay TUNEL reaction was performed using an
in situ cell death detection kit, according to manufacturer's
instructions. Briefly, the section was deparaffinized and
rehydrated with serial changes of xylene and ethanol.
Proteinase K 20 mg/L was applied to the section for 15 min. The
endogenous peroxidase was inhibited with 0.3 %
H2O2 for 5 min. The section was treated with the reaction mixture
containing TdT and biotinylated 16-dUTP for 1 h at 37 °C.
Labeled DNA was visualized with peroxidase-conjugated
antidigoxigenin antibody using 3,3'-diaminobenzidine as the
chromogen. TUNEL positive cells were detected according
to the instructions. Ten random fields per section were
analyzed, respectively, and the average value of the TUNEL
positive cardiomyocyte percentage was
calculated[22,23].
Western blot analysis The total cardiac protein was
extracted and Western blot analysis was performed as described
previously[6,24]. Briefly, the left ventricular tissues were
homogenized in a buffer containing Tris-HCl (pH 7.4) 10
mmol/L, NaCl 150 mmol/L, 1% Triton X-100, 1% sodium
deoxycho-late, 0.1% SDS, edetic acid 5 mmol/L,
phenyl-methylsulfonyl fluoride (PMSF) 1 mmol/L, aprotinin 0.28
kU/L, leupeptin 50 mg/L, benzamidine 1 mmol/L, and pepstatin A 7
mg/L. Protein concentration was determined by a
bicin-choninic acid (BCA) kit (Pierce, Rockford, IL, USA). Eighty
milligrams of protein from each sample was loaded onto 12%
SDS-PAGE gel and subjected to electrophoresis using a
constant current. The proteins were transferred to the
nitrocellulose membranes (Amersham, Arlington Height, IL, USA)
and incubated with rabbit anti-bcl-2 polyclonal antibody or
mouse anti-p53 monoclonal antibody in Tris buffered saline
containing 0.1% Tween-20 (TBST) and 5% nonfat dry milk
for 3 h. The membranes were washed and incubated with
horseradish peroxidase-conjugated secondary antibody
(Santa Cruz, USA) in TBST containing 5% nonfat dry milk
for 1 h. Immunoreactivity was detected by enhanced
chemolumine-scent autoradiography (ECL kit, Amersham,
USA) according to manufacturer's instructions. The results
were analyzed quantitatively using SigmaScan Pro 5.0.0 (SPSS
Inc, Chicago, Illinois, USA). The data were normalized with
respect to the ratios of actin detected on the same blot to
control for possible variations in protein loading.
Cell culture Newborn Sprague-Dawley rats, 1_2 d old,
were put into PBS after disinfecting. The heart tissues were
removed and put into cold D-Hanks' solution. After the
removal of the blood vessels and atria, the ventricles were
minced and incubated in 0.25 % trypsin at 37 °C for 10 min.
The supernate was discarded and the tissue was redigested
with trypsin for 10 min; an ice bath was then used to
terminate digestion, and the supernate was centrifuged at
700×g for 10 min. The sediment was resuspended in DMEM
containing 10 % fetal bovine serum. The residual tissues were
redigested for 4_6 cycles until single cell was obtained. Cell
suspensions were collected together and preplated in
culture flasks for 45 min at 37 °C in 95% air/5%
CO2 to remove fibroblasts. The unattached cells were counted and seeded
onto another culture flask at a density of
1.5×106/L. The medium was changed every 48 h. All experiments were
performed on 3_5 d cultures when synchronously contracting
cells were observed. The purity of the cardiomyocytes was
confirmed by anti-α sarcomeric actin
antibody[25,26].
Cell viability analysis The cardiomyocytes cultured
in 96-well plates were preincubated with EGCG 12.5_200
mg/L for 6, 12, 24, and 48 h. H2O2
200 µmol/L was added and incubated with the cells for additional 24 h. After
separating with the medium, the cells were washed twice with
PBS. DMEM containing MTT 0.5 g/L was appended to each
well and incubated at 37 °C for an additional 4 h. After the
medium was discarded, the remaining formazan crystals
were dissolved in 100 µL Me2SO. Absorbance at 570 nm
was measured by a DG3002 ELISA plate reader (Huadong
Electronic Company, Nanjing, China).
LDH release and MDA formation The cardiomyocytes
cultured in 24-well plates were preincubated with EGCG
12.5_200 mg/L for 24 h; H2O2
200 µmol/L was added and incubated with the cells for another 24 h. LDH activity in the
medium was measured by a LDH assay kit at 440 nm. MDA
content in the medium was measured by a MDA assay kit at
532 nm[27].
Flow cytometry analysis After pretreatment with EGCG
50 and 100 mg/L for 24 h, H2O2
200 µmol/L was added and incubated with the cells for an additional 24 h. Cell
morphological changes were observed by a phase contrast
micro-scope. For the flow cytometry analysis, the cells
(>1×106) were digested with 0.25% trypsin and collected by
centri-fugation. After washing twice with ice-cold PBS, the cells
were fixed in ice-cold 70% ethanol. After centrifugation, the
fixed cells were incubated with RNase 100 mg/L at 37 °C for
30 min and stained with 50 mg/L propidium iodide (PI) for 30
min. The cells were analyzed by EPICS XL flow cytometry
(Beckman Coutler, Califonia, USA). The hypodiploid
population of cells was considered apoptosis, and the apoptotic
rate was analyzed by Multicycle software (Beckman Coutler,
Califonia, USA)[28].
Statistical analysis Data were expressed
as mean±SD. One-way ANOVA in SPSS 10.0 software
(SPSS Inc, Chicago, Illinois, USA) was used for the statistical analysis. The
intergroup comparisons (post-hoc analysis) among the data
with equal variances were made by the LSD method, while
Tamhane's T2 method was used for the data with unequal
variances.
Results
Effects of EGCG on SBP and heart weight indices
In the rats with cardiac hypertrophy, compared with the sham-
operated group, the SBP of AC rats began to increase 1 week
after the operation, and increased progressively at 2_7 weeks
after the operation (Figure 1). The heart weight indices also
increased remarkably vs the sham-operated group. The HWI
and LVWI increased by 55.8% and 72.3%, respectively
(Figure 2). However, treatment with EGCG 25, 50, and 100
mg/kg for 6 weeks reduced SBP in a time-dependent manner
(F=0.2, 14.3, 52.9, 67.1, 64.3, 80.0, 100.0, 111.2 from 0 to 7
weeks postoperation, respectively). EGCG chronic treatment
also reduced HWI and LVWI compared with the AC group
(F=21.0 and 35.1, respectively). The HWI was reduced by
4.7%, 8.37%, and 17.7% at 3 doses respectively, whereas the
LVWI was reduced by 7.1%, 11.1%, and 21.3%, respectively.
Effects of EGCG on MDA content, and SOD and GSH-Px
activities in serum and the myocardium In the rats with
cardiac hypertrophy, the level of MDA content was increased,
and SOD and GSH-Px activities were reduced significantly
vs the sham-operated group, both in serum and in the
myocardium (P<0.01; Tables 1, 2). EGCG 25, 50, and 100 mg/kg
dose-dependently decreased MDA content, and increased
SOD and GSH-Px activities in serum and the myocardium
compared with the AC group.
Effects of EGCG on histological changes HE stain of the
heart tissues showed that compared with sham-operated
group, the hearts of the AC group displayed marked
structural abnormalities, cardiomyocyte hypertrophy, and
cellular fibrosis (Figure 3A_3D). Treatment with EGCG 50 and
100 mg/kg remarkably improved the histological changes in
the heart tissue vs the AC group. As demonstrated by VG
stain, significant intermuscular fibrosis was observed in the
hypertrophic hearts, while EGCG 50 and 100 mg/kg treatment
diminished the extent of fibrosis (Figure 3E_3H).
Effects of EGCG on cardiac apoptosis in the hypertrophic
myocardium In the hearts of the sham-operated group,
TUNEL-positive cells were nearly undetectable, but
numerical TUNEL-positive cells were observed in the myocardium
of rats with cardiac hypertrophy. Treatment with EGCG 50
and 100 mg/kg for 6 weeks showed a significant reduction in
the number of TUNEL-positive cells compared with the AC
rats (F=21.2; Figure 4).
Effects of EGCG on p53 and bcl-2 protein expression in
the myocardium In the rats with cardiac hypertrophy, the
level of the p53 protein increased, and the bcl-2 protein
decreased remarkably compared with the sham-operated
group. EGCG 50 and 100 mg/kg ig for 6 weeks decreased p53
protein expression and increased bcl-2 protein expression in
the hypertrophic myocardium vs the model group
(F=30.8 and 29.2, respectively; Figure 5).
Effects of EGCG on cell viability in
H2O2-induced injury The exposure of cardiomyocytes to
H2O2 200 µmol/L for 24 h
produced an obvious decrease in cell viability as measured
by MTT (P<0.01 vs the model group). When the cultures
were pretreated with EGCG 12.5_200 mg/L
for 6_48 h, the cell damage was greatly attenuated
(F=13.9, 13.5, 19.3, 7.5 from 6_48 h, respectively; Figure 6). The protective effects of
EGCG were shown at pretreatment of myocytes for 6 h,
reached the best at 24 h, and maintained its effect at 48 h. As
to the concentration, pretreatment with EGCG 12.5_100
mg/L concentration-dependently inhibited
H2O2-induced cardiomyocyte injury, but the effect of EGCG 200 mg/L was
reduced to a degree.
Effects of EGCG on LDH release and MDA
formation The exposure of cardiomyocytes to
H2O2 200 µmol/L for 24 h induced marked MDA formation and LDH leakage in
the culture medium (P<0.01; Table 3). Pretreatment with EGCG
12.5_200 mg/L for 24 h attenuated LDH release and MDA
formation in the culture medium.
Effects of EGCG on
H2O2-induced cardiomyocyte
apoptosis When exposed to
H2O2 200 µmol/L for 24 h,
the sizes of most cardiomyocytes were reduced obviously.
Cytoplasm shrinkage and nuclei pyknosis were found under
microscope, indicating distinct apoptosis of cardiomyocytes.
Pretreatment with EGCG 50 and 100 mg/L significantly
attenuated the morphological changes in cardiomyocytes (Figure
7A_7D). When cardiomyocyte apoptosis was quantified by
flow cytometry, the percentage of apoptotic cells increased
from 0.8%±0.7% in the control group to 15.9%±2.3% in the
H2O2 group (n=3,
P<0.01 vs the control group; Figure
7E-7F). Pretreatment with EGCG 50 and 100 mg/L for 24 h decreased
the apoptotic rate to 6.3%±3.1% and 3.6%±0.7%, respectively
(F=32.08, P<0.01 vs the
H2O2 group; Figure 7G_7H).
Discussion
Tea is one of the most consumed beverages in the world,
especially in Asian countries. Tea consumption may be
linked to low incidences of various pathological conditions,
including cardiovascular disease, diabetes, obesity, and
cancer. The principal active polyphenols in green tea
include EGCG, epigallocatechin (EGC), epicatechin (EC) and
epicatechin gallate (ECG), with EGCG being the most
abundant, and possessing the most potent antioxidative
activity. In our experiment, EGCG 25_100 mg/kg,
administered once a day for 6 weeks was used to treat rats with
cardiac hypertrophy. The dose was equivalent to the EGCG
content in approximately 8-23 cups (one cup=120 mL) of
green tea consumption everyday in humans (approximately
80 kg weight)[29].
Chronic pressure overload is the critical factor leading to
cardiac hypertrophy and even heart failure. In the
abdominal aortic constriction model, aortic pressure increased
immediately after AC, and cardiac hypertrophy occurred as an
adaptive response to the imposition of long-term pressure
overload on the heart[19,20,30]. In the present study, the
cardiac hypertrophy model was established by AC in the rats
for 7 weeks. The SBP increased progressively at 1_7 weeks
after aortic abdominal constriction. The HWI and LVWI
increased remarkably vs the sham-operated group.
Structural abnormality, cardiomyocyte hypertrophy, and fibrosis
were detected in the hypertrophic myocardium by
histological analysis. However, chronic treatment with EGCG 25_100
mg/kg dose-dependently reduced SBP, HWI, and LVWI, and
improved histological changes in the myocardium. All these
data supported our finding that EGCG was an effective
therapeutic agent against pressure overload-induced cardiac
hypertrophy.
In this paper, the effects of EGCG on rats with cardiac
hypertrophy were compared with the AC group, so we did
not detect the effect of EGCG on sham-operated rats. It is a
limitation of the experiment and we will try to investigate it in
the further experiments. Nevertheless, Li et
al reported[17] the beneficial effects of EGCF on hemodynamics in the AC
rat model with little effect on the sham-operated rats.
Mounting evidence strongly implicated that oxidative
stress played an important role in the genesis and process of
cardiac hypertrophy[10,11]. Oxidative stress leads to the
accumulation of lipid peroxidation products MDA in the heart,
and causes impaired cell function, while antioxidant enzyme
SOD and GSH-Px play great roles in cellular defense against
oxidative stress. In this study, to confirm the presence of
increased oxidative stress in cardiac hypertrophy, we
quantified myocardial and serum levels of MDA content, and SOD
and GSH-Px activities. The results showed that in the
pressure overload-induced cardiac hypertrophy rat model, the
level of MDA in serum and cardiac tissue increased
significantly, while the activities of SOD and GSH-Px was
greatly reduced vs the sham-operated control, indicating a
significant oxidative stress in cardiac hypertrophy. The
treatment with EGCG almost completely prevented the pressure
overload-induced decrement in SOD and GSH-Px levels, and
MDA formation both in serum and in cardiac tissue. These
results suggest that the protective effects of EGCG on
cardiac hypertrophy were correlated with amelioration of
pressure overload-induced oxidative stress.
As already stated, apoptosis is a critical factor during
transition from compensatory cardiac hypertrophy to heart
failure[4_6]. The introduction of the TUNEL method to
localize the 3' end of DNA in situ has been proposed as a useful
tool for the identification of apoptosis. It has been reported
that pressure overload in the murine heart leads to an
increase in TUNEL-positive
cardiomyocytes[2,6]. In the present study, a TUNEL analysis was used to detect apoptotic
myocytes in the hypertrophic myocardium. The results
showed that in the pressure overload-induced cardiac
hypertrophy rat model, TUNEL- positive cells increased
significantly compared with the sham-operated group, while
EGCG 50 and 100 mg/kg reduced the number of
TUNEL-positive cells remarkably. We also tested whether EGCG could
protect against cultured cardiomyocyte injury and apoptosis
from oxidative stress. H2O2, one major kind of ROS, can lead
to the formation of hydroxyl radicals mediated by
intracellular heavy metal ions through the Fenton reaction. Both
H2O2 and hydroxyl radicals induce severe intracellular oxidant
stress, which cause damage to various intracellular
biomacro-molecules and eventually result in apoptosis and necrosis
of cardiomyocyte[31]. In this experiment, when
cardiomyo-cytes were exposed to exogenous
H2O2, cell viability decreased significantly, and marked LDH release was observed,
indicating injury in the membrane integrality. Lipid
peroxidation products MDA formation greatly increased,
reflecting a significant oxidative stress. Pretreatment with
EGCG 12.5_200 mg/L greatly increased cell viability, reduced
LDH release and MDA formation in the culture medium.
These results suggest that EGCG exerted its antioxidant
effects in H2O2-induced cardiomyocyte injury. Moreover,
H2O2 induced distinct apoptosis in cardiomyocytes.
Morphological observation found cytoplasm shrinkage and nuclei
pyknosis, and the apoptotic rate increased markedly, while
preincubation with EGCG 50 and 100 mg/L significantly
improved myocyte morphological changes and reduced the
apoptotic rate in cardiomyocytes, indicating that EGCG
attenuated cardiomyocyte apoptosis induced by oxidative
stress.
Townsend et al[15] reported that green tea extract, as well
as EGCG, prevented ischemia/ reperfusion (I/R)-induced
cardiac myocyte apoptotic cell death, partly by reducing the
expression of the STAT-1 proapoptotic target gene and the
Fas receptor. However, in this paper we aimed at p53 cell
death signal in cardiac hypertrophy rather than the death
receptor pathway. Evidence shows that p53 plays an
important role in cardiac myocyte apoptosis in cardiac
hyper-trophy. Leri A et al[32] reported that the activation of the p53
and p53-dependent genes were critical in the modulation of
myocyte apoptosis in pacing-induced heart failure. The
upregulation of the local renin-angiotensin system in
stretched cardiomyocytes increased susceptibility of
myo-cytes to undergo apoptosis, coupled with the activation of
p53 and bcl-2 decrease[7]. To determine whether
stretch-induced activation of p53 is necessary for the upregulation
of the local renin-angiotensin system and angiotensin (Ang)
II-induced apoptosis, they infected myocytes with Adp53m
to prevent p53 stimulation, and found Adp53m interference
blocking the functions of endogenous wild-type p53,
reducing Ang II generation and myocyte apoptosis after
mechanical stretching. The research presents definitive proof that
p53 is an essential cofactor in the stimulation of proapoptotic
genes and the induction of cell death by mechanical
stress[33]. Therefore, we used Western blot analysis to detect p53 and
bcl-2 protein expression in the hypertrophic myocardium.
Results showed a significant decrease in bcl-2 and an
increase in the p53 protein in the hypertrophic myocardium
compared with the sham-operated control. In contrast, EGCG
treatment effectively upregulated the bcl-2 protein and
downregulated the p53 protein. It has been reported that
p53 can act to regulate the intracellular redox state and
induce apoptosis by a pathway dependent on ROS
production[5,8,34], whereas bcl-2 may function as an antioxidant to
prevent apoptosis[5]. Bcl-2 may decrease lipid peroxidation
by increasing cell resistance to ROS and blocking ROS
production[5]. Therefore, we proposed the antiapoptotic effect
of EGCG by inhibiting p53 induction and bcl-2 decrease might
be related, at least in part, to its antioxidant effects. We tried
to use RNA silencing technology to detect the effects of
EGCG on p53-mediated apoptosis, but failed to silence the
gene because of the difficulty of transfection into primary
neonatal rat cardiomyocytes. Further investigation is needed
to verify this proposal.
In summary, EGCG remarkably inhibits the formation of
cardiac hypertrophy with marked reductions in pressure
overload-induced oxidative stress and cardiac apoptosis.
Also, EGCG protects cultured cardiomyocytes from
hydrogen peroxide-induced injury and apoptosis. The mechanism
might be related to the inhibitory effects of EGCG on p53
induction and bcl-2 decrease.
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