Pharmacology & Pharmacy, 2013, 4, 619-627
Published Online November 2013 (http://www.scirp.org/journal/pp)
http://dx.doi.org/10.4236/pp.2013.48088
Open Access PP
619
Protective Effect of Catalpol on Myocardium in Rats with
Isoprenaline-Induced Myocardial Infarcts via Angiogenesis
through Endothelial Progenitor Cells and Notch1 Signaling
Pathway
Jing Zeng1, Feng Huang1, Yuangqing Tu1, Saichun Wu1, Manping Li1, Xi aoy un Tong2*
1College of Pharmacy, Jinan University, Guangzhou, China; 2Teaching & Research Section of Internal Medicine of Traditional Chi-
nese Medicine, College of Clinical Medicine, Yunnan University of TCM, Kuming, China.
Received September 20th, 2013; revised October 21st, 2013; accepted October 28th, 2013
Copyright © 2013 Jing Zeng et al. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Protective effect of catalpol on myocardium was studied in relation to endothelial progenitor cells, Notch1 signaling
pathway and angiogenesis in rats with isoprenaline (INN)-induced acute myocardial infarcts. To analyze the pathologi-
cal status and impact of catalpol on the rats, 3 weeks after intragastric gavage, the animals were verified for myocardial
infarcts with electrocardiogram and measured for enzyme activity of lactate dehydrogenase (LDH), malondialdehyde
(MDA), creatine kinase (CK) and superoxide dismutase (SOD) in myocardium, and further analyzed using HE and TTC
staining, as well as visual examination of infarct area. Flow cytometry study of endothelial progenitor cells (EPCs) in-
dicated that the EPCs were mobilized during infarction. The roles of Notch1 signaling pathway in angiogenesis of the
infracted animals were studied using immunohistochemistry analysis of RBPjκ and Western blot analysis of Notch1 and
Jagged1. Our results obtained from the rats treated with catalpol, positive drug and control showed that catalpol could
protect rats from infarction probably by mobilization of EPCs and activation of Notch1 signaling pathway.
Keywords: Myocardial Infarction; Endothelial Progenitor Cell; Notch1 Signaling Pathway; Angiogenesis; Catalpol
1. Introduction
Acute myocardial infarction caused by ischemic cardio-
myopathy is one of the major human diseases and has the
highest mortality and morbidity among all diseases. Pre-
vention and control of the disease is number one healthy
issue globally. Many options have become available to
treat the disease, such as thrombolytic therapy and per-
cutaneous coronary intervention. However, not all pa-
tients are suitable for these therapies due to non-com-
pliant and ineffectiveness in restoration of blood supply.
Therefore, search for new options to treat myocardial
infarction has been a hotspot in cardiovascular disease
research. Myocardial infarction decreases or blocks the
blood supply of coronary artery due to the damage in
coronary artery, resulting in severe and long-lasting
ischemia of myocardial muscle, and eventually ischemic
necrosis of the affected muscle. On the other hand, an-
giogenesis can improve the blood circulation in coronary
collateral artery and restore the supply of blood to the
ischemic myocardium, reducing the death of myocar-
dium cells. EPCs exist in bone marrow and peripheral
blood, which could differentiate into endothelial cells [1].
Since the isolation of EPCs from peripheral blood in
1997 by Ashahara, which could differentiate into endo-
thelial cells and are involved in angiogenesis, the roles of
EPCs in angiogenesis or vasculogenesis have attracted
considerable attention. Many studies have demonstrated
that EPCs play roles in the postnatal neovascularization
and restoration of injured blood vessel endothelium [2].
When induced by cytokines released by ischemic tissue,
EPCs can be mobilized from bone marrow to peripheral
blood to circulate, migrate, home to the injured area,
where they proliferate and differentiate into endothelial
cells to involve in blood restoration and neovasculariza-
tion [3]. Notch signaling is an important signal pathway
that extensively exists in vertebrates and invertebrates [4].
*Corresponding author.

Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via
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It has been shown that the differentiation of endothelial
cells is regulated by the pathway. In addition, it also par-
ticipates in the vascularization of adult. Therefore, a bet-
ter understanding of relationship between Notch signal
pathway and EPCs would be scientifically and clinically
important to improve neovascularization and restoration
of ischemic myocardium. Notch1 has been reported to
play a key role in angiogenesis [5]. EPCs were found to
decline dramatically in mice that had been knocked out
for Jagged1, the ligand of Notch1 receptor [6]. There-
fore, Jagged1 is believed to have an important role in
determining the number of EPCs and their mobilization
into blood for restoration of injured blood vessels [7].
Clinical data have shown that prescriptions that nourish
kidney and activate blood are effective in treatment of
acute myocardial infarction [8]. Previous animal studies
have demonstrated that the prescriptions improved the
release of bone marrow stem cells into peripheral blood,
leading to increase in number of CD34+ cells and resto-
ration of the injured muscles [9]. Catalpol, an iridoid
glucoside separated from the roots of Rehmannia gluti-
nosa, is the major active integrant in a Chinese medical
prescription that nourishes kidney and activates blood. It
has been shown to be neuroprotective in transient global
ischemia in gerbils [10]. However, little is known about
the role of catalpol in EPCs and neovascularization in
myocardium. In this study, we investigated the repair
process of blood vessel by catalpol and analyzed if
Notch1 signal pathway is involved in promoting the re-
lease of EPCs into peripheral blood and restoring ne-
ovascularization in damaged myocardium.
2. Materials and Methods
2.1. Animals
42 healthy adult SD rats, male, weighting 200 ± 20 g,
were purchased from Guangdong Experimental Animal
Center (quality assurance permit no. SCXK 2008-0002).
2.2. Reagents and Instruments
Catalpol was obtained from Medicine Inspecting Institute
of Guangdong province. The purity was confirmed by
HPLC to be 98%. Isoprenaline (INN) (lot no. CBC7466)
was purchased from Sigma. MDA detection kit (lot no.
20121010), LDH detection kit (lot no. 20121010), SOD
detection kit (lot no. 20121011) and CK detection kit (lot
no. 20121010) were purchased from Nanjing Jiancheng
Bioengineering Institute. Dimethyl benzene (lot no.
20120120), hematoxylin (lot no. 07H08A05) and neutral
balsam (lot no. 20111215) were purchased from Guang-
zhou Weijia technology company. PerCP-Cy5.5-CD34
and rat-anti Jagged1 antibody (sc-6011) were obtained
from Santa Cruz Biotechnology, INC. FITC conjugated
rabbit anti-VEGFR2/VEGFR2 antibody, rabbit anti-
CD133 antigen/PE and rat-anti Notch1 antibody were
from Cell Signaling Technology. BCA protein quantifi-
cation kit and RIPA lysis buffer (lot no. P0012 and
P0013B) were purchased from Biyuntian Biotechnology
Institute. APS, Acr-Bic and Tween 20 were from Amer-
sco. Tris-base, SDS and glycine were purchased from
Guangzhou Pubo Instrument Company. PVDF was from
Osmonics (USA). Protein marker (lot no. 00061924) was
from MBI Fermentas (Canada). Chemiluminescence so-
lution (ECL) was from PBP1001 (USA) (lot no. 26).
Multifunctional full wavelength micro plate reader (Syn-
ergy 2) was purchased from Biotek (USA), inverted mi-
croscope (TS-100F) was from Nilsson (Japan).
3. Experimental Contents
3.1. Animal Model
All experimental procedures were conducted in compli-
ance with institutional guidelines for the care and use of
laboratory animals in SPF laboratory, experimental ani-
mal research center of Jinan University, Guangzhou,
China.
The rats were randomly divided into the following six
groups with 7 animals each: control, model, positive, low
(10 mg/kg), middle (20 mg/kg) and high dose (40 mg/kg)
catalpol. For animals used in control and model groups,
they were gavaged for three weeks with physiological
saline and were injected subcutaneously with physio-
logical saline or INN (10 mg/kg) on 19th, 20th and 21th
day to induce acute myocardial infarction. Rats in posi-
tive group were gavaged with Simvastatin dissolved in
physiological saline (1.5 mg/kg, 1 mL/100g) for 3 weeks,
and then injected subcutaneously with INN on 19th, 20th
and 21th day (10 mg/kg) to induce acute myocardial in-
farction. For catalpol group rats were injected subcuta-
neously with INN on 19th, 20th and 21th day (10 mg/kg)
after 3 week gavage with catalpol (1 mL/100g).
3.2. Electrocardiogram
Within 4 hours after the last INN injection, the animals
were anesthetized by intraperitoneally injection with
10% chloral hydrate (0.35 mL/100g). 5 min later, electro-
cardiograms of the rats in all groups were simultaneously
made with electrocardiograph VII (paper speed 50 mm/s)
to record the change in EGG-ST segment and the patho-
logical T wave.
3.3. Enzyme Assays
22 days after the gavage, rats were anesthetized and
blood collected from abdominal aorta in two tubes for
each rat, one containing coagulant and the other contain-
ing EDTA as anticoagulant. The coagulant tubes were
left on rack for 2 h and then centrifuged at 4˚C for 20
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Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via
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min at 3500 rpm. The supernatants were collected to de-
termine the contents of LDH, SOD, CK and MDA. The
blood in the anticoagulant tubes was used immediately
for subsequent EPC flow cytometry analysis.
3.4. EPC Flow Cytometry
For each assay, a sample and blank tube was used. In the
blank tubes, only PerCP-Cy5.5-CD34 antibody was
added for gating, but not CD133+ and KDR antibody. In
the sample tubes, 5 µl each of the three antibodies was
added and mixed with 100 µl blood, incubated at the
dark for 20 min, lysed for at least 5 min by adding 1 ml
of erythrocyte lysis buffer till the blood solution was
completely transparent. The lysed blood samples were
pelleted at 1000 rpm for 5 min, washed with PBS,
votexed and centrifuged again. The pellets were resus-
pended in 200 µl PBS and used for flow cytometry assay.
3.5. TTC Staining
The rats were injected 20 ml of 1% TTC via abdominal
aorta following collecting the blood as described in pre-
vious section. After staining for 10 min, the chests were
opened and hearts taken to dissect for photograph. The
sections were fixed in formalin and photographed again
for better contrast.
3.6. HE Staining
Paraffin sections were dewaxed, stained hematoxylin
solution for 5 min, washed 1 min in running tap water.
They were then hydrated for 30 s in 75% hydrochloric
acid-alcohol and washed 2 min in water. After treated
with ammonia for 30 s and washed with water for 1 - 2
min, the slides were dehydrated through an alcohol gra-
dient, sealed with neutral balsam after clarified with di-
methyl benzene and viewed under a microscope for
pathological changes.
3.7. Immunohistochemistry
Paraffin sections were baked at 60˚C, dewaxed and hy-
drated through dimethyl benzene and ethanol serials. The
slides were finally washed with distilled water and incu-
bated in sodium citrate buffer in a microwave oven for 5
min at high power to restore antigen. After cooling down
at room temperature, they were washed two times for 5
min each with TBS and incubated in 3% H2O2 for 30 min.
The slides were then washed two time with TBS for 5
min each, and blocked with 10% goat serum for 30 min,
and reacted with 1:20 diluted first antibody (anti CD34
antibody). After incubation at 37˚C for 60 min or at 4˚C
overnight, the samples were washed two times with
TBST for 5 min each, and stained with DAB for 10 min.
After the staining, the slides were washed with running
tap water and stained with hematoxylin for 60 seconds,
washed 7 to 8 times and under running water for 3 min.
The slides were dehydrated through an ethanol and di-
methyl benzene serial, and sealed with neutral balsam
and observed under a microscope.
3.8. Western Blot Analysis
100 mg of −80˚C frozen stored myocardial tissue was
homogenated in 400 µL RIPA lysis buffer in an ice bath
for 2 min, and incubated in the ice bath for 15 min before
centrifugation at 4˚C for 10 min. The supernatants were
transferred to 1.5 mL Eppendorf tubes for protein quanti-
fication according to the BCA kit manual. 45 µg of pro-
tein was taken from each sample for electrophoresis and
transferred to the PVDF membranes after the electro-
phoresis. The membranes were blocked with TBST (10
mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1%
Tween-20) containing 5% skim milk powder incubated
and reacted with Notch1 antibody (1:1000 dilution) and
Jagged1 antibody (1:1000 dilution) overnight at 4˚C.
After washed with TBST, the membranes were incubated
with secondary antibody (1:2000 dilution) for 1 h,
washed and reacted with ECL for 1 min. The images
were exposed and captured on X-ray films.
4. Results
4.1. Electrocardiogram
As shown in Figure 1, compared with control group, rats
in model group had an abnormal raise in S-T segment.
Meanwhile, Simvatastin and catalpol at doses used could
reduce the elevation.
4.2. Enzyme Assays
Results showed that the activity of LDH (Figure 2) in
model group was 17111.1 U/L, significantly higher than
that of control (6105.5 ± 542.6 U/L, p < 0.01). This con-
firmed that the rats in the model group were infracted.
After given catalpol at the doses used, LDH activities
were reduced to 7343.1 ± 2110.1, 5352.9 ± 3070.3, and
3705.9 ± 624.5 U/L at low, middle and high dose, re-
spectively.
In the model group, the SOD level (Figure 3) was
lower than that in control (65.0 ± 6.6 vs. 7.1 ± 3.3 U/mL),
indicating that the antioxidation ability in the acutely
infracted muscle was reduced. After given catalpol, SOD
activity was increased slightly to 70.1 ± 8.1, 76.7 ± 4.6
and 81.1 ± 11.2 at the three doses levels, respectively.
As shown in Table 1, the CK level of rats (Figure 4)
in model group was significantly higher than that of con-
trol (12.2 ± 2.0 vs. 7.1 ± 3.3 U/mL, p < 0.01), indicating
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(a) (b)
(c) (d)
(e) (f)
Figure 1. Representative electrocardiograms of rats with INN-induced infraction. (a) Control; (b) INN; (c) Simvastatin; (d)
Catalpol (10 mg/kg); (e) Catalpol (20 mg/kg); (f) Catalpol (40 mg/kg).
**
**
**
**
##
0
5000
10000
15000
20000
25000
ControlModel SimvastatinLMH
catalpol
LDH activity in serum(U/L)122
Figure 2. Effect of catalpol on serum LDH activity in rats with myocardium infarction induced by INN (#p < 0.05, ##p < 0.01
vs control; *p < 0.05, **p < 0.01 vs model).
**
**
**
*
##
0
4
8
12
16
20
ControlModel SimvatastinLMH
Cat a lpo l
CK activity in serum(U/mL)11
Figure 3. Effect of catalpol on serum SOD activity of rats with myocardium infarction induced by INN (#p < 0.05, ##p < 0.01
vs control; *p < 0.05, **p < 0.01 vs model).
Data in Table 1 shows that MDA content in model
group was significantly higher than in control (p < 0.01),
indicating that there was an increase in serum MDA in
the infracted rats. After given catalpol, MDA levels (Fig-
re 5) were decreased to 4.6 ± 1.2, 4.5 ± 0.6 and 2.5 ±
that injury of the myocardial tissue had led to increased
CK activity. Catalpol treatments were found to reduce
the activities to 5.1 ± 0.7, 3.6 ± 0.2 and 2.8 ± 0.8 U/mL at
the three doses, respectively, in a dose-dependent man-
ner. u

Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via
Angiogenesis through Endothelial Progenitor Cells and Notch1 Signaling Pathway
623
Table 1. Effect of catalpol on LDH, SOD, CK and MDA activity of rats with INN-induced myocardium infarction.
Group n LDH U/L SOD U/mL CK U/mL MDA nmol/mL
Control 6 6246.4 ± 464.9 78.0 ± 10.1 7.0 ± 2.2 1.7 ± 0.3
Model 6 17602.8 ± 3846.5## 62.1 ± 14.7## 12.3 ± 1.7## 4.2 ± 0.9##
Simvastatin 6 5904.8 ± 3565.5** 84.2 ± 5.8** 9.7 ± 1.5* 2.0 ± 0.5**
CAT 10 mg/kg 6 10534.3 ± 3473.1** 68.6 ± 6.5 6.0 ± 0.8** 4.4 ± 1.0*
CAT 20 mg/kg 6 4152.0 ± 3545.5** 77.2 ± 4.0* 4.1 ± 0.5** 2.9 ± 0.4**
CAT 40 mg/kg 6 3983.8 ± 949.1** 81.1 ± 8.4* 3.0 ± 0.5** 2.9 ± 0.6**
## **
**
0
15
30
45
60
75
90
105
120
135
ControlModel SimvastatinLMH
Catalpol
SOD activity in seru
m
（U/m
L
）
Figure 4. Effec t of catalpol on serum CK activity of ra ts with myocardium infarction induced by INN (#p < 0.05, ##p < 0.01 vs
control; *p < 0.05, **p < 0.01 vs model).
**
**
**
##
0
2
4
6
8
10
ControlModel SinvastatimLMH
Catalpol
MDA content in serum
（
nmol/ml
）
Figure 5. Effect of catalpol on the serum content of MDA in of rats with myocardium infarction induced by INN (#p < 0.05,
##p < 0.01 vs control; *p < 0.05, **p < 0.01 vs model).
1.9 nmol/mL at the three dose levels, respectively.
4.3. EPC Flow Cytometry
In this study, gating was made for CD34. The CD34
positive events were counted for CD133+/VEGFR2+
double positive cells and expressed as percentage. As
shown in Figure 6, the peripheral blood EPC counts for
CD34+/CD133+/VEGFR2+ events were higher in model
than in control. After catalpol treatments, the counts in-
creased slightly at all dose levels to 0.51%, 0.97% and
3.22%, respectively.
4.4. TTC Staining
As shown in Figure 7, model rats had larger white areas
than control after TTC staining. Rats receiving Simvas-
tatin and catalpol had smaller white areas, indicating that
the infract areas were reduced.
4.5. HE Staining
In normal rats, the myocardial cells were regularly ar-
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(a) (b) (c)
(d) (e) (f)
**
**
**
##
0
1
2
3
4
5
6
7
ControlModel SimvastatinLMH
Catalpol
EPCs(%)
Figure 6. Effect of catalpol on the number of EPCs in peripheral blood of rats with myocardial infarction induced by INN. (a)
control; (b) ISO; (c) Simvastatin; (d) Catalpol (10 mg/kg); (e) Catalpol (20 mg/kg); (f) Catalpol (40 mg/kg), (#p < 0.05, ##p <
0.01 vs control; *p < 0.05, **p < 0.01 vs model).
ranged with intact morphology and unchanged nuclei.
The staining was uniform with clearly striated muscle
fibers and without swollen and necrosed cells (Figure
8(a)). In the infracted rats, arrangements of myocardial
cells were disrupted, cardiac muscle fiber swollen with
increased cytoplasm acidophilia. The nuclei were seen
shrunk or broken, and invasioned by inflammatory cells
(Figure 8(b)). Myocardial striated muscle disappeared
and nucleus become dissolved, resulting in necrosed
myocardial muscle. In comparison with catalpol, Sim-
vastatin treated rats had smaller amount of inflammatory
cell infiltration and leaking of red blood cells (Figure
8(c)). At low and middle catalpol doses, there were some
inflammatory cell infiltration with partially dissolved myo-
cardial fibers and increased acidophilia (Figures 8(d)
and (e)). At high catalpol dose, myocardial cells were
mostly intact with more regularly arranged muscle fibers
and less inflammatory exudate (Figure 8(f)).
4.6. Immunohistochemistry
Immunohistochemistry study showed that there were
slight brown staining in rats in model but not in control
groups, indicating that RBPjκ were expressed slightly
higher in the myocardial muscle of infracted rats than in
control (Figures 9(a) and (b)). Simvastatin treatment re-
sulted in deeper brown staining (Figure 9(c)). Increased
staining was seen at middle and high dose of catalpol
(Figures 9(d)-(f) ) but not at low dose, although the stain-
ing was not as obvious as in Simvastatin-treated rats.
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(a) (b) (c) (d) (e) (f)
Figure 7. TTC staining of myocardium tissues from rats
with INN-induced infraction. (a) Control; (b) INN; (c) Sim-
vatastin; (d) Catalpol (10 mg/kg); (e) Catalpol (20 mg/kg); (f)
Catalpol (40 mg/kg).
(a) (b) (c)
(d) (e) (f)
Figure 8. Effect of catalpol on tissue structure of rats with
INN-induced myocardial infraction (H&E, 200×). (a) Con-
trol; (b) INN; (c) Simvastatin; (d) Catalpol (10 mg/kg); (e)
Catalpol (20 mg/kg); (f) Catalpol (40 mg/kg).
(a) (b) (c)
(d) (e) (f)
Figure 9. Effect of catalpol on RBPjκ expression in cardiac
tissue of rats with myocardial infarction induced by INN. (a)
Control; (b) INN; (c) Simvastatin; (d) Catalpol (10 mg/kg);
(e) Catalpol (20 mg/kg); (f) Catalpol (40 mg/kg).
4.7. Western Blot Analysis
To investigate the molecular mechanism of catalpol-in-
duced mobilization of bone marrow-derived EPCs to
peripheral blood in the infracted rats, we measured the
expression of Notch1 and Jagged1 in the Notch signaling
pathway in the myocardial tissues using Western blot
analysis. As shown in Figure 10, Notch1 was found
downregulated in the infracted rats, and it increased
slightly after given catalpol. Jagged1 was also reduced
remarkably in the infracted rats and increased after
catalpol treatments at all dose levels used. The increase
was found to be dose-dependent. These findings indi-
cated that changes in expression of Notch1 and Jagged1
were consistent each other.
5. Discussion
In this study, we injected INN subcutaneously to prepare
the infract rat model. This is a widely used method.
Myocardium infarction is a common and severe disease
clinically. One of the most important methods to examine
the myocardial injury is thorough the detection of bio-
markers in blood [11,12]. In normal condition, LDH, an
important enzyme involved in energy metabolism, exists
widely in cardiac tissues. When the tissues are injured,
the enzyme is released into blood, resulting in high se-
rum LDH level. SOD plays important role in balancing
the oxidation and antioxidation activities, and is a major
oxygen free radical scavenger. The final product of lipid
oxidation is MDA, which is an indirect indictor of cell
injury, and therefore can be used to measure the damage
in the cardiac tissues. Results from our study indicated
that in the infracted rats, serum activities of LDH and CK
increased significantly, while those of SOD reduced sig-
nificantly. After feeding with medium or high dose of the
prescription, the activities and levels of LDH, CK, SOD
and MDA were all returned to normal, indicating that
catalpol was able to scavenge oxygen free radicals, re-
duce the production of oxidized products from lipids and
the damage in cardiac tissues. TTC staining can visualize
the infract areas. Using TTC staining, we found that
catalpol effectively reduced the infract areas and improve
the pathological status of the infracted tissues. We also
GADPH
Notch1
Jagged1
(a) (b) (c) (d) (e) (f)
Figure 10. Effects of catalpol on the expression of Notch1
and Jagged1 in cardiac tissue of rats with myocardial in-
farction induced by INN. (a) Control; (b) INN; (c) Simvas-
tatin; (d) Catalpol (10 mg/kg); (e) Catalpol (20 mg/kg); (f)
Catalpol (40 mg/kg).
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showed that the size of infracting areas observed by TTC
was in line with the data from the enzyme and biomarker
assays. These results all showed that catalpol is an effec-
tive protective agent to myocardial ischemia.
EPCs are the endothelial progenitor cells. They are
involved in embryonic vasculogenesis, angiogenesis after
birth and repair of endothelial injury in blood vessels
[13,14]. Studies have shown that EPCs in bone morrow
trend to home to ischemic tissues. Once arriving in the
ischemic tissues, they differentiate into myocardial cells
and endothelial cells, to repair the impaired tissues. Nor-
mally, EPCs account for 0.1% of the peripheral blood.
When ischemia occurs, bone morrow-derived EPCs are
mobilized to enter peripheral blood at an amount that is
not sufficient for the repair. Therefore, promotion of an-
giogenesis in myocardium tissue through clinical treat-
ments is currently the hotspots of research and practice in
treatment of myocardial ischemia. How to increase the
proliferation and differentiation of EPCs is a new direc-
tion in treatment of coronary heart diseases. Clinically,
ischemic diseases, particularly coronary heart diseases,
are always associated with one or more risk factors for
cardiovascular system. The risks are negatively related to
the number of EPCs, which are considered as prognosis
indicator for coronary heart diseases [15]. There are a
number of methods to determine the amounts of EPCs. In
most studies, CD34+, VEGFR-2+, and CD133+ are three
mostly frequently used indicators. In this study, we in-
vestigated the CD34+, VEGFR-2+, and CD133+ events
in peripheral blood, and found that EPCs were mobilized
to peripheral blood in the infracted rats with or without
drug treatment. These findings confirmed that EPCs are
released from bone marrow to peripheral blood when the
rats are stressed with dramatic shocks such as ischemia.
Notch signal pathway is first discovered in Drosophila,
and made up of receptors (Notch1, Notch 2, Notch 3, and
Notch 4), ligands (Jagged1, Jagged 2, Dll-1, Dll-3 and
Dll-4) and DNA binding protein CSL. It has been shown
that the differentiation of endothelial cells is mainly
regulated via Notch signal pathway, and the endothelial
cells have shown the potential to differentiate into artery
and vein before blood perfusion [16]. Notch/Jagged1 is a
newly discovered important angiogenesis factor. Western
blot analysis indicated that the expression of Notch1 re-
ceptor in the myocardium was reduced remarkably in the
infracted rats and in the normal rats [17]. Feeding of the
rats with the drugs increased the expression, in a dose
dependent way in case of catalpol, where high and me-
dium doses were better than low dose. Therefore, we
speculate that catalpol may activate Notch signal path-
way to promote the differentiation and proliferation of
endothelial cells in the infracted rats, and to improve the
oxygen supply to ischemic and injured myocardial tis-
sues, resulting in protection to myocardium and reduced
infracted area.
6. Acknowledgements
This research was supported by National Natural Science
Foundation of China (No. 81060295).
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