Am J Physiol Heart Circ Physiol 298: H1166H1176, 2010.

First published January 29, 2010; doi:10.1152/ajpheart.01186.2009.

Cardiotonic pills, a compound Chinese medicine, protects

ischemia-reperfusion-induced microcirculatory disturbance and myocardial
damage in rats
Na Zhao,1 Yu-Ying Liu,1 Fang Wang,1 Bai-He Hu,1 Kai Sun,1 Xin Chang,1 Chun-Shui Pan,1 Jing-Yu Fan,1
Xiao-Hong Wei,1 Xiang Li,1 Chuan-She Wang,1 Zhi-Xin Guo,1 and Jing-Yan Han1,2
1

Tasly Microcirculation Research Center, Health Science Center, and 2Department of Integration of Chinese and Western
Medicine, School of Basic Medical Sciences, Peking University, Beijing, China
Submitted 11 December 2009; accepted in final form 29 January 2010

Zhao N, Liu YY, Wang F, Hu BH, Sun K, Chang X, Pan CS,

Fan JY, Wei XH, Li X, Wang CS, Guo ZX, Han JY. Cardiotonic
pills, a compound Chinese medicine, protects ischemia-reperfusioninduced microcirculatory disturbance and myocardial damage in rats.
Am J Physiol Heart Circ Physiol 298: H1166 H1176, 2010. First
published January 29, 2010; doi:10.1152/ajpheart.01186.2009.Cardiotonic pills (CP) is a compound Chinese medicine widely used in
China, as well as other countries, for the treatment of cardiovascular
disease. However, limited data are available regarding the mechanism of
action of CP on myocardial function during ischemia-reperfusion (I/R)
injury. In this study, we examined the effect of CP on I/R-induced
coronary microcirculatory disturbance and myocardial damage. Male
Sprague-Dawley rats were subjected to left coronary anterior descending
branch occlusion for 30 min followed by reperfusion with or without
pretreatment with CP (0.1, 0.4, or 0.8 g/kg). Coronary blood flow,
vascular diameter, velocity of red blood cells, and albumin leakage were
evaluated in vivo after reperfusion. Neutrophil expression of CD18,
malondialdehyde, inhibitor-B, myocardial infarction, endothelial expression of intercellular adhesion molecule 1, apoptosis-related proteins,
and histological and ultrastructural evidence of myocardial damage were
assessed after reperfusion. Pretreatment with CP (0.8 g/kg) significantly
attenuated the I/R-induced myocardial microcirculatory disturbance, including decreased coronary blood flow and red blood cell velocity in
arterioles, increased expression of CD18 on neutrophils and intercellular
adhesion molecule 1 on endothelial cells, and albumin leakage from
venules. In addition, the drug significantly ameliorated the I/R-induced
myocardial damage and apoptosis indicated by increased malondialdehyde, infarct size, myocardial ultrastructural changes, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive myocardial cells, inhibitor-B degradation, and expression of Bcl-2, Bax, and
caspase-3 in myocardial tissues. The results provide evidence for the
potential role of CP in preventing microcirculatory disturbance and
myocardial damage following I/R injury.
myocardial microcirculatory disturbances; Salvia miltiorrhiza; Panax
notoginseng; apoptosis-related proteins

the leading cause of death worldwide, and 3.8 million men and 3.4 million women die of the
disease each year (34). Prevention and management of myocardial ischemia-reperfusion (I/R) injury is a key step in
coronary heart disease surgery and recovery (22). Although
choices of intervention are available in the treatment of this
disorder, significant challenge remains in reducing the morbidity and mortality due to complication of I/R injury.

CORONARY HEART DISEASE IS

Address for reprint requests and other correspondence: J.-Y. Han, Dept. of
Integration of Chinese and Western Medicine, School of Basic Medical Sciences,
Peking Univ., 38 Xueyuan Road, Beijing 100191, Peoples Republic of China
(e-mail: hanjingyan@bjmu.edu.cn).
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I/R exerts multiple insults in the microcirculation that are

often manifested as endothelial cell dysfunction, enhanced
adhesion of leukocyte, macromolecular efflux, production of
reactive oxygen species, and mast cell degranulation, which are
followed by decreased coronary blood flow (24). The release of
ROS and myocardium damage are exaggerated by leukocyte
activation and emigration from microvessels. Moreover, the
production of ROS has been demonstrated to be a crucial event
in the degradation of inhibitor-B (IB), and the activation
and translocation to nucleus of nuclear factor-B (NF-B),
resulting in the expression of various adhesion molecules and
apoptosis factor (14, 35). It has been reported that I/R-elicited
myocardial apoptosis is mediated, at least in part, by caspase-3,
Bcl-2, and Bax (6). Although ischemic preconditioning is
known to protect the myocardial tissue from I/R injury (3), the
clinical application of such an approach is limited for obvious
ethical and practical reasons. On the other hand, pharmacological agents that produce the pathophysiological responses mimicking preconditioning (prophylactic) have been considered as
alternative means for reducing ischemic insults. Several cardioprotective drugs, including ROS scavengers, calcium channel blockers, adenosine, and nicorandil, have recently been
evaluated in clinical trials; their protective effects are insufficient, however (21). One of the reasons for these unsatisfactory
results may reside in the multifactorial mechanisms of reperfusion injury and the lack of therapy that can simultaneously
target multiple pathways (21).
Cardiotonic pills (CP) is a pharmaceutical preparation consisting of ingredients extracted from Salvia miltiorrhiza (SM),
Panax notoginseng (PN), and Borneol. It has been widely used
in China, Korea, Russia, Cuba, and Vietnam for the prevention
and management of ischemic heart diseases. Increasing studies
have been published evaluating the pharmacology of SM and
PN. Recent research demonstrates that SM increases myocardial capillary density and reduces infarct size and peroxide
production after myocardial infarction in rats (23, 26). Our
laboratory has previously demonstrated that pretreatment with
3,4-dihydroxy-phenyl lactic acid, a major ingredient of SM
aqueous extract, improves microcirculatory function by reducing peroxide production, leukocyte adhesion, and albumin
leakage in rat mesentery following I/R (9). Another component
of CP, PN, is known to protect the brain and liver from
ischemic injury, which is ascribed to its inhibitory effect on
inflammation by attenuating tumor necrosis factor (TNF-)
production and Bcl-2, caspase-1, and caspase-3 expression
(25). Pretreatment with ginsenoside Rb1, a major ingredient of
PN, is able to attenuate myocardial I/R injury, an effect partly

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mediated by activating the phosphatidylinositol 3-kinase/AKTdependent pathway. Furthermore, evidence is accumulating

that supports the beneficial effect of CP in treating microcirculatory disturbance and cell damage imposed by I/R or other
insults. Our previous study demonstrated that CP could inhibit
gut I/R-induced adhesion of leukocytes to hepatic sinusoid, the
activation of platelets and leukocytes, and the formation of
thrombus (11, 31). Other studies show that CP can inhibit
photochemical reaction-induced venous thrombosis in the rat
mesentery by reducing the expression of adhesion molecules,
such as CD31, in platelets and vascular endothelial cells. CP is
also reported to ameliorate TNF--induced apoptosis in the
myocardium (31). Besides, there are numerous clinical trials on
CP for the treatment of angina pectoris (37). However, no
study has been published regarding the use of CP as a prophylactic drug to prevent cardiac microcirculatory disturbance and
myocardial damage induced by I/R.
The purpose of this study was to investigate whether pretreatment with CP improves coronary microcirculation and
reduces myocardial damage on I/R challenge. The potential
mechanisms underlying its protective effects were examined.
MATERIALS AND METHODS

Animals. Male Sprague-Dawley rats, weighing 240 260 g, were

purchased from the Animal Center of Peking University Health
Science Center (Beijing, certificate no. SCXK 2002 0001). The
animals were fasted for 12 h before the experiment, while allowing
free access to water. All animals were handled according to the
guidelines of the Peking University Health Science Center Animal
Research Committee, and the surgical procedures and experimental
protocol were approved by Peking University Biomedical Ethics
Committee Experimental Animal Ethics Branch.
Drugs. CP (batch number: 200605027) was obtained from Tasly
Pharmaceutical (Tianjing, China), which was prepared from waterethanol extract of SM, PN, and borneol under the guidelines of Good
Manufacturing Practice and Good Laboratory Practice verified by the
Chinese, Australian, and US Government agencies. The concentration
of the major compounds for quality control of the CP was analyzed by
HPLC (18). One pill of CP contains 9 mg of SM, 1.76 mg of PN, 0.5
mg of borneol, and 13.74 mg of polyethylene glycol.
Animal model and drug administration. Rats were anesthetized
with urethane (1.25 g/kg) by intramuscular injection. After tracheotomy, the animals were ventilated with a positive-pressure respirator
(ALC-V8, Shanghai, China). The chest was opened between the
second and fourth ribs by left thoracotomy. The heart was quickly
exposed, and a 3 0 silk ligature was placed around the left coronary
artery (12 mm region under the boundary of pulmonary artery

pyramid and left auricle of heart). A small polyethylene tube was

placed between the ligature and myocardial tissues. For the I/R group,
the coronary artery was occluded by tightening the ligature for 30 min,
and reperfusion was achieved by releasing the tension applied to the
ligature (24a). In the sham group, the animals underwent the same
surgical procedures, except that the silk passing around the left
coronary artery was not tied. In the CP I/R group, CP dissolved in
saline was given through intragastric administration 90 min before
ischemia at the volume of 4 ml/kg, with the final concentration of 0.1,
0.4, and 0.8 g/kg, which were 1.2-, 4.9-, and 9.9-fold higher than that
administered to humans in the clinic, respectively, for the three CP
pretreatment groups. A total of 195 animals were included and
randomly distributed into the sham, I/R, CP (0.1 g/kg) I/R, CP (0.4
g/kg) I/R, and CP (0.8 g/kg) I/R groups, with 39 animals in each
group. See Table 1 for further details.
Diameter of coronary arterioles and venules and RBC velocity in
the vessels. The hemodynamics of coronary microvessels (25 40 m
in diameter) was observed using an upright microscope (BX51WI,
Olympus) connected with a high-speed video camera (APX, Photon
Fastcam-ultimate) under epi-illumination. The heart was kept warm
and moist by continuous superfusion with saline solution at 37C, but
without infusion with any solution. Images were transmitted onto a
monitor (20PF5120, Philips) and DVD videocassette recorder (DVR560H, Philips). The red blood cell (RBC) velocity in the venule and
arteriole was recorded for 4 s at a rate of 500 frames/s, and the stored
images were replayed at a rate of 25 frames/s. The RBC velocity and
vessel diameter were determined using Image-Pro Plus 5.0 software
(Media Cybernetic) before ischemia (baseline), 30 min after ischemia,
and 30 and 60 min after reperfusion, respectively. Results were
expressed as percentages of the baseline (10).
FITC-albumin leakage from coronary venules. FITC-albumin
(Sigma, 50 mg/kg) was administrated via the femoral vein at 60 min after
reperfusion. Fluorescent images were acquired under an excitation light
(455-nm wavelength) that was irradiated from a mercury burner (100 W)
to an upright fluorescence microscope (DM-LFS, Leica, Germany) at 23
min after injected FITC-albumin. The fluorescent intensity in the venule
and extravenular interstice was measured with Image-Pro Plus 5.0 software (Media Cybernetic). The ratio of fluorescent intensity outside and
inside the venule was calculated (16).
Myocardial blood flow. After left thoracotomy, myocardial blood
flow (MBF) was measured by using Laser-Doppler Perfusion Imager
(PeriScan PIM3, Perimed, Sweden) equipped with a computer before
and after ischemia and 5, 10, 30, 60 min after reperfusion. All images
were evaluated with the software LDPIwin 3.1 (Perimed, Sweden).
Results were expressed as percentages of the baseline (36).
Expression of CD18 on neutrophils. Sixty minutes after reperfusion, 10 ml blood were collected via the abdominal aorta and anticoagulated with 3.8% sodium citrate. To determine the expression of
CD18 on neutrophils, 50 l blood were taken and incubated with 1 g

Table 1. The number of animals for different experimental groups and various parameters

Groups

RBC Velocity and

Albumin Leakage

Myocardial Blood Flow,

CD18 on Neutrophils and
Myocardial Infarct Size

MDA
Level

TUNEL
Assay

Histological and
Immunohistochemistry
Evaluation

Ultrastructure
Examination

IB

Total

Sham
I/R
CP 0.1 I/R
CP 0.4 I/R
CP 0.8 I/R
Total

6
6
6
6
6
30

6
6
6
6
6
30

6
6
6
6
6
30

6
6
6
6
6
30

6
6
6
6
6
30

3
3
3
3
3
15

6
6
6
6
6
30

39
39
39
39
39
195

The same animals were used for determination of red blood cell (RBC) velocity and venular diameter and albumin leakage, and the same animals were used
for myocardial blood flow, CD18 on neutrophils and myocardial infarct size. MDA, malondialdehyde; TUNEL, terminal deoxynucleotidyl transferase-mediated
dUTP nick-end labeling; Sham, sham group; I/R, ischemia-reperfusion group; CP 0.1 I/R, pretreatment with cardiotonic pills (CP) at 0.1 g/kg plus I/R group;
CP 0.4 I/R, pretreatment with CP at 0.4 g/kg plus I/R group; CP 0.8 I/R, pretreatment with CP at 0.8 g/kg plus I/R group. For ultrastructure examination,
only 3 animals were involved in each group, while 6 animals were evaluated in each group for each of the remaining parameters.
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Fig. 1. The effect of cardiotonic pills (CP)

pretreatment on red blood cells (RBC) velocity in coronary arteriole (CA) and venule (CV)
in rat after ischemia-reperfusion (I/R). A: the
cardiac coronary microcirculation. Bar 100
m. B: the time course of change in RBCs
velocity in CA. C: the time course of change
in RBCs velocity in CV. Sham, sham group;
I/R, I/R group; CP 0.1 I/R, pretreatment
with CP at 0.1 g/kg plus I/R group; CP 0.4
I/R, pretreatment with CP at 0.4 g/kg plus I/R
group; CP 0.8 I/R, pretreatment with CP at
0.8 g/kg plus I/R group. Values are means
SE from 6 animals. *P 0.05 vs. sham group.
#P 0.05 vs. I/R group.

FITC-labeled antibody against CD18 (BD) for 20 min at room

temperature in the dark. RBCs were lysed with hemolysin (BD), and
the samples were washed twice with PBS. The mean fluorescence
intensity of CD18 was accessed with a flow cytometer (FACS Calibur,
BD). Neutrophils were then sorted by characteristic forward/sidescatter expression, as reported. Five thousand neutrophils were evaluated for each sample (27).
Malondialdehyde level in myocardial tissue. The level of malondialdehyde (MDA) in the myocardium was measured as an indicator
of lipid peroxidation. Animals were killed at 60 min of reperfusion.
The myocardial tissue was dissected from the surrounding infarction
areas of the left ventricle and homogenized in ice-cold buffer (0.1 mM
C12H22O11, 1.5 M MgCl2, 50 mM Tris) with a weight-to-volume ratio
of 1:9. The homogenate was centrifuged at 8,000 rpm for 15 min at
4C. The MDA level was determined according to the manufacturers
instruction (Nanjing Jiancheng Institute of Biotechnology). Protein

concentration was measured by the Coomassie blue protein-binding

assay using bovine serum albumin as a standard (32).
Myocardial infarct size. Sixty minutes after reperfusion, the heart
was rapidly excised and sliced parallel to the atrioventricular groove
into five sections (1 mm thick) from the apex of the ligation site. The
myocardial slices were incubated for 15 min at 37C in a 0.375%
solution of 2,3,5-triphenyltetrazolium chloride in phosphate-buffered
saline, and then photographed as digital images (Digital Sight DS5M-U1, Nikon). Total infarct area was analyzed by Image-Pro Plus
5.0 software (Media Cybernetic). Myocardial infarct size was expressed as a percentage of the left ventricle (33).
Myocardial damage. Ninety minutes after reperfusion, the heart
was cut from approximately the middle one-third, between the apex
and the ligation point, and fixed through perfusion with 4% paraformadehyde. The fixed tissues were embedded in OCT and cut into 6-m
sections. Damaged cardiac myocytes were detected by using a cell

Fig. 2. The effect of pretreatment with CP on

FITC-albumin leakage from rat CV wall 60
min after reperfusion. A: the fluorescence images of albumin leakage from the rat CV wall.
Bar 50 m. A1: sham group; A2: I/R group;
A3: CP 0.1 g/kg I/R group; A4: CP 0.4 g/kg
I/R group; A5: CP 0.8 g/kg I/R group. B: the
quantification of albumin leakage from rat CV
wall in various groups. Values are means SE
from 6 animals. *P 0.05 vs. sham group.
#P 0.05 vs. I/R group.

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Fig. 3. The effect of CP pretreatment on coronary blood flow (CBF) of rats subjected to I/R.
A: the CBF images acquired by Laser-Doppler
Perfusion Imager in sham group (a), I/R group
(b), CP 0.1 g/kg I/R group (c), CP 0.4 g/kg
I/R group (d), and CP 0.8 g/kg I/R group (e)
at baseline (1) and 0 (2), 30 (3), and 60 min (4)
after reperfusion. Flux is represented on a color
scale from dark blue (low flux) to red (high
flux). B: time course of changes in CBF in
various groups. Values are means SE from 6
animals. *P 0.05 vs. sham group. #P 0.05
vs. I/R group.

death detection kit (Roche). The total number of nuclei (blue) and the
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei (green) in each field were observed
using a laser confocal microscope system (Radiance2100, Bio-Rad;
Axiovert 200, Zeiss). Five fields were selected from the surrounding
infarction areas of the left ventricle, and the number of TUNELpositive nuclei was automatically calculated with Image-Pro Plus 5.0
software (Media Cybernetic) (28).
Histological and immunohistochemistry evaluation of myocardial
tissues. Hearts were removed from approximately the middle onethird between the apex and the ligation point at 90 min after reperfusion, fixed in 4% formalin, and further prepared for paraffin sectioning. The paraffin sections (5 m) were either stained with hematoxylin and eosin, or incubated with an antibody against ICAM-1
(BD), caspase-3 (Santa Cruz), Bcl-2 (Santa Cruz), or Bax (NOVUS)
after being blocked with bovine serum albumin. The samples were
then incubated with a biotinylated secondary antibody followed by
avidin-biotin-peroxidase complex. Positive staining was visualized
with diaminobenzidine. The images were captured by a digital camera
connected to a microscope (BX512DP70, Olympus). Five fields were
selected from the surrounding infarction areas of the left ventricle, and
the mean optical density was measured using Image-Pro Plus 5.0
software (Media Cybemetrics) (2).
Myocardial ultrastructure. Fresh myocardial tissues (2 mm3) were
excised from the surrounding infarction areas of the left ventricle at 90
min after reperfusion. Tissues were fixed with 3% gluteraldehyde and
postfixed with 1% osmium tetroxide. The specimens were processed
for ultrathin sections. The sections were stained with uranium acetate
and lead citrate, and the ultrastructural changes of myocardial tissue
were then evaluated by a transmission electron microscope (JEM1230, Tokyo, Japan) (36).
Western blot assay for myocardial tissue IB. At 60 min after
reperfusion, 100-mg myocardial tissue sample was taken from the
surrounding infarction areas of the left ventricle and stored at 80C
before use. Whole protein was extracted by protein extraction kit
(Applygen Technologies) and mixed with 2 electrophoresis sample
buffer. After electrophoresis on 10% polyacrylamide gels, the separated proteins were transferred from the gels to polyvinylidene difluoride membrane. The membrane with target protein was scissored and
incubated with the antibody against IB (Calbiochem) after being
blocked with 5% nonfat dry milk. The membrane was then washed
and incubated with a horseradish peroxidase-conjugated second antibody. The membranes were analyzed using the enhanced chemiluminescence system, according to the manufacturers protocol and exposure in the dark box. The protein signal was quantified by scanning
densitometry in the X-film by bio-image analysis system (Image-Pro
plus 5.0, Media Cybermetrics). The results from each experimental
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group were expressed as relative integrated intensity compared with

that from the sham group (12).
Statistics. All parameters are expressed as means SE. Statistical
analysis was performed using one-way ANOVA, followed by Tukey
test for multiple comparisons. A probability of 0.05 was considered
to be statistically significant.
RESULTS

Diameter of, and RBC velocity in, coronary arterioles and

venules. In the beating heart, coronary microvessels and RBC
moving inside the vessels were clearly observed under the microscope equipped with a high-speed video camera (Fig. 1A). At
baseline, the diameters of the coronary arterioles of the sham
group, I/R group, CP 0.1 I/R group, CP 0.4 I/R group, and
CP 0.8 I/R group were 15.25 1.26, 15.56 0.41, 15.63
0.92, 16.17 1.01, and 15.75 1.52 m, and the coronary
venules were 44.67 1.13, 46.2 1.98, 46.03 2.78, 46.25
2.28, and 45.42 0.93 m, respectively. There was no significant
difference in diameters at baseline of either coronary arterioles or
venules among the five groups. No significant alteration in the
diameter of either coronary arterioles or venules was observed in
all groups over the period of examination (data not shown).
The time course of RBC velocity change in coronary arterioles is illustrated in Fig. 1B. In the sham group, there was no
significant change in RBC velocity in arterioles throughout the
observation period. In the I/R group, RBC velocity in arterioles
significantly decreased in the beginning of reperfusion and
gradually recovered thereafter. Pretreatment with CP at the
dose of 0.8 g/kg significantly attenuated the I/R-induced RBC

Fig. 4. Influence of pretreatment with CP on the expression of the adhesion

molecule CD18 on peripheral neutrophils of rats subjected to I/R. Values are
means SE from 6 animals. *P 0.05 vs. sham group. #P 0.05 vs. I/R group.

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Fig. 5. The influence of pretreatment with CP on the expression of ICAM-1 on vascular endothelial cells in myocardium of rats after I/R. A: representative
photomicrographs of immunohistochemistry staining for ICAM-1. A1: the location of the sample for ICAM-1 immunostaining; A2: sham group; A3: I/R group; A4: CP
0.1 g/kg I/R group; A5: CP 0.4 g/kg I/R group; A6: CP 0.8 g/kg I/R group. Bar 100 m. Arrow indicates ICAM-1-positive vascular endothelium. B:
quantitative analysis of ICAM-1-positive staining in various groups. Values are means SE from 6 animals. *P 0.05 vs. sham group. #P 0.05 vs. I/R group.

velocity decrease from 30 min after reperfusion, whereas no

effect was observed for lower doses of CP on this alteration.
Figure 1C shows the time course of change in RBC velocity
in coronary venules. Similar to that in coronary arterioles, the
RBC velocity in coronary venules in the sham group remained
nearly constant throughout the observation period. By contrast,
the RBC velocity in venules was significantly decreased in the I/R
group immediately after reperfusion and remained at a low level
until 60 min of reperfusion. Pretreatment with CP at 0.8 g/kg, but
not at 0.4 and 0.1 g/kg, significantly attenuated the I/R-elicited
RBC velocity decrease from 30 min after reperfusion.
Albumin leakage from coronary venules. Pretreatment with
CP prevented FITC-labeled albumin leakage from coronary
venules evoked by I/R, with representative images illustrated in
Fig. 2A. No albumin leakage was detected in the sham group (A1),

Fig. 6. Influence of pretreatment with CP on malondialdehyde (MDA) level in

the myocardial tissue of rats subjected to I/R. Values are means SE from 6
animals. *P 0.05 vs. sham group. #P 0.05 vs. I/R group.
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whereas apparent leakage was observed in the I/R group (A2),

which was abated by pretreatment with CP (0.8 g/kg) (A5).
The leak response in different groups was quantified, as
shown in Fig. 2B. Compared with the sham group, the I/R

Fig. 7. The effect of pretreatment with CP on myocardial infarct size of rats after
I/R. A: representative slice of left ventricle (LV) stained by 2,3,5-triphenyltetrazolium chloride in sham group (a), I/R group (b), CP 0.1 g/kg I/R group (c), CP
0.4 g/kg I/R group (d), and CP 0.8 g/kg I/R group (e). The infarct
myocardium is revealed as pale white. B: the quantitative analysis of infarct size
in various groups presented as percentage of LV. Values are means SE from 6
animals. *P 0.05 vs. sham group. #P 0.05 vs. I/R group.

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group displayed significant increase in extravasation of albumin from coronary venules at 60 min after reperfusion. Pretreatment with CP attenuated the I/R-induced albumin leakage
from coronary venules in a dose-dependant manner, although a
significant effect was observed only for the 0.8 g/kg CP group.
Changes in MBF. Figure 3A shows the color images acquired by the Laser-Doppler Perfusion Imager in five groups at
different time point, where the different magnitude of MBF is
indicated by distinct color, with the red color representing the
highest MBF. No obvious difference in MBF at baseline was
observed among the five groups (a1, b1, c1, d1, and e1). As
expected, however, manipulation of ischemia gave rise to an
apparent decrease in MBF (b2, c2, d2, and e2) compared with
the sham group (a2). This decrease in MBF was recovered to
a great extent during reperfusion in the 0.8 g/kg CP pretreatment group (e3 and e4), whereas no significant recovery was
observed by pretreatment with CP at 0.1 g/kg (c3 and c4) or 0.4
g/kg (d3 and d4).
The time courses of MBF changes in the five groups are plotted
in Fig. 3B. As noticed, in the I/R group, the MBF decreased to
65% of baseline after ischemia, which was reversed to 82% of
baseline from 5 to 10 min after reperfusion and then gradually
reduced to 77% of baseline at the end of 60 min of observation.
MBFs in CP pretreatment groups followed a time course similar
to that in the I/R group, but with significant difference in that

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MBF was reversed from 106 to 99% of baseline from 5 to 30 min

after reperfusion in 0.8 g/kg CP pretreatment and to 91% of
baseline 5 min after reperfusion in 0.4 g/kg CP pretreatment
group, presenting a beneficial action on the MBF disorders
evoked by I/R.
Expression of CD18 on neutrophils. The expression of the
adhesion molecule CD18 on neutrophils was determined by
flow cytometer 60 min after reperfusion; the result is presented
as fluorescence intensity in Fig. 4. The fluorescence intensity of
CD18 was enhanced by I/R, and the increased CD18 expression was significantly attenuated by pretreatment with 0.8 g/kg
of CP, but not a lower dose of CP (0.4 or 0.1 g/kg).
ICAM-1 expression on the endothelium of coronary blood
vessels. Figure 5A shows the representative images of immunohistochemistry staining of ICAM-1 in myocardial tissues in
various conditions. Positive staining of ICAM-1 was obvious
on the endothelium surface of myocardial blood vessels in the
I/R group (A3), as well as in 0.1 and 0.4 g/kg of CP pretreatment groups (A4 and A5). In contrast, 0.8 g/kg of CP pretreatment apparently attenuated the I/R-induced expression of
ICAM-1 (A6).
A quantitative evaluation of ICAM-1 expression on the
endothelium of myocardial blood vessels in different groups is
presented in Fig. 5B. After I/R, ICAM-1 expression in myocardial tissues was significantly higher than that in the sham

Fig. 8. The effects of pretreatment with CP on myocardial injury. A: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-stained
myocardium of rats from sham (A1), I/R (A2), pretreatment with CP at 0.1 g/kg I/R (A3), pretreatment with CP at 0.4 g/kg I/R (A4), and pretreatment with
CP at 0.8 g/kg I/R (A5) group. Arrows indicate TUNEL-positive cells. Arrowheads indicate normal nuclei. Bar 50 m. B: representative photomicrographs
of immunohistochemistry staining for caspase-3 of rat myocardium in sham (B2), I/R (B3), pretreatment with CP at 0.1 g/kg I/R (B4), pretreatment with CP
at 0.4 g/kg I/R (B5), and pretreatment with at CP 0.8 g/kg I/R (B6) group. Arrows indicate caspase-3-positive staining. Bar 100 m. B1: the location
of the samples for caspase-3 immunohistochemistry staining. C: the quantitative analysis of TUNEL-positive cells. D: quantitative analysis of caspase-3 activity.
Values are means SE from 6 animals. *P 0.05 vs. sham group. #P 0.05 vs. I/R group.
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group, and the increased expression was significantly reduced

by pretreatment with CP at the dose of 0.8 g/kg. Lower dose
(0.1 or 0.4 g/kg) of CP did not show any effect on ICAM-1
expression on the endothelium.
MDA level in the myocardium. To address the tissue oxidative damage, the MDA level in surrounding infarction areas of
the left ventricle myocardial tissues was accessed 60 min after
reperfusion. As shown in Fig. 6, the level of MDA observed at
60 min of reperfusion was significantly increased in the I/R
group compared with the sham group. Pretreatment with all
three doses of CP reduced the I/R-enhanced MDA production
with statistic significance.
Myocardial infarct size. Slices of the hearts from the various
groups were stained by 2,3,5-triphenyltetrazolium chloride for
evaluation of myocardial infarct size, and the representative
images are illustrated in Fig. 7A. As expected, myocardial
tissue slice from the sham group exhibited no evidence of
infarction (Fig. 7A, a). In contrast, noticeable infarct areas
were observed in myocardial tissue slice in the I/R group
(Fig. 7A, b), which was reduced in the animals receiving CP
pretreatment, particularly at 0.8 g/kg (Fig. 7A, e). A quantitative analysis of the infarct area further confirms the result
(Fig. 7B).

Tissue damage and apoptosis-related protein expression in

myocardial tissues. To determine the effect of pretreatment
with CP on myocardial apoptosis after I/R, experiments were
conducted on myocardium 90 min after reperfusion by virtue
of TUNEL staining and immunohistochemistry of caspase-3,
Bcl-2, and Bax. Figure 8A shows the images of TUNEL
staining in surrounding infarction areas of the left ventricle
myocardium from the various groups. In the sham group, few
TUNEL-positive cells were seen in the myocardium (A1). In
contrast, a large number of TUNEL-positive cells were observed in the I/R group (A2). Noticeably, the number of
TUNEL-positive cells in the myocardium of CP I/R groups
was decreased at all of the three doses tested (CP 0.1, 0.4, and
0.8 g/kg; A3, A4, and A5, respectively). Figure 8C is the
statistical results of TUNEL-positive cells in surrounding infarction areas of the left ventricle myocardium from various
groups, which is consistent with the qualitative survey.
The immunohistochemistry and corresponding quantification for caspase-3 in surrounding infarction areas of the left
ventricle myocardial tissues from the five groups are shown in
Fig. 8, B and D, respectively. A large number of caspase-3positive cells were observed in myocardial tissues in the I/R
group (B3), which is in distinct contrast to that in the sham

Fig. 9. The effect of pretreatment with CP on the expression of Bcl-2 and Bax in myocardium of rats after I/R. A: representative photomicrographs of
immunohistochemistry staining for Bcl-2 (A) and Bax (B) in sham (2), I/R (3), pretreatment with CP at 0.1 g/kg I/R (4), pretreatment with CP at 0.4 g/kg
I/R (5), and pretreatment with CP at 0.8 g/kg I/R (6) group. A1: the location of sample for Bcl-2 immunohistochemistry staining. Bar 100 m. B1: the
location of sample for Bax immunohistochemistry staining. Arrows indicate immunohistochemistry staining positive cells. Arrowheads indicate nuclei of cardiac
muscle cells. C: quantitative analysis of Bcl-2 expression in rat myocardium in different groups. D: quantitative analysis of Bax expression in rat myocardium
in different groups. Values are means SE from 6 animals. *P 0.05 vs. sham group. #P 0.05 vs. I/R group.
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group (B2). The number of caspase-3-positive cells was decreased by pretreatment with CP at 0.8 g/kg (B6). CP at 0.1 or
0.4 g/kg did not show any effect (Fig. 8D).
Figure 9 illustrates the expression of apoptosis-related proteins Bcl-2 and Bax in surrounding infarction areas of the left
ventricle myocardial tissues. As noticed from Fig. 9, A and C,
the expression of Bcl-2 was depressed by I/R challenge.
Pretreatment with CP at 0.8 g/kg, but not at 0.1 or 0.4 g/kg,
upregulated Bcl-2 expression. On the contrary, the expression
of Bax was increased remarkably by I/R and downregulated by
pretreatment with CP at 0.8 g/kg (Fig. 9, B and D).
Histology. Figure 10 presents the result of histological examination of surrounding infarction areas of the left ventricle
myocardial tissues in different groups. Compared with the
sham group (Fig. 10B), distinct alterations occurred in the
surrounding infarction areas of myocardial tissues from the I/R
group, including disruption of myocardial fibers, tissue edema,
and neutrophil infiltration (Fig. 10C). Of notice, all of the
I/R-induced alterations were ameliorated by pretreatment with
CP (Fig. 10, DF), especially at the dose of 0.8 g/kg.
Changes in myocardial ultrastructure. The ultrastructures of
surrounding infarction areas of the left ventricular myocardial
tissues from different groups were examined. Figure 11A
presents the representative electron micrographs of capillaries
with surrounding tissues. The endothelium of capillaries in the
sham group was preserved well, and both the capillary and
surrounding tissue displayed normal ultrastructural features
(Fig. 11A, A1). The I/R injury led to an apparent swelling of the
endothelium with a large number of caveolaes in endothelial
cells and prominent edema in the vascular surroundings (Fig.
11A, A2). At 0.4 and 0.8 g/kg of CP, the I/R-evoked endothelial
swelling and surrounding tissue edema were conspicuously
ameliorated (Fig. 11A, A4 and A5).
The ultrastructure of cardiac muscle cells in each condition
was examined as well; the representative electron micrograms
are presented in Fig. 11B. In the sham group (B1), cardiac
myofibrils stood regularly arranged with well-preserved myofilaments, and mitochondria occupied the cytoplasm between

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myofibrils with densely packed cristae. The I/R challenge

provoked a dramatic injury in cardiac muscle cells (B2), as
indicated by disrupted myofibrils and ruptured mitochondria.
Pretreatment with CP at each of the three doses attenuated the
ultrastructural alterations induced by I/R (B3, B4, B5).
IB degradation in the myocardium. To examine the effect
of CP on IB regulation, Western blot assay was performed for
myocardial tissues from different groups. As shown in Fig. 12, I/R
induced significant IB degradation at 60 min of reperfusion
compared with the sham group, pretreatment with CP at 0.4 and
0.8 g/kg significantly inhibited IB degradation elicited by I/R
challenge, while 0.1 g/kg CP did not affect the level of IB in the
myocardium following I/R.
DISCUSSION

In the present study, we reported for the first time the in vivo
data demonstrating that pretreatment with CP significantly
attenuates the I/R-induced cardiac impairments, including decreased RBC velocity in coronary microvessels, decreased
coronary blood flow, increased expression of CD18 on neutrophils and ICAM-1 on endothelium, albumin leakage across the
venular wall, and myocardial injury. Furthermore, the results
of TUNEL staining and caspase-3 expression indicate that
myocardium injury observed in this study is attributed, at least
partly, to apoptosis mediated by Bcl-2 and Bax.
The beneficial effect of CP on microcirculation has been
demonstrated in gut I/R-induced hepatic injury (11). However,
no study has been reported to address its action on myocardial
microcirculatory dysfunction after I/R, despite the fact that it
has long been used for the prevention and treatment of I/Rrelated heart diseases. In the present study, intravital microscopy was applied, enabling visualization in real time of the
dynamics of coronary microcirculation in beating hearts. The
results are consistent with the findings from the Laser-Doppler
Perfusion Imager study, suggesting that pretreatment with CP
significantly attenuates the decreases in coronary blood flow,
as well as arteriolar/venular flow after I/R. Diminished micro-

Fig. 10. Effect of pretreatment with CP on

histology of myocardial tissue of rats after I/R.
The tissue was stained by hematoxylin and
eosin. A: location of the samples for histology examination. B: sham group. C: I/R
group. D: pretreatment with CP at 0.1 g/kg
I/R group. E: pretreatment with CP at 0.4
g/kg I/R group. F: pretreatment with CP
at 0.8 g/kg I/R group. a: Disrupted
myocardial fiber. b: Edema. c: Infiltrated
neutrophils. Bar 100 m.

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Fig. 11. Influence of pretreatment with CP on the ultrastructure of myocardial tissues of rats after I/R. A: the representative electron micrographs of capillary
in myocardial tissues of rats from different groups. a: Vascular endothelium. b: Caveolae. c: Perivascular edema. B: the representative electron micrographs of
cardiac muscle cells of rat from different groups. d: Myofibrils. e: Mitochondrion. f: Ruptured mitochondrion. 1, 2, 3, 4, and 5: Sham group, I/R group,
pretreatment with CP at 0.1 g/kg I/R group, pretreatment with CP at 0.4 g/kg I/R group, and pretreatment with CP at 0.8 g/kg I/R group, respectively.

vascular flow in the ischemic area after reperfusion is usually

referred to as low reflow, a phenomenon resulting from diverse
insults where leukocyte recruitment plays a pivotal role (8, 15).
The accumulation of leukocytes in microvessels after I/R not
only plugs the blood flow, but also causes endothelial injury
and barrier dysfunction: both effects contribute to low reflow
(24). Thus inhibition of leukocyte adherence to the vascular
endothelium exerts protective effects against I/R-induced microcirculatory disorders. For this purpose, monoclonal antibodies against leukocyte adhesion molecules have been tested in
clinical trials, although the data are negative in general (21).

Fig. 12. Effects of CP on I/R-induced IB degradation in rat myocardium. A: the

representative Western blotting picture of the IB protein in sham group, I/R
group, CP 0.1 g/kg I/R group, CP 0.4 g/kg I/R group, and CP 0.8 g/kg I/R
group at 60 min after reperfusion. B: semiquantitative analysis of IB protein
content. Values are means SE from 6 animals. *P 0.05 vs. sham group.
#P 0.05 vs. I/R group.
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While the molecular details underlying the protective effect of

CP against I/R-induced microcirculatory disturbance found in
this study need to be interrogated in more depth, its inhibitory
effect on leukocyte adhesion molecule expression may serve as
an important factor in its mechanism of action.
The present study revealed that pretreatment with CP reduces the infarct size and the number of TUNEL-positive cells
and improves myocardial structure, pointing to the potential of
CP in protecting the myocardium from I/R injury. Myocardium
injury after I/R is caused by multiple factors, including oxidative stress, depletion of high energy stores due to hypoxia, and
cytotoxic substances released from infiltrated leukocytes, including cytokines, chemotactic factors, and proteinases (1, 7,
19, 30). Considering the fact that SM, one of the major CP
ingredients, was demonstrated to have antioxidant ability (20), it
is reasonable to propose that CP exerts its protective effect on the
myocardium through mechanisms involving suppressed ROS production, which may, in turn, reduce leukocyte adhesion and
infiltration and thus improve oxygen supply to the cardiac muscle
cells. This notion is further supported by the data that the I/R
injury-induced MDA production, an indicator of lipid peroxidation, was attenuated by administration of CP.
It is well recognized that necrosis and apoptosis are two major
types of cell death in tissues subject to I/R (29). This study does
not discriminate the relative contribution of each type to infarct
size in the myocardium. Nevertheless, I/R-induced apoptosis of
cardiac myocytes was attenuated by pretreatment with CP, as
evidenced by the decrease in the number of TUNEL-positive cells
and caspase-3 expression. Furthermore, the increase of Bcl-2 and
decrease of Bax indicates that the intrinsic apoptotic pathway is at
least one of the targets that CP acts on (5). More studies that
determine the activity of caspase-8 are required to clarify whether
or not the extrinsic apoptotic pathway is involved in this process.

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I/R stimulates the production of peroxides that induce the

degradation of IB, and then activation of NF-B, resulting
in expression of various adhesion molecules and apoptosisrelated proteins (14, 35). Studies using a model of myocardial
I/R injury have shown that blockade of NF-B reduces infact
size and protects myocytes from ischemic insult (17). Watersoluble fraction from SM was reported to inhibit the TNF-
induced translocation of NF-B from the cytoplasm to the
nucleus on human umbilical vein endothelial cells (4). Our
results agree with previous studies that the IB was degradation induced by myocardial I/R and further propose that CP
inhibits IBa degradation. Nonetheless, the underlying mechanism needs to be further elucidated.
In China, CP is currently used for treatment of angina in the
clinic by administration for 4 wk at a dose of 0.75
gkg1day1. In the present study, the doses of CP used were
0.1, 0.4, and 0.8 g/kg, which were designed to be 1.2-, 4.9-, and
9.9-fold higher, respectively, than the equivalent effective dose
for treatment of human angina. The results of the present study
revealed that administration of CP at 0.1 and 0.4 g/kg may
attenuate the increase in MDA level and the number of
TUNEL-positive cells in rat myocardium, alleviating morphological disorder in myocardial tissue induced by I/R, while
administration of CP at a dose of 0.8 g/kg attenuates I/Relicited rat myocardium injury and cardiac microcirculation
disturbance as well. No report is available as yet regarding
whether or not pretreatment with CP by a single dose may
attenuate I/R-induced cardiac microcirculation disturbance and
myocardial tissue injury, and, if yes, what is the effective dose.
Nonetheless, the present result suggests that, if CP is intended
for use of preventing I/R-induced cardiac microcirculation
disturbance and myocardial tissue injury, the dose should be
higher than that for treatment of angina.
In summary, pretreatment with CP (0.8 g/kg) attenuates
I/R-induced coronary microcirculatory disturbance and albumin leakage, which may be attributed to its inhibitory effect on
CD18 and ICAM-1 expression. Furthermore, CP may protect
the myocardium from damage following I/R injury by suppressing the intrinsic apoptotic pathway. The protective effects
of CP on rat coronary microcirculatory disturbance and myocardial damage may be related to inhibiting degradation of
IB.
GRANTS
This work was supported, in part, by the International Science and Technology Cooperation Project from the Ministry of Science and Technology of
the People, s Republic of China (2006DFA32340).
DISCLOSURES
No conflicts of interest are declared by the author(s).
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