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FEBS Letters 580 (2006) 13911397

Anthocyanins from soybean seed coat inhibit the expression of


TNF-a-induced genes associated with ischemia/reperfusion in
endothelial cell by NF-jB-dependent pathway and reduce rat
myocardial damages incurred by ischemia and reperfusion in vivo
Hye Jung Kima, Irina Tsoya, Jung Min Parka, Jong Il Chungb, Sung Chul Shinc, Ki Churl Changa,*
a

Department of Pharmacology, School of Medicine and Institute of Health Sciences, Gyeongsang National University, Republic of Korea
b
Department of Agronomy, Research Institute of Life Science, Gyeongsang National University, Republic of Korea
c
Department of Chemistry, Research Institute of Life Science, Gyeongsang National University, Republic of Korea
Received 8 November 2005; revised 19 January 2006; accepted 20 January 2006
Available online 26 January 2006
Edited by Beat Imhof

Abstract We examined the inhibition of the expression of some


inammatory genes associated with ischemia-reperfusion (I/R)
injury by anthocyanins isolated from black soybean seed coat in
tumor necrosis factor-alpha (TNF-a)-treated bovine aortic endothelial cells. In addition, its potential use on I/R-injury was investigated using rats subjected to 30-min occlusion of left descending
coronary artery followed by 24-h reperfusion. Western blot
analysis and luciferase activity assay showed that anthocyanins
inhibited TNF-a-induced vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and cyclooxygenase-2 levels, which
is through NF-jB-dependent pathway. Further, anthocyanins
protected myocardiac injury from I/R in rats. It is suggested that
anthocyanins from black soybean seed coat can be used as a useful drug to modulate cardiovascular disorder.
 2006 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: Atherosclerosis; Anthocyanins; Cyclooxygenase-2;
Ischemic reperfusion injury; Intracellular adhesion molecule-1;
Vascular cell adhesion molecule-1; NF-jB; Tumor necrosis
factor-a

1. Introduction
Myocardial damage due to reperfusion of ischemic tissue is
caused primarily by proinammatory cytokine, tumor necrosis
factor-alpha (TNF-a) [1]. TNF-a is produced by the cardiac
myocytes following ischemia. Inltrating neutrophils also play
a crucial role for the production of TNF-a in myocardial ischemia and reperfusion (I/R)-injury. The rapid release of TNF-a, a
cytokine found in reperfused myocardium [2], contributed to
the transcriptional activation of intracellular adhesion molecule-1 (ICAM-1). They suggest that early cleavage of TNF-a
triggers a cascade of NF-jB activation and ICAM-1 induction
in response to postischemic reperfusion. In fact, NF-jB activa*
Corresponding author. Fax: +82 55 759 0609.
E-mail address: kcchang@gsnu.ac.kr (K.C. Chang).

Abbreviations: COX-2, cylooxygenase-2; ICAM-1, intracellular adhesion molecule-1; I/R, ischemic reperfusion; VCAM-1, vascular adhesion molecule-1; TBS-T, Tris-buered saline/Tween 20

tion can occur after ischemia and reperfusion (I/R) of the heart,
as shown in a study by Chandrasekar and Freeman [3]. Other
factors besides enhanced ICAM-1 expression on the endothelial surface may have contributed to enhanced polymorphonuclear (PMN) leukocytes adhesion after prolonged reperfusion,
especially induction of P-selectin [4] and E-selectin [5], adhesion
molecules mediating rolling as the rst step of PMN adhesion.
Anthocyanins are polyphenols responsible for many of the
fruit and oral colors, basic skeleton of which is 2-phenylbenzopyrylium or avylium glycoside. Anthocyanins are
especially abundant in the epidermis palisade layer of the black
soybean seed coat [611]. Three main anthocyanins, i.e.,
cyanidin-3-glucoside, delphinidin-3-glucoside and petunidin3-glucoside, were characterized in black soybean seed coats
[79,11]. To date, 17 naturally occurring anthocyanins have
been known. Although there is considerable current interest
in various health-promoting benets of anthocyanins such as
potential antioxidant eects, reduction of the risk of coronary
heart disease and prevention of some chronic diseases [12
14], its eect on TNF-a induced upregulation of endothelial
adhesion molecules such as ICAM-1 and vascular cell adhesion
molecule-1 (VCAM-1), which are important factors for aggravating of myocardial I/R-injury, has not been elucidated. In this
study, we investigated our hypothesis that anthocyanins may
attenuate adhesion molecules expression such as ICAM-1 and
VCAM-1, and inammatory gene such as cyclooxygenase-2
(COX-2) in TNF-a-treated endothelial cells and so that it can
reduce I/R-induced injury in vivo. Actually, inammatory cytokines or mediators (TNF-a or LPS) increased VCAM-1 and
ICAM-1 expression in dierent organs of mouse model [15].

2. Materials and method


2.1. Materials
DMEM, fetal bovine serum, penicillin, streptomycin, and glutamine
were supplied by Gibco-BRL (Rockville, MD). PRO-PREP protein
extract solution and ECL Western blotting detection reagents were
supplied by iNtRON Biotechnology (Sungnam, Korea). Ketamine
was purchased from Yuhan Corporation (Seoul, Korea). Xylazine
was obtained from Bayer Korea Ltd. (Seoul, Korea). Anti-ICAM-1,
anti-VCAM-1, anti-COX-2, and anti-phospho-IjBa antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other
chemicals including MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl
tetrazolium bromide) were supplied by Sigma (St. Louis, MO).

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2006.01.062

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2.2. Plant materials
Soybean (Glycine max (L.) Merr.) accessions with black seed coat
were planted at the experimental eld of Gyeongsang National University in May 2004. Plots were harvested when the plants in the plot
reached maturity. Harvested seed was air-dried to a seed moisture content of about 8.0%. The dried seeds were stored at 4 C until they were
used. The seed coats of soybean accessions were peeled manually.
2.3. Anthocyanins extraction and purication
The seed coats of soybean accessions (200 g) were extracted for 24 h at
4 C with methanol. The extraction was repeated three times. After concentration under reduced pressure, the extract was diluted to a total volume of 200 ml and partitioned against ethyl acetate (3 200 ml). The
solution containing anthocyanins was concentrated to 100 ml. The solution was subjected to an Amberlite XAD-7 (Aldrich, St. Louis, MO) column and washed with deionized water and eluted with methanol
containing 1% HCl. The solvent was vaporized under reduced pressure
and the purple sticky solids dissolved in 50 ml of 30% aqueous methanol
containing 1% HCl. The solution was applied to a column packed with
Sephadex LH-20 (Amersham Biosciences, Sweden) and eluted using
30% aqueous methanol containing 1% HCl. Cyanidin-3-glucoside, delphinidin-3-glucoside, and petunidin-3-glucoside were isolated from
Seed Coats of Black Soybean and used as anthocyanin source. The compositions of anthocyanin consisted of cyanidin-3-glucoside (72%),
delphinidin-3-glucoside (20%) and petunidin-3-glucoside (6%).
2.4. Cell culture
Bovine aortic endothelial cells (BAECs) were purchased from American Type Culture Collection (Arlington, VA). Cells were cultured in
100 mm dish and grown in DMEM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated FBS in a
humidied atmosphere of 5% CO2. BAECs were plated at a density
of 1 107 cells/100 mm dish. Cells used were between passages 6 and 14.
2.5. Cell viability assay
The cell viability was determined colorimetrically using the MTT reagent. Cells at the exponential phase were seeded at 104 cells/well in 24well plates. After dierent treatments, 20 ll of 5 mg/ml MTT solution
was added to each well (0.1 mg/well) and incubated for 4 h. The supernatants were aspirated and the formazan crystals in each well were dissolved in 200 ll dimethyl sulfoxide (DMSO) for 30 min at 37 C and
570 nm was read on the Microplate Reader (Bio-Rad, Hercules, CA).
2.6. Western blot analysis
Cellular compartment was extracted according to the minor modication of the procedure described previously [16]. Briey, cells were
washed with ice-cold PBS (pH 7.4) and lysed in buer A [10 mM
HEPES (pH 7.9), 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT),
5 lM leupeptin, 2 lM pepstatin A, 1 lM aprotinin, and 20 lM phenylmethylsulfonyl uoride] by repeated freezing and thawing. Nuclear
and cytoplasmic fractions were separated by centrifugation at
1000 g. The supernatant (cytoplasmic extract) was obtained by further centrifugation at 10 000 g for 15 min. The pellets were washed
once with buer A, and resuspended in buer B [10 mM TrisCl (pH
7.5), 0.5% deoxycholate, 1% Nonidet P-40, 5 mM EDTA, 0.5 mM
DTT, 5 lM leupeptin, 2 lM pepstatin A, 1 lM aprotinin, and
20 lM phenylmethylsulfonyl uoride]. The suspension was agitated
for 30 min at 4 C and centrifuged at 10 000 g for 20 min. The
supernatant fraction containing nuclear proteins was collected. For
isolation of total cell extracts, cells were lysed in PRO-PREP protein
extract solution. The sample was centrifuged at 13 000 rpm 20 min
at 4 C. Protein concentration was determined by Bradford method.
An equal volume of 2 SDS sample buer (0.1 M Tris-CI, 20% glycerol, 4% SDS, and 0.01% bromophenol blue) was added to aliquot of
the supernatant fraction from the lysates and the samples were boiled
for 5 min. Aliquots of 30 lg of protein were subjected to 10% SDS
polyacrylamide gel electrophoresis for 1 h 30 min at 110 V. The separated proteins were transferred to PVDF membrane for 2 h at
20 mA with S.D. Semi-dry Transfer Cell (Bio-Rad). The membranes
were blocked with 5% non-fat milk in Tris-buered saline (TBS) containing 0.05% Tween 20 (TBS-T) for 2 h at room temperature. The
membranes were then incubated with anti-ICAM-1, anti-VCAM-1,
anti-COX-2, anti-phospho-IjBa, or anti-NF-jB (p65) antibody at
1:500 concentration (4 lg/ml) in 5% skimmed milk in TBS-T over-

H.J. Kim et al. / FEBS Letters 580 (2006) 13911397


night at 4 C and the bound antibody was detected by horseradish
peroxidase (HRP) conjugated anti-goat IgG. The membranes were
washed and then developed using a Western Blotting Luminol Reagent system and autoradiography.
2.7. Transfection
NF-jB-luciferase constructs (consensus NF-jB binding sequence
was cloned into the pGL3 basic luciferase expression vector) were
kindly provided by Dr. G. Koretzky (University of Pennsylvania)
and the dominant negative IjBa mutant plasmid (pcDNA/IjBa-SR)
was a gift from Dr. H. Nakshatri (Indiana University School of Medicine, Indianapolis, IN). Transient transfections were performed using
Lipofectin (Gibco-BRL) according to the manufacturers protocol.
Briey, 5 105 cells were plated in 60 mm dish plate the day before
transfection and grown to about 70% conuence. Cells were transfected with empty vector (pGL3 and/or pcDNA3), 1 lg of NF-jB-luciferase + 0.5 lg of pRL-TK-luciferase, or 1 lg of pcDNA/IjBa-SR and/or
1 lg of NF-jB-luciferase + 0.5 lg of pRL-TK-luciferase. Transfections were allowed to proceed for 12 h. The transfected cells were
washed with 4 ml of PBS and then stimulated with 10 ng/ml TNF-a.
The cells were continually cultured in serum-free DMEM until they
were harvested. Luciferase activity was normalized using pRL-TKluciferase activity (Renilla luciferase activity) in each sample.
2.8. Luciferase assay
After experimental treatments, the cells were washed twice with cold
PBS, lysed in a passive lysis buer provided in the dual luciferase kit
(Promega, Madison, WI), and assayed for luciferase activity using
TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) according
to the manufacturers protocol. All transfections were done in triplicate. Data were presented as a ratio between Firey and Renilla luciferase activity.
2.9. Myocardial ischemia and reperfusion injury
Adult male Sprague-Dawley rats (weighing 220250 g) were anesthetized with ketamine (30 mg/kg) and xylazine (6 mg/kg). The trachea was
cannulated with a catheter and articial respiration was provided by a
respirator with a frequency of 60 strokes/min, and a tidal volume of
2 ml to maintain normal PO2 and PCO2. Rectal temperature was monitored with a rectal probe and maintained within 36.5 and 37 C. Coronary occlusion and reperfusion were performed as described by
Zingarelli et al. [17]. The chest was opened by a cut along the left side
of the sternum through the ribs. The animal was rotated to expose
the left ventricle. Ligation proceeded with a 6-0 silk suture passed with
a tapered needle underneath the left anterior descending branch (LAD)
of the left coronary artery. After 30 min occlusion, reperfusion occurred by cutting the knot on top of the tubing with a surgical blade.
One group of rats underwent this surgical procedure with the exception
of LAD occlusion and reperfusion, and served as a sham (S) control.
All animals were maintained in accordance with the Guide for the Care
and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The protocol was approved before the animal study by the Animal Research
Committee of the Gyeongsang National University, Korea.
2.10. Experimental protocol
Rats were randomly assigned to one of the three groups: Group A,
sham (n = 20); Group B, ischemia (30 min) and subsequent reperfusion
(24 h) and treatment with placebo (Water 0.2 ml/200 g, n = 32); and
Group C, pretreatment with anthocyanins (25 mg/kg; n = 32, 50 mg/
kg; n = 32, 100 mg/kg, n = 32) before I/R injury. Anthocyanins or
water were administered orally (p.o.) 24 h prior to I/R injury.
2.11. Tissue preparation
After completion of the experiment according to the protocols,
hearts from each group were rapidly harvested, and left ventricles were
removed and used for both histological and biochemical studies.
2.12. Quantication of myocardial injury
The heart was excised from the thorax rapidly and the greater vessels
were removed. The left ventricle was separated from the heart and was
weighed. It was sliced parallel to the atrioventricular groove to 23 mm

H.J. Kim et al. / FEBS Letters 580 (2006) 13911397


thick sections and the slices (7 pieces per heart) were incubated in 1%
TTC (2,3,5-triphenyl tetrazolium chloride) solution prepared in pH 7.4
phosphate buer for 30 min at 37 C. In viable myocardium, TTC is
converted by dehydrogenase enzymes to a red formazan pigment that
stains tissue dark red. The infarcted myocardium that does not take
TTC stain, where the dehydrogenase enzymes are drained o, remains
pale in color. The pale necrotic myocardial tissue was separated from
the stained portions and weighed on an electronic balance. The sizes of
the corresponding area were calculated as a percent normalized to the
weight of the slice and expressed as ratio of the infarcted infracted area
(INF) to left ventricle (LV) using GS-710 Calibrated Imaging Densitometer and Quantity One 1-D Analysis Software (Bio-Rad).

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and were incubated for 24 h. As shown in Fig. 1, anthocyanins


even at high concentration did not inuence cell viability in
cultured BAEC by MTT assay. Instead, anthocyanins protected BAECs from H2O2-induced cytotoxicity. Cell viability
was decreased by 1 mM H2O2 (about 68%), which is signicantly increased by pretreatment of anthocyanins in a concentration dependent manner (1, 10, 50, 100 lg/ml).

2.13. Statistical evaluations


Values are expressed as means S.D. The signicance of the dierence from the respective controls for each experimental test condition
was assayed by using students t test for each pair of experiments.
P 6 0.05 was regarded as a signicant dierence.

3. Results
3.1. Cytoprotective eect of anthocyanins in BAEC under
cytotoxicity by H2O2
To examine cell viability of BAEC as the response to anthocyanins, cells were treated with anthocyanins dose-dependently

Fig. 1. Cytoprotective eect of anthocyanins on H2O2-induced injury


in BAEC. (A) The eect of anthocyanins (ANT) on cell viability was
measured in a concentration-dependent manner by MTT. Cells were
incubated with anthocyanins for 24 h. (B) Cells were incubated with
H2O2 (1 mM) for 24 h. Anthocyanins (ANT) were pretreated for 24 h
before incubation of cells with 1 mM of H2O2 for 24 h. It clearly
showed that anthocyanins signicantly increased the cell viability
against H2O2, which was also concentration dependent. Data represent
means S.D. of three independent experiments (signicance compared
with the control, ** P < 0.01; signicance compared with H2O2,

P < 0.05, P < 0.01).

Fig. 2. Inhibition of TNF-a-induced ICAM-1, VCAM-1, or COX-2


expression by anthocyanins in BAEC. Cells were pretreated with
anthocyanins (ANT, 10, 50, 100 lg/ml) for 24 h and then treated with
TNF-a (10 ng/ml) for 6 h. After treatment, protein was extracted from
the cells and ICAM-1, VCAM-1, or COX-2 protein level was
determined by Western blot analysis as described in Section 2 (A).
The band intensities were assessed by scanning densitometry (B). Data
are presented as means S.D. of three independent experiments. Oneway analysis of variance was used to compare the multiple group
means followed by NewmanKeuls test (signicance compared to the
control, ** P < 0.01; signicance compared with TNF-a, P < 0.05,

P < 0.01) (control level = 1).

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3.2. Inhibitory eect of anthocyanins on the expression of


ICAM-1, VCAM-1, or COX-2 as well as phosphorylation
of IjBa by TNF-a in BAEC
To investigate whether anthocyanins could exert antiinammatory eect on endothelial cells, inammatory
markers (ICAM-1, VCAM-1, and COX-2) involved in cardiovascular disorder such as atherosclerosis and ischemic
heart disease were determined after stimulation with TNF-a
in BAEC. Endothelial cells were pretreated with various concentrations of anthocyanins for 24 h and next cotreated with
TNF-a for 6 h. As the concentration of anthocyanins increases, ICAM-1, VCAM-1, and COX-2 expressions induced
by TNF-a were diminished (Fig. 2). Phosphorylation of
IjBa, the inhibitors of NF-jB activity, by kinases results in
the degradation of IjB with release of NF-jB, which translocates to the nucleus where it is active in the regulation of a
transcription gene. Thus, we determined if anthocyanins prevent the phosphorylation of IjBa by TNF-a. As shown in
Fig. 2, anthocyanins dose dependently prevented the phosphorylation of IjBa (Fig. 2).

H.J. Kim et al. / FEBS Letters 580 (2006) 13911397

in the regulation of a transcription gene. Thus, we determined


if anthocyanins prevent the phosphorylation of IjBa by TNFa. As shown in Fig. 3, anthocyanins dose dependently prevented the phosphorylation of IjBa (Fig. 3B). Moreover,
TNF-a increased NF-jB-luciferase activity by approximately
2.6-folds higher than that of the untreated control (Fig. 4A).
However, when the mutant pcDNA/IjBa-SR was transfected,
it did not change the luciferase activity by TNF-a (Fig. 4B). In
addition, anthocyanins eciently inhibited NF-jB-luciferase

3.3. Inhibition of TNF-a-induced NF-jB activity and IjBa


phosphorylation by anthocyanins
Inammation-related genes such as COX-2 and adhesion
molecules (ICAM-1 and VCAM-1) contain binding sites for
a number of important transcription factors, including NFjB [1820]. NF-jB is activated by various inammatory stimuli including TNF-a. Therefore, to determine whether NF-jB
activated by TNF-a is inhibited by treatment of anthocyanins,
we performed NF-jB translocation experiments and luciferase
reporter assay. As shown in Fig. 3A, TNF-a caused NF-jB
(p65) translocation into nucleus, which is inhibited by pretreatment with anthocyanins concentration-dependently. In addition, anthocyanins concentration-dependently inhibited the
translocation of NF-jB (p65) from cytosol to the nucleas.
Phosphorylation of IjBa, the inhibitors of NF-jB activity,
by kinases results in the degradation of IjB with release of
NF-jB, which translocates to the nucleus where it is active

Fig. 3. Inhibitory eect of anthocyanins on TNF-a-induced NF-jB


translocation into nucleus (A) and IjBa phosphorylation (B) in
BAEC. Cells were pretreated with anthocyanins (ANT, 10, 50, 100 lg/
ml) for 24 h and then treated with TNF-a (10 ng/ml) for 6 h. After
treatment, nuclear, cytoplasmic fractions or total cell lysates were
extracted and protein level was determined by Western blot analysis as
described in Section 2. Data were conrmed by two independent
experiments.

Fig. 4. Inhibition of TNF-a-induced NF-jB-luciferase activity by


anthocyanins in BAEC. Cells were transfected with empty vector or
1 lg of NF-jB-luciferase + 0.5 lg of pRL-TK-luciferase (A, C), and
empty vector or 1 lg of pcDNA/IjBa-SR and 1 lg of NF-jBluciferase + 0.5 lg of pRL-TK-luciferase (B). Cells were allowed to
recover for 24 h and then treated with 10 ng/ml of TNF-a with/without
anthocyanins (ANT, 50 lg/ml). Cells were harvested 6 h posttreatment. Luciferase activities are presented as the fold activation relative
to that of the untreated cells. Data are presented as means S.D. of
three independent experiments. One-way analysis of variance was used
to compare the multiple group means followed by NewmanKeuls test
(signicance compared to the control, ** P < 0.01; signicance compared with TNF-a, P < 0.05, P < 0.01) (control level = 1).

H.J. Kim et al. / FEBS Letters 580 (2006) 13911397

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activity by TNF-a (Fig. 4C). These results suggest that anthocyanins inhibit NF-jB activation by TNF-a through prevention of IjBa phosphorylation.
3.4. Anthocyanins reduce infarct size by I/R injury
We then investigated the protective eect of anthocyanins on
the I/R injury. Sham group rats underwent on surgical procedure without LAD occlusion that showed no infarct area,
whereas rat I/R hearts showed clearly increased infarct area,
32 4.5%. Anthocyanins also dose-dependently decreased infarct size in rat I/R hearts in vivo. As shown in Fig. 5, the results clearly show that dose-dependent administrations of
anthocyanins (25, 50 and 100 mg/kg) signicantly reduced
the percent of infarct area (INF)/area at left ventricle (LV)
from 32 4.5% to 15.1 3.5%, 13.2 2.8%, 8.8 1.2%,
respectively.

4. Discussion

Fig. 5. Protection of myocardial ischemia and reperfusion injury by


anthocyanins in vivo. Myocardial ischemia and reperfusion injury were
induced as described in Section 2. (A) Sham, (B) I (30 min) + R (24 h),
(C) I/R + anthocyanins (ANT, 25, 50, 100 mg/kg). (D) The results were
conrmed by four independent experiments. The percent of infarcted
area (INF) at left ventricle (LV) is presented as the fold activation
relative to that of the sham group. Data are presented as means S.D.
of four independent experiments. One-way analysis of variance was
used to compare the multiple group means followed by Newman
Keuls test (signicance compared to sham, ** P < 0.01; signicance
compared with ischemia/reperfusion (I/R), P < 0.01). Ischemia and
reperfusion signicantly increased myocardiac injury in the left
ventricles compared to sham group, in which the administration of
anthocyanins concentration dependently and statistically signicantly
decreased myocardiac injury.

We clearly showed that anthocyanins concentration-dependently inhibited TNF-a induced expression of those genes
associated with I/R-injury, such as ICAM-1, VCAM-1, and
COX-2 in BAECs. A major site of action of TNF-a for these
eects is the vascular endothelium, where it induces inammatory responses by enhancing adhesion molecule expression and
secretion of inammatory mediators. Because TNF-a has been
implicated in the pathogenesis of myocardial dysfunction in I/
R-injury, we, therefore, used TNF-a as proinammatory cytokine in the present study. Many previous investigations indicated that inhibition of the expression of TNF-a eectively
reduced myocardial I/R-injury [21]. This benecial eect
against TNF-a on I/R-injury does not limit to cardiac cells. Indeed, treatment of rats with anti-TNF-a antibodies also prevented tissue injury and lethality after intestinal I/R-injury
[22]. I/R-injury is associated with the coordinated activation
of a series of cytokines and adhesion molecules. ICAM-1
and VCAM-1 are responsible for monocyte adhesion and increased vascular inammation. ICAM-1 and VCAM-1 mediate transmigration of leukocytes to the endothelium leading
to endothelial damage. In addition, COX-2 expression is increased in endothelial cells of human atherosclerotic plaques
[2325], resulting in the activation of MMPs [26]. A critical element in the regulation of these genes involves the transcription
factor NF-jB [27]. NF-jB is activated by a vast number of
agents, including TNF-a. The genes regulated by the NF-jB
family of transcription factors are diverse and include those involved in the inammatory response (COX-2), cell adhesion
and growth control [2830]. Furthermore, NF-jB activation
has been demonstrated in various models of experimental I/
R [31,32]. Additionally, Li et al. [33] demonstrated that inhibition of NF-jB activation in cardiac cell from rat heart downregulated TNF-a mRNA expression during myocardial
ischemia. The present study showed that a natural anthocyanins extract from black soybean with known antioxidant properties [34] regulated TNF-a-induced VCAM-1, ICAM-1, and
COX-2 levels through NF-jB dependent pathway. These
ndings suggest that anthocyanins may have benecial eect
for the protection of endothelial cells when administered
during myocardial ischemia or I/R-injury. Furthermore, we
conrmed that the expression of ICAM-1, VCAM-1, and other

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inammatory mediators (COX-2) are major targets for TNF-a


in endothelial cells and these genes are associated with I/Rinjury, because anthocyanins signicantly reduced the myocardial damage due to I/R in vivo (see below). Because TNF-a is
reportedly expressed during ischemia and can induce vascular
inammation leading to endothelial dysfunction, we hypothesized that this inammatory cytokine may play a pivotal role
in I/R injury-induced coronary endothelial dysfunction. This
hypothesis is working in vivo as shown in vitro, that is, anthocyanin administration indeed reduced infarct size in I/R rat
in vivo. Although we did not investigate that the principal molecule of the I/R-injury in vivo was TNF-a in the present study,
we proved this potentiality by showing that administration of
anthocyanins signicantly prevented the I/R-injury in rats subjected to ischemia (30 min) and reperfusion (24 h). Furthermore, recent clinical investigation by Blancke et al. [35]
supported that systemic inammation, in particular high level
of TNF-a, is strongly associated with the occurrence of reperfusion injury after successful recanalization in patients with
acute myocardial infarction. However, further study is needed
to identify that anthocyanins reduce inammatory cells in
endothelium under I/R injury in vivo. To the best of our knowledge, this is the rst study to examine that anthocyanins from
black soybean have protective eects on the myocardiac infarction and endothelial adhesion molecule expression, which is
through inhibition of NF-jB activation.
Black soybeans have popularly been utilized as a food and
medicinal material for a long time with low price. Since anthocyanins have antioxidant eect along with downregulation of
expression of genes associated with I/R in the present study,
it can be more useful for the treatment of cardiovascular disorders. Although the exact mechanism by which anthocyanins
prevent the expression of adhesion molecules remained to be
elucidated, they can be used as good materials to modulate
or prevent the cardiovascular related disorder such as
atherosclerosis and ischemic injury. We clearly showed that
anthocyanins attenuate adhesion molecules expression such
as ICAM-1 and VCAM-1, and inammatory gene such as
COX-2 in TNF-a-treated endothelial cells by preventing the
phosphorylation of IjBa and thereby the inhibition of activation of NF-jB. Most importantly, it reduced I/R-induced
myocardial injury in vivo. Taken together, in this study, we
suggested the possibility that anthocyanins from black
soybean can be used or developed as useful drug to modulate
cardiovascular disorder.
Acknowledgment: This work was supported by a grant (CG 3122) from
Functional Genomics Center (CFGC), 21C Frontier R&D Project,
Ministry of Science & Technology, Korea.

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