Cardioprotection of Vitexin On Myocardial Ischemia

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Cardioprotection of Vitexin on Myocardial Ischemia/Reperfusion

Injury in Rat via Regulating Inflammatory Cytokines and MAPK
Pathway
Article in The American Journal of Chinese Medicine · November 2013

DOI: 10.1142/S0192415X13500845 · Source: PubMed
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The American Journal of Chinese Medicine, Vol. 41, No. 6, 1251–1266
© 2013 World Scientific Publishing Company
Institute for Advanced Research in Asian Science and Medicine
DOI: 10.1142/S0192415X13500845
Cardioprotection of Vitexin on Myocardial

Ischemia/Reperfusion Injury in Rat via
Regulating Inflammatory Cytokines and
Am. J. Chin. Med. 2013.41:1251-1266. Downloaded from www.worldscientific.com
MAPK Pathway
Liu-Yi Dong,*,a Sheng Li,*,a Yi-Lan Zhen,*,a Ya-Nan Wang,* Xu Shao† and Zhi-Gang Luo‡
by 86.51.26.16 on 06/22/14. For personal use only.
*Department of Pharmacology, Anhui Medical University
Key Laboratory of Anti-Inflammatory and Immunopharmacology of Ministry of Education

Key Laboratory of Chinese Medicine Research and
Development of State Administration of Traditional Chinese Medicine
Hefei 230032, China
†
Hefei Qi-Xing Medicine and Technology Co. Ltd
Hefei 230088, China
‡
Department of Cardiology
First Affiliated Hospital of Anhui Medical University
Hefei 230022, China
Abstract: This study was conducted to demonstrate myocardial protective effects and poss-
ible underlying mechanisms of vitexin on myocardial ischemia/reperfusion (I/R) injury in
rats. Occluding the anterior descending artery for 30 min and restoring blood perfusion for
60 min in rat established a model of myocardial I/R. The elevation of the ST segment of
Electrocardiograph (ECG) was observed. The infarct size of the rat heart was assessed by
triphenyltetrazolium chloride staining (TTC). LDH, CK, SOD activities and MDA content
were determined. An immunohistochemical analysis was applied to measure the expression
of myocardial NF-Bp65 and TNF-. ERK/phospho-ERKand c-Jun/phospho-c-Jun protein
expression was examined via Western Blot. Vitexin significantly reduced the elevation of the
ST segment of ECG and myocardial infarct size. LDH and CK activities and MDA content
were attenuated in serum, while SOD activity was markedly enhanced. Vitexin significantly
attenuated I/R-induced increases of myocardial NF-B and TNF-. Moreover, Western Blot
analysis presented that vitexin markedly enhanced the expression of phospho-ERK and
weakened the expression of phospho-c-Jun compared to I/R group. The significant protective
Correspondence to: Dr. Liu-Yi Dong, Department of Pharmacology, Anhui Medical University, No. 81 Meishan
Road, Hefei, Anhui 230032, China. Tel: (þ86) 551-6511-6866, E-mail: dongliuyi@aliyun.com; or Dr. Zhi-Gang
Luo, Department of Cardiology, First Affiliated Hospital of Anhui Medical University, No. 81 Meishan Road,
Hefei 230022, China. E-mail: luo2343@sina.com
a
These authors contributed equally to this work.
1251
1252 L.-Y. DONG et al.
effect against myocardial ischemical/reperfusion injury in rat, which is exhibited by vitexin,

may be related to its antioxidative and anti-inflammatory effects by regulating inflammatory
cytokines and the MAPK pathway.
Keywords: Heart; Vitexin; Ischemical/Reperfusion; Inflammatory Cytokines; Antioxidation;

MAPK.
Introduction
Despite the extensively studied therapeutic strategies implemented to protect the ischemic
myocardium, cardiovascular disease culminating in myocardial infarction and heart failure
remains a leading cause of morbidity and mortality.
Prolonged cardiac ischemia leads to irreversible cell death. Therefore, reperfusion is
essential in order to salvage tissue from inevitable necrosis. Although restoration of blood
flow is necessary, it also augments a localized oxidative burst and regional inflammatory
response that can provoke further myocardial damage named “reperfusion injury”
(Marchant et al., 2012). Ischemia and reperfusion (I/R) occurs in a wide range of situations,
such as vascular reflow after contraction, thrombolysis treatment, transluminal coronary
angioplasty, and hypovolemic shock with resuscitation. The underlying mechanisms of I/R
injury have not been fully explained. Cardiac I/R injury is a multi-factorial process, and
associates with a series of factors such as oxidative stress, inflammatory cytokines, etc.
Numerous studies have shown that apoptosis is one of the most important causes of
damage after myocardial I/R (Lee et al., 2006). Considerable evidence implicates reactive
oxygen species (ROS) as an initial cause of I/R injury (Venardos et al., 2007). The
progression of heart failure is postulated to involve the continual loss of functional cardiac
myocytes through pathways of cell death, including necrosis, apoptosis, and autophagy
(Diwan and Dorn, 2007; Shaw and Kirshenbaum, 2008). Cardiac myocyte-specific over-
expression of TNF- in transgenic mouse provoked dilated cardiomyopathy and heart
failure. This suggested that activation of the death receptor pathway by TNF- is harmful
to the heart (Kubota et al., 1997). As an important contributor, inflammation is the
pathophysiology of cardiac I/R injury. Acute and chron inflammatory events, a con-
sequence of I/R, have profound effects on the functional deterioration of the heart
(Marchant et al., 2012). A complex process called myocardial ischemia/reperfusion (I/R)
injury, which involves the generation and release of reactive oxygen species and inflam-
matory cytokines, were implied in all cases above (Vinten-Johansen, 2004).
Plenty of traditional Chinese medicine herbs have protective effects against myocardial
ischemia/reperfusion injury, especially the flavonoids (Chan et al., 2011; Wang et al.,
2011). They are polyphenolic compounds that isolated from various plants. Furthermore,
they are significant components of various foods of plant origin. Vitexin, identified as
apigenin-8-C-β-D-glucopyranoside, is a flavonoid compound found in Anthurium versi-
color, Cucumis sativus L. (Cucurbitaceae), and Acer palmatum (Aceraceae) (Aquino et al.,
2001; McNally et al., 2003; Kim et al., 2005). Early studies have shown that vitexin has
VITEXIN CARDIOPROTECTION ON MYOCARDIAL ISCHEMIA/REPERFUSION 1253
the hypotensive effect and anti-inflammatory action (Prabhakar et al., 1981). Recent
research suggested the potential use of vitexin in the treatment for diseases such as cancer
(Choi et al., 2006).
In our previous work, it was implied that vitexin has a protective effect against cardi-
omyocytes anoxia/reoxygenation injury and myocardial I/R injury in isolated heart of rat
(Dong et al., 2008, 2011). The underlying mechanisms cannot be fully explained and there
is limited study about the cardio-protective effect of vitexin in vivo. Therefore, we tended
to investigate the probable mechanism of action and therapeutic effect of vitexin on
I/R-induced myocardial injury in vivo in our current study.
Materials and Methods
Drugs and Reagents
Vitexin, a flavone glycoside, was obtained from Hefei Qi-xing Medicine and Technology
Co., Ltd. Puerarin injection was provided by Beijing Concord pharmaceutical factory.
Hydral was bought from Shanghai Bai-he chemical plant in China. 2,3,5-triphenylte-
trazolium chloride (TTC) and Evans blue were products of Sigma Chemical Co. Lactate
dehydrogenase (LDH), creatine kinase (CK), superoxide dismutase (SOD) and mal-
ondialdehyde (MDA) assay kits were purchased from NanJing JianCheng Bioengineering
Institute. Rabbit polyclonal antibody of (phospho-) ERK and (phospho-) c-Jun were pur-
chased from Bioworld Technology Inc. The rabbit polyclonal antibody of NF-Bp65 and
TNF- was purchased from Beijing Zhong-Shan Biotechnology Co. ltd. A Diamino-
benzidine (DAB) staining kit was provided by Beijing Zhong-Shan Biotechnology Co.,
Ltd. Other reagents were all from commercials sources.
Animals and Experimental Protocol
Healthy male adult Sprague-Dawley (SD) rats (weighing 230 20 g), purchased from the
Experimental Animal Center of Anhui Medical University (Licence: SCXK (Wan) 2011-
001). This investigation has been carried out in accordance with the regulations stipulated
by Anhui Medical University Animal Care Committee which follows the protocol outlined
in the Guide for the Care and Use of Laboratory Animals published by the US National
Institute of Health (NIH publication No. 85-23, revised 1996), and the experimental pro-
cedures used in this research were approved by the Animal Experimentation Ethics
Committee of Anhui Medical University. These animals were adapted to their environment
for one week before being used in experimentation. They were housed at room temperature
(232 C) with a 12-hour light/dark cycle, and given standard chow and water ad libitum
for the duration of the study.
The 96 rats were randomly divided into the following six groups with 16 rats in each
group: (1) sham group, sham-operated without occludes the artery, (2) I/R group, I/R alone
with normal saline (NS) treatment, (3) I/R þ puerarin group, puerarin was administered
intravenously at a dose of 30 mg/kg, beginning at coronary ischemia and again at
reperfusion, (4) I/R þ vitexin 6 mg/kg group, vitexin was administered intravenously at a
dose of 6 mg/kg, beginning at coronary ischemia and again at reperfusion, (5) I/R þ vitexin
3 mg/kg group, vitexin was administered intravenously at a dose of 3 mg/kg, beginning at
coronary ischemia and again at reperfusion, (6) I/R þ vitexin 1.5 mg/kg group, vitexin was
administered intravenously at a dose of 1.5 mg/kg, beginning at coronary ischemia and
again at reperfusion. After the reperfusion, blood samples were collected from the
abdominal aortic and centrifuged, and then serum were collected and frozen at 80 C until
analysis. The mortality of rats for the experiment is approximately 10%. Ninety-six rats
were involved in the final analysis.
Myocardial Ischemia/Reperfusion Model
Rats were anesthetized with an intraperitonal injection of chloral hydrate at a dose of

300 mg/kg (10%, 0.3 mL/100g), and then fixed in a supine position. Electrocardiograph
(ECG) electrodes were subcutaneously connected in all limbs. Lead electrocardiogram

(ECG) II was continuously monitored throughout the experiment. The trachea was intu-
bated through a midline neck incision and mechanically ventilated with ambient air using a
volume-controlled rodent ventilator (respiratory rate was maintained at 50–70 strokes/min,
tidal volume, 8 ml/kg). The chest was opened through a left thoracotomy in the fourth
intercostal space followed by a pericardiotomy to expose the heart. For the I/R model, a
6–0 black silk suture was passed around the left anterior descending (LAD) coronary
artery, 2 mm from the tip of the left auricle. The ends of suture were pulled through a piece
of plastic tube to form a snare that occludes the artery when tightened and clamped.
Successful ischemia was notarized by the appearance of regional epicardial cyanosis over
the myocardial surface and myocardial infarction performance of ECG (elevation of the ST
segment). After 30 min of ischemia, the coronary artery was reperfused for 60 min by
releasing the clamp. The rats of the sham group were sham-operated without I/R and
underwent the same procedures. The mortality for the experiment is approximately 10%.
Measurement of the Elevation of the ST Segment of ECG
During the experiment, ECG electrodes were connected subcutaneously in all limbs of rats.
Lead electrocardiogram (ECG) II was continuously monitored and recorded. The
elevations of the ST segment of basic, ischemia for 30 min, reperfusion for 30 min and
reperfusion for 60 min were measured respectively using the image analysis software.
Measurement of Infarct Volume and Area at Risk
At the end of reperfusion, 48 rats were sacrificed and their hearts were removed. The LCA
was occluded again and the ascending aorta was cannulated with a 25-gauge tubing
adapter. 0.25% Evans blue dye was perfused into the aorta and coronary arteries, allowing
the dye to stain the non-ischemic portion of the heart. The risk zone was identified as the
region lacking blue staining. TTC staining was used to identify the area with infarct. The
hearts were cross-sectioned into 2-mm thick transverse sections. Then incubated in 1% TTC
PBS solution, which had a pH of 7.4, at 37 C for 15 min and subsequently fixed in 10% PBS-
buffered formalin overnight at 4 C. Ischemic myocardium, which is still viable, stains red
with TTC, whereas the necrotic myocardium does not stain and appears white or pale. Both
sides of each TTC-stained tissue slice were photographed again with the digital camera.
Infarct size-IS (white) and the area at risk-AAR (red and white) from each section were
measured using an image analyzer. The percentage of infarction size (IS) against the area at
risk (AAR) was calculated using the image analysis software (Sigma Scan program 4).
Detection of Creatine Kinase (CK), Lactate Dehydrogenase (LDH),

Superoxide Dismutase (SOD) and Malondialdehyde (MDA)
To measure the activities of creatine kinase (CK) and lactate dehydrogenase (LDH), the
concentration of malondialdehyde (MDA) and superoxide dismutase (SOD) supernatant of
blood was collected and centrifuged for 5 min at 1500 g. Experiments were carried out with
the corresponding commercial kits according to the manufacturer’s instructions. Then was
detected respectively at 440 nm and 340 nm by spectrophotometry.
Immunohistochemical Analysis
Another 48 rats were sacrificed and their hearts were obtained for immunohistochemistry and
Western Blot detection. The protein expression of NF-Bp65 and TNF- was determined by
immunohistochemistry. Paraffin-embedded myocardial tissue blocks were sectioned at 3 m.
Sections were deparaffinaged in xylene and dehydrated through graded ethanol, then washed
thrice with phosphate-buffered saline (PBS) for 5 min. The sections were treated with 3%
H2 O2 for 10 min at room temperature to quench endogenous peroxidase activity. After the
sections were blocked with 5% normal goat serum in PBS for 30 min, they were incubated in
primary antibodies for 1 h at 37 C. The purpose of the PBS buffer solution was to replace the
antibody in the negative control group. After three washes in PBS, the sections were incu-
bated with biotinylated secondary antibody for 30 min, and followed by incubation with
streptavidin-biotin complex for 60 min at 37 C. The sections were enveloped with gelatin and
observed under an immunofluorescence microscopy after the reaction with a diamino-
benzidine (DAB) reagent. For each group, three random tissue sections (four fields per
section) were photographed and examined. The total area of positive track point in each visual
field and the mean optical density (OD), which related to immunohistochemical staining
intensity, was calculated by image analysis software. Finally, quantitative immunohisto-
chemical assessments for myocardial NF-Bp65 and TNF- expression were performed as a
ratio of the total area of positive track point to the mean optical density.
Western Blot Analysis
The hearts of rats were harvested and washed with ice-cold normal saline (NS) ð4 C), then
cut pieces and placed in lysis buffer containing: [Hepes 20 (pH 7.7), MgCl2 2.5,
dithiothreitol (DTT) 0.5, EDTA 0.1, (-glycerophosphate 20, Sodium orthovanadate 0.1,
NaCl 75 and leupeptin 4 (g/ml, phenyl-methylsulfonyl fluoride (PMSF) 20 (g/ml, TritonX-
100 0.05% (v/v)], homogenized. The buffer was placed on ice for 30 minutes. After
centrifugation for 5 min at 12,000 rpm at 4 C, supernatant samples (200 l) were collected.
The protein concentration of each sample was determined using a BCA protein assay kit.
To detect p-ERK/ERK and p-c-Jun/c-Jun, samples containing equal amounts of protein
(40 g) were electrophoresed and separated on 12% SDS polyacrylamide gels. Wet elec-
trophoresis (120 min at 100 mA) transferred the gels to polyvinylidene difluoride (PVDF)
membranes. The PVDF membranes were blocked overnight at 4 C in 5% milk to reduce
nonspecific binding. After incubation for 2 h at room temperature with a 1:1000 dilution of
primary antibodies, the membranes were washed and exposed to secondary antibodies
(goat anti-mouse, goat anti-rabbit) immunoglobulin G conjugated with horseradish per-
oxidase for 2 h. Proteins were detected using enhanced chemiluminescence ECL Western
blotting detection reagent.
Statistical Analysis
All data were expressed as meanS.D. All statistical analyses were performed using SPSS
15.0 for Windows software. One-way analysis of variance (ANOVA) was used to compare
groups. A level of p < 0:05 was considered to be statistically significant.
Results
Effect of Vitexin on the Elevation of the ST Segment of ECG
The elevation of the ST segment was significantly ðp < 0:05, p < 0:01) enhanced in I/R
group 30 min after ischemia, 30 and 60 min after reperfusion compared with sham group.
Vitexin 6 mg/kg and puerarin have a significant (p < 0:05, p < 0:01) inhibiting effect
against the elevation of the ST segment 30 and 60 min after reperfusion compared with I/R
group (Figs. 1A and 1B).
Effect of Vitexin on Myocardial Infarct Volume
Compared with the sham group, the myocardial infarct volume of MI/R group significantly
(p < 0:01) improved. This proved the injury induced by ischemia/reperfusion in vivo.
Administration of vitexin reduced the infarct volume to a different extent. Treatment with
6 mg/kg of vitexin (29.53 4.69%) was most effective, reducing infarct volume by 40%
compared with MI/R group (Fig. 2).
Effect of Vitexin on the Activities of LDH, CK, SOD, and the Content of MDA in Serum
The activities of LDH, CK, SOD, and MDA content in the study groups are shown in
Table 1. There were significant (p < 0:01) increases in LDH and CK inthe I/R group
(A)
(B)
Figure 1. Effect of vitexin on the elevation of the ST segment of ECG. (A) Typical segments of ECG of each
group on basic, ischemia 30 min, and reperfusion 30 and 60 min. (B) The elevation of the ST segment.
## p < 0:01, # p < 0:05 compared to the sham group; **p < 0:01, *p < 0:05 compared to MI/R group. Each
group consisted of eight rats (n ¼ 8). VT: vitexin; PUE: puerarin; I: Ischemia; R: Reperfusion.
(a) (b) (c) (d) (e) (f)
(A)
(B)
Figure 2. Effect of vitexin on myocardial infarct volume after myocardial ischemia/reperfusion (I/R) injury in rats.
(A) Representative photos of myocardial infarct volume. (a) Sham group, (b) I/R group, (c) puerarin group, (d)
vitexin 1.5 mg/kg, (e) vitexin 3 mg/kg, (f) vitexin 6 mg/kg. (B) Histogram of the percentage of myocardial infarct
volume. ## p < 0:01 compared to the sham group; **p < 0:01 compared to I/R group. Each group consisted of
eight rats (n ¼ 8). I/R: ischemia/reperfusion, VT: vitexin; PUE: puerarin.
compared to the sham group, which indicated injury on cytomembrane of cardiocytes after
reperfusion leading to the leakage of LDH and CK. These parameters significantly
decreased ( p < 0:05, p < 0:01) in those groups treated with vitexin and puerarin in
comparison with I/R group. Compared with sham group, the activity of SOD was sig-
nificantly (p < 0:01) reduced in I/R group, while SOD activities increased significantly
(p < 0:05, p < 0:01) in vitexin and puerarin groups compared with I/R group. In addition,
the content of MDA was increased in I/R group compared with sham group, and decreased
in the vitexin 6, 3 mg/kg and puerarin groups compared with I/R group.
Table 1. Effects of Vitexin on the Activities of LDH, CK, SOD and the Content of MDA in Serum in
MI/R Rats
Group Doses (mg/kg) LDH (U/L) CK (U/ml) SOD (U/ml) MDA (nmol/ml)
Sham — 4508.6 508.3 0.95 0.47 150.5 6.1 8.2 0.6

I/R — 5807.2 135.3 ## 1.76 0.52 ## 122.2 18.7 ## 10.9 1.2 ##
VT 6 4586.3 568.2 ** 1.15 0.55 * 149.4 4.4 ** 8.4 0.5 **
VT 3 4709.5 488.2 ** 1.40 0.38 147.6 8.4 ** 8.6 1.0 **
VT 1.5 5048.2 507.2 ** 1.72 0.52 143.4 8.2 ** 10.6 1.2
PUE 30 4815.1 412.3 ** 1.22 0.58 * 135.6 7.6 * 9.0 0.4 **
Note: ## p < 0:01 compared tothe sham group; *p < 0:05, **p < 0:01 compared to MI/R group. Each group
consisted of eight rats. I/R: ischemia/reperfusion, VT: vitexin; PUE: puerarin.
Effect of Vitexin on the Protein Expression of NF-Bp65 and TNF-

The areas of yellow and brown in the cytoplasm were the areas of positive expression of
NF-Bp65 and TNF- protein. As shown, a very small amount of NF-Bp65 and TNF-
was detected in myocardial tissue of the sham group. This significantly ( p < 0:01)
increased in I/R group. Vitexin and puerarin significantly ( p < 0:05, p < 0:01) reduced the
protein expression of NF-Bp65 and TNF- compared to I/R group (Figs. 3 and 4).
Effect of Vitexin on MAPK Signaling Pathways
Protein expression in each group was quantified with Western Blotting analysis, to define
the mechanisms by which vitexin inhibits I/R induced cardiocyte apoptosis. Puerarin and
vitexin 6 treatment (3 mg/kg) increased phospho-ERK expression significantly compared
to the I/R group (p < 0:01). While the expression of phospho-c-Jun was significantly
increased in I/R group, an administration of puerarin and vitexin inhibited the increase of
phospho-c-Jun (Fig. 5) ( p < 0:01).
Discussion
Coronary artery thrombosis obstruction usually causes myocardial ischemia. However,

reperfusion also seems to activate a series of reactions. Myocardial ischemia /reperfusion
that follows can cause injuries similar to or even worse than ordinary ischemia. This
phenomenon is called myocardial ischemia/reperfusion injury (I/R).
A primary active ingredient drawn from the traditional Chinese medicine, Ge-gen
(Radix Puerariae, RP), which has been widely applied in treatment of myocardial and
cerebral ischemia, is puerarin [4H-1-benzopyran-4-one, 8-b-D-glucopyranosyl-7-hydroxy-
3-(4-hydroxyphenyl), C21 H20 C9 ], one of three major isoflavonoid compounds (Xie et al.,
2003; Xu et al., 2005; Hsiang et al., 2011). Zhang et al. found that puerarin can reduce the
infarct area in the rat heart with myocardium infarction. It can also reduce induced
angiogenesis in the nonischemic and ischemic myocardium, which was one of the
mechanisms of curing coronary artery diseases (Zhang et al., 2006). Our previous study
(a) (b) (c)

(d) (e) (f)

(A)
(B)
Figure 3. Effect of vitexin on the expression of NF-B. (A) Expression of NF-B was analyzed by immuno-
histochemistry. (a) Sham group, (b) I/R group, (c) puerarin group, (d) vitexin 1.5 mg/kg, (e) vitexin 3 mg/kg, (f)
vitexin 6 mg/kg. (B) OD value of NF-B activity was measured. ## p < 0:01 compared to the sham group;
**p < 0:01, *p < 0:05 compared to MI/R group. Each group consisted of eight rats (n ¼ 8). I/R: ischemia/
reperfusion, VT: vitexin; PUE: puerarin.
indicated that puerarin has a similar cardioprotection against myocardial I/R injury in
isolated rat heart. Thus, puerarin was used as a positive control drug.
In our present study, we discovered that vitexin has a significant protective effect
against myocardial I/R injury in the rat heart in vivo. Vitexin 6, 3, 1.5 mg/kg significantly
lowered the elevation of the ST segment of ECG, and reduced myocardial infarct size. The
decreased levels of cardiac marker enzymes further support this. In this study, the increase
of CK and LDH activities in the reperfused hearts implied the deleterious effects to
(a) (b) (c)

(d) (e) (f)

(A)
(B)
Figure 4. Effect of vitexin on the expression of TNF-. (A) Expression of TNF- was analyzed by immuno-
histochemistry. (a) Sham group, (b) I/R group, (c) puerarin group, (d) vitexin 1.5 mg/kg, (e) vitexin 3 mg/kg and
(f) vitexin 6 mg/kg. (B) OD value of TNF- activity was measured. ## p < 0:01 compared to the sham group;
**p < 0:01, *p < 0:05 compared to I/R group. Each group consisted of eight rats (n ¼ 8). I/R: ischemia/reper-
fusion, VT: vitexin; PUE: puerarin.
cardiocytes during ischemia/reperfusion. After the treatment with vitexin, CK and LDH
activities decreased significantly in serum. When there are oxidative stress responses, the
content of MDA, which is a kind of metabolic product of lipid peroxidation, will rise. In
contrast, free radical scavengers SOD has been provided with the efficacy of antioxidants.
The treatment with vitexin also decreased MDA content and enhanced SOD activity. This
reveals the anti-oxidation of vitexin against myocardial ischemia/reperfusion injury.
(A)
(B)
Figure 5. Effect of vitexin on phospho-ERK and phospho-c-Jun expression after myocardial ischemia/reperfusion.
(A) Western blot analysis of p-ERK/ERK and p-c-Jun/c-Jun expression. Beta-actin was a loading control. (a)
Sham group, (b) I/R group, (c) puerarin group, (d) vitexin 1.5 mg/kg, (e) vitexin 3 mg/kg and (f) vitexin 6 mg/kg.
(B) Gray value of Western blot analysis of p-ERK/ERK and p-c-Jun/c-Jun expression. ## p < 0:01 compared to
the sham group; **p < 0:01, *p < 0:05 compared to I/R group. Each group consisted of 8 rats (n ¼ 8). I/R:
ischemia/reperfusion, VT: vitexin; PUE: puerarin.
Plenty of studies have documented a potent role for the nuclear factor -B (NF-Bp65)
family of transcription factors in the regulation of cardiac myocyte survival. They are
documented through the through repression of apoptotic cell death triggered by hypoxia or
ischemic myocardial injury (Mustapha et al., 2000; Maekawa et al., 2002; Misra et al.,
2003). Furthermore, p50 mice display protection from TNF- induced cardiomyopathy
and improved cardiac function following myocardial infarction (Kawamura et al., 2005;
Kawano et al., 2006). Recent studies have indicated that prolonged activation of NF-
Bp65 seems to be detrimental and promotes heart failure by eliciting signals that trigger
chronic inflammation through enhanced expressions of cytokines such as TNF-, inter-
leukin-1, and interleukin-6, leading to endoplasmic reticulum stress responses and cell
death (Gordon et al., 2011). Activated NF-Bp65 translocates to the nucleus from cyto-
plasm and mediates the transcription of a series of proteins, including pro-inflammatory
proteins such as TNF- (Gaur and Aggarwal, 2003; Csiszar et al., 2005). Indeed, activation
of NF-Bp65 down-stream of the TNF- is known to provoke apoptosis. Our study
suggested that vitexin inhibited NF-Bp65 activity, thus inhibiting its downstream
regulation of gene expression.
TNF-, which involved in the occurrence and development of cardiovascular diseases

including atherosclerosis, myocardial ischemia/ reperfusion injury and heart failure, also
has an important role. Studies have shown that TNF- can increase the expression ability
of other inflammatory cytokines (such as IL-6 and IL-1), which enhance the TNF-
-induced myocardial dysfunction (Kumar et al., 1996; Meldrum, 1998). Sustained, high
concentration expression of TNF- also contributes to structural alterations in the failing
heart, such as cardiac hypertrophy, increased cardiomyocyte apoptosis and cardiac fibrosis.
Thus, TNF- exerts some of its deleterious effects through direct modification of mito-
chondrial function, thereby starting the apoptotic pathway (Kleinbongard et al., 2010). In
our study, a significant increase in TNF- concentration was observed in rat after myo-
cardial I/R. The application of vitexin suppressed TNF- protein production obviously,
which further illustrates the cardi-protective effect of vitexin.
Reactive oxygen species mediate the mitochondrial permeability transition pore (MPTP)
opening, leading to mitochondrial membrane rupture, cytochrome c release and apoptosis
(Ferdinandy et al., 2007; Murphy and Steenbergen, 2011). During ischemia, mitochondria
produce ROS and then an extra burst of ROS generation took place at reperfusion. The
oxidative stress response occurred, and the imbalance of oxidation/ antioxidant system was
broken, resulting in myocardial tissue injury. In addition, ROS activates a variety of
inflammatory molecular cascades (Meldrum et al., 1998; Meldrum, 1998). So far, three
major MAPKs signaling pathways in mammalian cells have been identified, including
ERK1/2, p38 protein and c-Jun N-terminal protein kinases (JNKs)/stress-activated protein
kinases (SAPKs). They are activated in response to myocardial I/R (Abe et al., 2000). There
are conflicting effects between the activations of ERK1/2 (beneficial) and p38 MAPKs-
JNKs (deleterious) on post-ischemic myocardial apoptosis and the recovery of cardiac
function (Yue, 2000). Several studies show that the activation of ERK1/2 is conducive to
cell survival and have a protective compensatory effect on antioxidant imbalance, whereas
p38 MAPK and JNK can promote apoptosis (Fischer et al., 2005; Wang et al., 2006; Yang
et al., 2006). This notion is further proved by Jin et al. who reported on ERK1/2 was
involved in the anti-apoptotic effect of antioxidants in I/R-induced myocardial injury in rat,
in vivo (Jin et al., 2008). In the cardiovascular system, ERK1/2 is activated by growth factors,
cytokines and so on, thereby mediating cell survival as well as cytoprotection (Wang et al.,
1998) Sugden and Bogoyevitch 1995. By comparison, JNK is activated by cellular stresses,
including oxidative stress, and they are considered to be associated with cardiomyocyte
apoptosis and cardiomyopathy (Kyriakis and Avruch, 1996). Moreover, numerous surveys
have shown that MAPK plays a key, mediating role in disease states, characterized by
inflammation (Stambe et al., 2003). In the present study, we showed that the level of
phosphorylated ERK1/2 was increased while the level of phosphorylated c-jun was decreased
in the heart by vitexin, which revealed the antioxidation and anti-inflammatory effect of
vitexin during myocardial I/R injury via MAPK signal pathway.
In summary, vitexin has a protective effect against myocardial I/R injury in rat heart
in vivo, which may be associated with the antioxidation and inhibition of the release of
inflammatory cytokines via attenuating expression of NF-Bp65 and TNF-, as well as the
up-regulation of phospho-ERK and the down-regulation of phospho-c-Jun expression.
Acknowledgments
This work was supported by a grant from the National Major Program of New Drug during
the 11th Five year Plan Period, P.R. China (No. 2009ZX09102-311). The authors
acknowledge the help of the staff members of Department of Pharmacology, Anhui
Medical University.
References
Abe, Y., S. Matsumoto, K. Kito and N. Ueda. Cloning and expression of a novel MAPKK-
like protein kinase, lymphokine-activated killer T-cell-originated protein kinase, specifi-

cally expressed in the testis and activated lymphoid cells. J. Biol. Chem. 275: 21525–21531,
2000.
Aquino, R., S. Morelli, M.R. Lauro, S. Abdo, A. Saija and A. Tomaino. Phenolic constituents and
antioxidant activity of an extract of Anthuriumversicolor leaves. J. Nat. Prod. 64: 1019–1023,
2001.
Chan, E., X.X. Liu, D.J. Guo, Y.W. Kwan, G.P. Leung, S.M. Lee and S.W. Chan. Extract of
Scutellaria baicalensis Georgi root exerts protection against myocardial ischemia-reperfusion
injury in rats. Am. J. Chin. Med. 39: 693–704, 2011.
Choi, H.J., J.S. Eun, B.G. Kim, S.Y. Kim, H. Jeon and Y. Soh. Vitexin, an HIF-1 alpha inhibitor, has
anti-metastatic potential in PC12 cells. Mol. Cells 22: 291–299, 2006.
Csiszar, A., K.E. Smith, A. Koller, G. Kaley, J.G. Edwards and Z. Ungvari. Regulation of bone
morphogenetic protein-2 expression in endothelial cells: role of nuclear factor-kappaB acti-
vation by tumor necrosis factor-alpha, H2 O2 , and high intravascular pressure. Circulation 111:
2364–2372, 2005.
Diwan, A. and G.W. Dorn 2nd. Decompensation of cardiac hypertrophy: cellular mechanisms and
novel therapeutic targets. Physiology (Bethesda) 22: 56–64, 2007.
Dong, L.Y., Z.W. Chen, Y. Guo, X.P. Cheng and X. Shao. Mechanisms of vitexin preconditioning
effects on cultured neonatal rat cardiomyocytes with anoxia and reoxygenation. Am. J. Chin.
Med. 36: 385–397, 2008.
Dong, L.Y., Y.F. Fan, X. Shao and Z.W. Chen. Vitexin protects against myocardial ischemia/
reperfusion injury in Langendorff-perfused rat hearts by attenuating inflammatory response
and apoptosis. Food Chem. Toxicol. 49: 3211–3216, 2011.
Ferdinandy, P., R. Schulz and G.F. Baxter. Interaction of cardiovascular risk factors with myocardial
ischemia/reperfusion injury, preconditioning, and postconditioning. Pharmacol. Rev. 59: 418–
458, 2007.
Fischer, S., M. Wiesnet, D. Renz and W. Schaper. H2 O2 induces paracellular permeability of porcine
brain-derived microvascular endothelial cells by activation of the p44/42 MAP kinase path-
way. Eur. J. Cell Biol. 84: 687–697, 2005.
Gaur, U. and B.B. Aggarwal. Regulation of proliferation, survival and apoptosis by members of the
TNF superfamily. Biochem. Pharmacol. 66: 1403–1408, 2003.
Gordon, J.W., J.A. Shaw and L.A. Kirshenbaum. Multiple facets of NF-B in the heart: to be or not
to NF-B. Circ. Res. 108: 1122–1132, 2011.
Hsiang, S.W., H.C. Lee, F.J. Tsai, C.C. Tsai, C.H. Yao and Y.S. Chen. Puerarin accelerates per-
ipheral nerve regeneration. Am. J. Chin. Med. 39: 1207–1217, 2011.
Jin, Y.C., K.J. Kim, Y.M. Kim, Y.M. Ha, H.J. Kim, U.J. Yun, K.H. Bae, Y.S. Kim, S.S. Kang, H.G.
Seo, J.H. Lee and K.C. Chang. Anti-apoptotic effect of magnolol in myocardial ischemia and
reperfusion injury requires extracellular signal-regulated kinase1/2 pathways in rat in vivo.
Exp. Biol. Med. (Maywood) 233: 1280–1288, 2008.
Kawamura, N., T. Kubota, S. Kawano, Y. Monden, A.M. Feldman, H. Tsutsui, A. Takeshita and
K. Sunagawa. Blockade of NF-kappaB improves cardiac function and survival without
affecting inflammation in TNF-alpha-induced cardiomyopathy. Cardiovasc. Res. 66: 520–529,
2005.
Kawano, S., T. Kubota, Y. Monden, T. Tsutsumi, T. Inoue, N. Kawamura, H. Tsutsui and
K. Sunagawa. Blockade of NF-B improves cardiac function and survival after myocardial
infarction. Am. J. Physiol. Heart Circ. Physiol. 291: H1337–1344, 2006.
Kim, J.H., B.C. Lee, J.H. Kim, G.S. Sim, D.H. Lee, K.E. Lee, Y.P. Yun and H.B. Pyo. The isolation
and antioxidative effects of vitexin from Acer palmatum. Arch. Pharm. Res. 28: 195–202,
2005.
Kleinbongard, P., G. Heusch and R. Schulz. TNF-alpha in atherosclerosis, myocardial ischemia/
reperfusion and heart failure. Pharmacol. Ther. 127: 295–314, 2010.
Kubota, T., C.F. McTiernan, C.S. Frye, S.E. Slawson, B.H. Lemster, A.P. Koretsky, A.J. Demetris
and A.M. Feldman. Dilated cardiomyopathy in transgenic mice with cardiac-specific over-
expression of tumor necrosis factor-alpha. Circ. Res. 81: 627–635, 1997.
Kumar, A., V. Thota, L. Dee, J. Olson, E. Uretz and J.E. Parrillo. Tumor necrosis factor alpha and
interleukin 1beta are responsible for in vitro myocardial cell depression induced by human
septic shock serum. J. Exp. Med. 183: 949–958, 1996.

Kyriakis, J.M. and J. Avruch. Sounding the alarm: protein kinase cascades activated by stress and
inflammation. J. Biol. Chem. 271: 24313–24316, 1996.
Lee, Y.S., Y.J. Kang, H.J. Kim, M.K. Park, H.G. Seo, J.H. Lee, H.S. Yun-Choi and K.C. Chang.
Higenamine reduces apoptotic cell death by induction of heme oxygenase-1 in rat myocardial
ischemia-reperfusion injury. Apoptosis 11: 1091–1100, 2006.
Maekawa, N., H. Wada, T. Kanda, T. Niwa, Y. Yamada, K. Saito, H. Fujiwara, K. Sekikawa and
M. Seishima. Improved myocardial ischemia/reperfusion injury in mice lacking tumor necrosis
factor-alpha. J. Am. Coll. Cardiol. 39: 1229–1235, 2002.
Marchant, D.J., J.H. Boyd, D.C. Lin, D.J. Granville, F.S. Garmaroudi and B.M. McManus.
Inflammation in myocardial diseases. Circ. Res. 110: 126–144, 2012.
McNally, D.J., K.V. Wurms, C. Labbe, S. Quideau and R.R. Belanger. Complex C-glycosyl fla-
vonoid phytoalexins from Cucumis sativus. J. Nat. Prod. 66: 1280–1283, 2003.
Meldrum, D.R., C.A. Dinarello, J.C. Cleveland Jr, B.S. Cain, B.D. Shames, X. Meng and A.H.
Harken. Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac injury by a
P38 mitogen-activated protein kinase-dependent mechanism. Surgery 124: 291–296, 1998.
Meldrum, D.R. Tumor necrosis factor in the heart. Am. J. Physiol. 274: R577–595, 1998.
Misra, A., S.B. Haudek, P. Knuefermann, J.G. Vallejo, Z.J. Chen, L.H. Michael, N. Sivasu-
bramanian, E.N. Olson, M.L. Entman and D.L. Mann. Nuclear factor-kappaB protects the
adult cardiac myocyte against ischemia-induced apoptosis in a murine model of acute myo-
cardial infarction. Circulation 108: 3075–3078, 2003.
Murphy, E. and C. Steenbergen. What makes the mitochondria a killer? Can we condition them to be
less destructive? Biochim. Biophys. Acta 1813: 1302–1308, 2011.
Mustapha, S., A. Kirshner, D. De Moissac and L.A. Kirshenbaum. A direct requirement of nuclear
factor-kappaB for suppression of apoptosis inventricular myocytes. Am. J. Physiol. Heart Circ.
Physiol. 279: H939–945, 2000.
Prabhakar, M.C., H. Bano, I. Kumar, M.A. Shamsi and M.S. Khan. Pharmacological investigations
on vitexin. Planta Med. 43: 396–403, 1981.
Shaw, J. and L.A. Kirshenbaum. Molecular regulation of autophagy and apoptosis during ischemic
and non-ischemic cardiomyopathy. Autophagy 4: 427–434, 2008.
Stambe, C., R.C. Atkins, P.A. Hill and D.J. Nikolic-Paterson. Activation and cellular localization
of the p38 and JNK MAPK pathways in rat crescentic glomerulonephritis. Kidney Int. 64:
2121–2132, 2003.
Sugden, P.H. and M.A. Bogoyevitch. Intracellular signalling through protein kinases in the heart.
Cardiovasc. Res. 30(4): 478–492, 1995.
Venardos, K.M., A. Perkins, J. Headrick and D.M. Kaye. Myocardial ischemia–reperfusion injury,
antioxidant enzyme systems, and selenium: a review. Curr. Med. Chem. 14: 1539–1549, 2007.
Vinten-Johansen, J. Involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion
injury. Cardiovasc. Res. 61: 481–497, 2004.
Wang, C.Z., S.R. Mehendale, T. Calway and C.S. Yuan. Botanical flavonoids on coronary heart
disease. Am. J. Chin. Med. 39: 661–771, 2011.
Wang, J., Y.H. Shen, B. Utama, J. Wang, S.A. LeMaire, J.S. Coselli, G.M. Vercellotti and X.L.
Wang. HCMV infection attenuates hydrogen peroxide induced endothelial apoptosis-invol-
vement of ERK pathway. FEBS. Lett. 580: 2779–2787, 2006.
Wang, X., J.L. Martindale, Y. Liu and N.J. Holbrook. The cellular response to oxidative stress:
influences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem.

J. 333: 291–300, 1998.
Xie, R.Q., J. Du and Y.M. Hao. Myocardial protection and mechanism of puerarin injection on
patients of coronary heart disease with ischemia/reperfusion. Zhongguo Zhong Xi Yi Jie He Za
Zhi 23: 895–897, 2003.
Xu, X., S. Zhang, L. Zhang, W. Yan and X. Zheng. The Neuroprotection of puerarin against cerebral
ischemia is associated with the prevention of apoptosis in rats. Planta Med. 71: 585–591,
2005.
Yang, B., T.N. Oo and V. Rizzo. Lipid rafts mediate H2O2 prosurvival effects in cultured endothelial
cells. FASEB J. 20: 1501–1503, 2006.
Yue, G.K. Retrieval of stratospheric aerosol size distributions and integral properties from simulated
lidar backscatter measurements. Appl. Opt. 39: 5446–5455, 2000.
Zhang, S., S. Chen, Y. Shen, D. Yang, X. Liu, A.C. Sun-Chi and H. Xu. Puerarin induces angio-
genesis in myocardium of rat with myocardial infarction. Biol. Pharm. Bull. 29: 945–950,
2006.
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Cardioprotection of Vitexin on Myocardial Ischemia/Reperfusion

Article in The American Journal of Chinese Medicine · November 2013

Liu-Yi Dong Yilan Zhen

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Cardioprotection of Vitexin on Myocardial

*Department of Pharmacology, Anhui Medical University

Key Laboratory of Anti-Inﬂammatory and Immunopharmacology of Ministry of Education

effect against myocardial ischemical/reperfusion injury in rat, which is exhibited by vitexin,

Keywords: Heart; Vitexin; Ischemical/Reperfusion; Inﬂammatory Cytokines; Antioxidation;

Materials and Methods

Drugs and Reagents

Animals and Experimental Protocol

Myocardial Ischemia/Reperfusion Model

Rats were anesthetized with an intraperitonal injection of chloral hydrate at a dose of

(ECG) electrodes were subcutaneously connected in all limbs. Lead electrocardiogram

Measurement of the Elevation of the ST Segment of ECG

Measurement of Infarct Volume and Area at Risk

Detection of Creatine Kinase (CK), Lactate Dehydrogenase (LDH),

Superoxide Dismutase (SOD) and Malondialdehyde (MDA)

Western Blot Analysis

Effect of Vitexin on the Elevation of the ST Segment of ECG

Effect of Vitexin on Myocardial Infarct Volume

(a) (b) (c) (d) (e) (f)

Sham — 4508.6 508.3 0.95 0.47 150.5 6.1 8.2 0.6

consisted of eight rats. I/R: ischemia/reperfusion, VT: vitexin; PUE: puerarin.

Effect of Vitexin on the Protein Expression of NF-Bp65 and TNF-

Effect of Vitexin on MAPK Signaling Pathways

Coronary artery thrombosis obstruction usually causes myocardial ischemia. However,

(a) (b) (c)

(d) (e) (f)

(a) (b) (c)

(d) (e) (f)

TNF-, which involved in the occurrence and development of cardiovascular diseases

like protein kinase, lymphokine-activated killer T-cell-originated protein kinase, speciﬁ-

septic shock serum. J. Exp. Med. 183: 949–958, 1996.

inﬂuences of mitogen-activated protein kinase signalling pathways on cell survival. Biochem.

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