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Introduction
Danshen, the dried roots of the medicinal plant
Salvia miltiorrhiza, is a traditional Chinese herbal medicine used for the
treatment of coronary heart disease. Depside salts from
S miltiorrhiza are novel drugs in which magnesium lithospermate B
(MLB, Figure 1) and its analogues are the active components. The pharmacological activities of MLB, which is a major
aqueous extract ingredient of Danshen, have been extensively investigated. MLB has been shown to reduce the size of
myocardial infarction[1] as Danshen
does[2]. Further studies investigated the involving mechanisms in several aspects. First,
it was speculated that the antioxidative and radical scavenging property of MLB played an essential role in its cardioprotective
efficacy[3_6]. Second, MLB was reported to stimulate the production of nitric oxide in endo-thelial
cells[7_9], possibly via the induction of constitutive NOS (cNOS)
expression[7]. Third, MLB was demonstrated to
behave in a Ca2+ antagonistic fashion
voltage-dependently[10] and inhibited extracellular calcium
influx[8,9]. In addition, MLB suppressed apoptosis induced
by myocardial
ischemia/reperfusion[11,12], probably through the inhibition of
the c-Jun N-terminal kinase 3 (JNK 3)
activity[11] and stress-activated protein (SAP) kinase
activity[12]. However, there has been no direct evidence on
its effect on coronary micro-circulation.
Laser Doppler flowmetry, which offers a continuous
real-time measurement of blood cell perfusion in the
microcirculatory beds of tissues, is a prospective method to be applied
for myocardial microvascular perfusion assessment. Ahn
et al[13] demonstrated that their data of the local myocardium
perfusion correlated well with coronary sinus blood flow in
the empty-beating hearts of pigs. Sidi et
al[14], who considered movement artifacts from the contraction of myocardium,
found that the local changes in flow measured by laser
Doppler flowmetry correlated with the regional myocardial blood
flow changes obtained by means of radioactive microspheres,
but not with global contractility which was evaluated by
first-time derivative of left ventricle pressure (LV
dp/dt). Klassen et
al[15] proposed a method to overcome the limits in
measuring regional phasic myocardial red cell flux during a
cardiac cycle using a laser Doppler velocimeter by inserting
the probe into the beating heart in various locations. They
suggested that the fiber tip moved in concert with the
contracting muscle fibers with little tethering of myocardial action,
thus only registering movements from the red blood cells.
They also confirmed their hypothesis subsequently in the
canine myocardium[16]. Therefore, it appears feasible to
apply the laser Doppler technique quantitatively in evaluating
the responses of drugs to myocardial microperfusion.
In the present study, we employed the laser Doppler
flowmetry system to investigate the effect of S
miltiorrhiza depside salts on myocardial microperfusion. We found that
S miltiorrhiza depside salts improved coronary
microcircula-tion, as well as cardiac output (CO).
Materials and methods
Drugs and reagents S miltiorrhiza depside salts which
contain MLB (³80.0%) were provided by the Department of
Phytochemistry, Shanghai Institute of Materia Medica
(Shanghai, China). Nifedipine (Sigma, USA) was dissolved
in a 5% DMSO/physiological saline solution, and the other
drugs involved were dissolved in physiological saline only.
Drug concentrations were adjusted to yield an injection
volume of 0.1 mL/100 g of body weight.
Experimental preparation Male Sprague-Dawley (SD)
rats (Certificate No SCXK 2003_0003, Shanghai SLAC
Laboratory Animal Co, Shanghai, China) weighing 250_300 g were
housed in cages (2 per cage), maintained at 25 °C with 12 h of
light, and were allowed free access to water and standard rat
chow. The rats were anesthetized using sodium
pentobarbital (40 mg/kg, ip), and additional doses were given to
maintain anesthesia when necessary. Body temperature was
maintained at 37 °C with a thermostat-controlled operation table
(Harvard Apparatus, USA). A catheter (PE-10 fused with
PE-50) filled with heparin/saline (2 mg/mL) for measurement of
mean arterial pressure (MAP) was introduced into the
abdominal aorta through the right femoral artery and
connected to a pressure transducer (Powerlab MLT 844,
ADInstruments, Australia). A second catheter for drug
administration was inserted in the right femoral vein. The
trachea was cannulated and connected to a respirator (HX-200,
Chengdu Taimeng Technology Co, China). Arterial blood
gases (i-STAT Portable Clinical Analyzer, Abbott
Labora-tories, Abbott Park, IL, USA) were kept within the normal
range by adjusting ventilation volume and/or rate as needed.
All experimental protocols involving the use of animals were
approved by Animal Care and Use Committee of the Chinese
Academy of Sciences.
Determinations of CO A left-sided thoracotomy was
performed from the second to the fourth intercostal space to
expose the lungs in the thoracic cavity, and the heart was
suspended in a pericardial cradle. After the ascending aorta
had been gently isolated from the pulmonary artery by blunt
dissection, the ultrasonic perivascular flow probe (2SB) of
ultrasonic Doppler flowmetry (T206, Transonic Systems Inc,
Ithaca, NY, USA) was mounted on the root of the ascending
aorta, with the sterile surgilube jelly (E Fougera & Co, USA)
filling the probe window to get rid of air bubbles, which
permitted the transmission of the ultrasound signal.
Additional jelly, loaded by a 20 mL syringe, was injected into the
flow probe lumen if necessary.
Determinations of myocardial
microperfusion To
assess microcirculation, a needle probe (MNP110, length 25
mm, diameter 480 µm) of laser Doppler flowmetry (Powerlab
ML191, ADInstruments, Australia) for microcirculation was
inserted into a specially-designed holder which was glued
to the epicardium of the left ventricular myocardium close to
the left anterior descending coronary artery where no
obvious large vessels lay. The orientation of probe was
appropriately adjusted against the holder until a stable,
high-quality signal for laser Doppler flowmetry was obtained. The
laser Doppler flowmetry signal was recorded as arbitrary
blood perfusion units (BPU), a relative unit scale defined by
reference to a controlled motility standard. Due to the
flexibility of the optical fiber and the plastic harmonious
movement of the light probe accompanying the beating heart,
virtually no tethering of the myocardium took place.
Protocol The doses of S
miltiorrhiza depside salts were selected based on the pilot study and the
literature[1_4]. After thoracotomy with instrumentation, the rats underwent a
recovery period of 30 min for hemodynamics stabilization.
The test drugs were injected intravenously, and no animal
received more than 1 agent, except those who received
vehicle control administration. After each drug was
intravenously administered within 15 s, the changes of all
para-meters, recorded simultaneously by a computer using the
chart software version 5.4.1 (ADInstruments, Australia), were
measured for 30 min consistently.
Criteria for an acceptable experiment The rats whose
MAP was below 80 mmHg after stabilization were excluded,
as well as those who failed to maintain stable within 1 h
post-operatively. It was essential that arterial blood gases
remained within the physiological range, and standardized
respiratory monitoring was performed.
Calculation of hemodynamics The flow-probe cable was
connected to a model T206 flow meter (Transonic Systems
Inc, USA) to measure CO[12], which was equal to the
ascending aortic flow and was expressed as flow per kg of body
weight (CI). MAP was automatically calculated from femoral
artery pressure by the software. Systemic vascular
resistance (SVR) was determined using the following formula:
SVR=MAP/CI. Cardiac inotropy was estimated using the
maximal velocity of flow increase
(df/dtmax)[27]. Myocardial
microperfusion, detected by laser Doppler flowmetry, was
quantified by change percentage due to the temporal and
spatial microheterogeneity of the myocardial blood
flow[13], as well as the small region of the myocardium under
observation. The mean values for each determination were
analyzed over a 10 s period.
Data statistical analysis Results were expressed as
mean±SD. Parameters at the same time point were compared
with the vehicle control by unpaired Student's
t-test. Student's paired t-test was used for comparison of the
parameters with their corresponding baseline values. A value
of P<0.05 was considered significant.
Results
Feasibility of laser Doppler flowmetry for myocardial
microperfusion assessment Myocardial microperfusion
profile was measured for up to 30 min without intervention.
Ascending aortic flow, femoral artery pressure, and
myocardial microperfusion were recorded for approximately 10 min
while no intervention occurred (Figure 2). When a
satisfactory positioning of the probe was made, a distinct, regular,
consistent pattern of myocardial microperfusion waveform
was observed which remained constant during control and
changed correspondingly, following an intervention, as that
of other parameters. Ventilation played a pivotal role in the
behavior of hemodynamics, and myocardial microperfusion
varied correspondingly as well (data not shown). No
alteration in either the pattern or value of myocardial
microperfu-sion was observed as long as ascending aortic flow and
femoral artery pressure were constant (Figure 2).
The myocardial microperfusion profile recorded by a
laser Doppler flowmetry device behaved in a phasic fashion
throughout the cardiac cycle (Figures 2, 3). The
relationships between ascending aortic flow, femoral artery pressure,
and myocardial microperfusion in cardiac cycles are shown
(Figure 3). As we know, diastole originated at the same time
point as when the ascending aortic flow returned to the
baseline and was indicated by the closure of the aortic valve.
The blood perfusion waveform, which increased with the
femoral artery pressure and decreased gradually after
reaching a peak in diastole, showed a dicrotic notch, as seen in the
femoral artery pressure to some extent. The peak of
ascending aortic flow from which CO was calculated coincided with
the nadir of the myocardial microperfusion waveform where
the blood flow started to increase until the early period of
diastole (Figure 3). The combined curves showed that the
myocardial microperfusion was predominately diastolic and
its phase was opposite to the ascending aortic flow.
Representative vasodilators, nifedipine and captopril,
were selected for system test and confirmation. The mean
basic values of myocardial microperfusion observed in each
group for 75 µg/kg nifedipine, 5 mg/kg captopril, and vehicle
control were 471±58 BPU, 458±74 BPU, and 445±58 BPU,
respectively. Our data showed that intravenous
administration of both 75 µg/kg nifedipine and 5 mg/kg captopril
exerted an increasing effect on myocardial microperfusion,
as well as on CO (Table 1). Intravenous administration of 75
µg/kg nifedipine increased myocardial microperfusion by
7.1%±2.9% (P<0.01) maximally, and 5 mg/kg captopril
improved myocardial microperfusion 4 min after drug
intervention with a peak increase of 9.5%±4.6%
(P<0.01). Typical tracings of CO, artery pressure, and myocardial
microperfu-sion after the intravenous administration of 75 µg/kg
nifedi-pine are shown in Figure 4.
Effects of S miltiorrhiza depside salts on
hemodynamics Intravenous administration of S
miltiorrhiza depside salts
resulted in a significant immediate increase in CO and cardiac
inotropy, as well as a fall in SVR (Figure 5).
S miltiorrhiza depside salts (30 mg/kg and 60 mg/kg) increased
CI by 12.2%±6.3% (P<0.01) and 20.1%±3.5%
(P<0.01) from the baseline, respectively, which was in a dose-dependent
manner. S miltiorrhiza depside salts (30 mg/kg and 60 mg/kg)
caused an increase in cardiac inotropy with a peak value of
21.7%±6.8% (P<0.01) and 50.5%±18.9%
(P<0.01) 0.5 min
after injection, respectively. However, 30 mg/kg
S miltior-rhiza depside salts showed a depressant effect 10_20 min
after injection. A slight fall in MAP appeared 10 min after 30
mg/kg S miltiorrhiza depside salts were intravenously
administered, with a significant decrease at the 15 min time
point (Figure 5C). Conversely, 60 mg/kg S
miltiorrhiza depside salts exerted an increasing effect on MAP in the
initial period by 17.1%±9.4% (P<0.01
vs baseline) maximally. Cardiac inotropy dropped significantly and reverted to the
baseline soon after.
Effects of S miltiorrhiza depside salts on myocardial
microperfusion The effect of S
miltiorrhiza depside salts (30 mg/kg and 60 mg/kg) on myocardial microperfusion in
the LV was measured in 7 open-chest anesthetized rats
(Figure 6). Myocardial microperfusion maximally
increas-ed by 6.3%±2.9% (P<0.01) and 9.6%±4.0%
(P<0.01) for 30 mg/kg and 60 mg/kg S
miltiorrhiza depside salts, respectively. The profile of myocardial microperfusion with
S miltiorrhiza depside salts intervention returned to the
baseline in no more than 15 min.
Discussion
Coronary microcirculation plays a pivotal role in
determining the supply of oxygen and nutrients to the myocardium.
Analyses of coronary microcirculation are important for
understanding the pathophysiology of ischemic hearts and
the effect of cardioprotective agents. Although the
cardio-protec-tive property of MLB has been studied extensively,
as well as those of S miltiorrhiza depside salts, there is still
no report concerning their effect on coronary
micro-circulation. To our knowledge, we demonstrated here for the
first time that S miltiorrhiza depside salts could improve
myocardial microperfusion using laser Doppler flowmetry,
as well as CO.
A number of strategies have been previously employed
to optimize the laser Doppler system to overcome movement
artifacts so that it can be used for myocardial microvascular
measurement[13_19]. In the present study, we proposed that
the probe was satisfactorily positioned when a distinct and
regular waveform pattern was observed during baseline
measurements and was altered following intervention, as
suggested by Klassen et al[15]. Kiyooka
et al[20] reported that red blood cell flow in the epimyocardium capillaries was
predominant during either systole or diastole, acting as the
watershed between diastolic arterial and systolic venous
flows. Besides, the diameters of subepicardial arterioles
remained basically constant during the entire cardiac
cycle[17]. In our research, data showed that the phasic profile of
myocardial microperfusion by laser Doppler flowmetry, which
reflected both arterial inflow and venous outflow, was
predominately diastole, which was identical to previous
studies[17,20]. Furthermore, the representative vasodilator nifedipine was
studied in our system and showed significant increasing
effects on myocardial microperfusion as
reported[21,22], so did captopril as predicted, which in turn convinced us that
the laser Doppler system was sensitive enough to the
responses of coronary microvascular to drugs for
measure-ment.
In our study, the improving effect of intravenously
administered S miltiorrhiza depside salts on CI was in a
dose-related manner. After a bolus injection of
S miltiorrhiza depside salts, we observed significant increases in CI and
myocardial microperfusion prior to minor changes of MAP,
which suggested that S miltiorrhiza depside salts might
improve cardiac function directly.
Yokozawa et al[23] reported that oral administration of
MLB produced a significant reduction in blood pressure in
rats with sodium-induced hypertension. The studies by
Kamata et al[24,25] also showed that intravenous injection of
lithospermic acid B (10_30 mg/kg) decreased the blood
pressure in a dose-dependent manner and behaved as an
endothelium-dependent vasodilator. In the current study, the
effect of 30 mg/kg S miltiorrhiza depside salts on MAP was
identical to previous studies, however, 60 mg/kg
S miltior-rhiza depside salts resulted in a marked increase in MAP in
the initial period of observation when CI was promoted
significantly. We presumed that it resulted from the
overwhelming CO increasing effect which consequently
concealed the vasodilation property of S
miltiorrhiza depside salts.
In conclusion, S miltiorrhiza depside salts improve
myocardial microperfusion, as well as CO, which provides
further evidence for the treatment of S
miltiorrhiza depside salts on coronary heart diseases.
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