Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via Angiogenesis through Endothelial Progenitor Cells and Notch1 Signaling Pathway

doi:10.4236/pp.2013.48088

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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.

Email: lanmaoma[email protected], *[email protected]

Received September 20th, 2013; revised October 21st, 2013; accepted October 28th, 2013

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

Angiogenesis through Endothelial Progenitor Cells and Notch1 Signaling Pathway

620

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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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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Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via

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(a) (b)

(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).

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).

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

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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**

## **

105

120

135

ControlModel SimvastatinLMH

Catalpol

SOD activity in seru

（U/m

）

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).

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)

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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