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Introduction
Danshen, the dried roots of medicinal plant Salvia
miltiorrhiza, is one of the most popular Chinese herbal products
widely used in many medicine preparations and formulae taken by people in certain Asian countries. Traditionally regarded
as an effective medicine for eliminating blood stasis, relieving pain, promoting blood flow, stimulating menstrual discharge,
and relaxing the mind, Danshen has been extensively used in the treatment of coronary heart disease, heart stroke, myocardial
infarction, menstrual disorder, and other cerebrovascular
diseases[1]. During the past decade, water-soluble
components of Danshen have attracted escalating attention on the
basis of their reported medicinal potency, although some
lipid-soluble constituents, such as tanshi-nones, of this herb
have been conventionally considered as the active
ingredients[2_4]. Among the water-soluble com-ponents, magnesium
lithospermate B (MLB), a derivative of caffeic acid tetramer,
is present as the major soluble ingredient in Danshen, and
has been demonstrated to possess several medicinal effects,
such as vasodilating, antihypertensive, antioxidative, and
free radical scavenging
activities[5_10].
Cardiac glycosides, such as ouabain and digoxin, have
been applied to the treatment of congestive heart failure for
more than 2 centuries since William Withering published his
famous monograph in 1785[11]. The molecular mechanism
responsible for the therapeutic effect of cardiac glycosides
lies in their reversible inhibition on the α-subunit of the
membrane-bound Na+,K+-ATPase, mainly, but not exclusively,
located in human myocardium[12,13]. This inhibition leads to
the accumulation of sodium in cardiac cells which are
enforced to promote the sodium-calcium exchange system in
the cell membrane, thus causing a higher level of
intracellular and myocardial calcium. The elevated intracellular
calcium leads to an increased inotropism, accentuating the force
of myocardial contraction by increasing the velocity and
extent of sarcomere shortening, thus translating into
increased stroke work for a given filling volume of pressure.
Moreover, cardiac glycosides were recently demonstrated
to provide neuroprotection against ischemic stroke in a
cortical brain slice-based compound screening
platform[14].
In light of the consentaneous utilization of Danshen and
cardiac glycosides in cardiac therapy, we wondered if any
constituents extracted from this Chinese herbal product might
possess medicinal potency similar to that of cardiac
glycosides via the same molecular mechanism. In this study,
partial similarity of molecular configuration was assumed in
chemical structures between MLB and ouabain, and their
potency of inhibition on
Na+,K+-ATPase activity of a
commercial product as well as in purified membrane fractions
from rat tissues was examined and compared.
Neuropro-tection of MLB against ischemic stroke was
also examined in a brain slice assay model.
Materials and methods
Structure comparison of ouabain and MLB The
structure of ouabain is obtained from
DrugBank[15] with accession number APRD00135. The structure of MLB resembling
ouabain was constructed by exploring the possible rotamers
by rotating the single bonds of MLB, and then the structure
was minimized with molecular mechanics and an ab
initio method (B3LYP/6-31G) using the Gaussian 03 package
(Gaussian, Inc, Pittsburgh PA, USA) consecutively. The
3-D structures of ouabain and MLB were displayed
using RasWin Molecular Graphics Windows Version 2.6 (Stevenage,
Hertfordshire, UK).
Extraction and purification of MLB Dried roots (Danshen)
of Salvia miltiorrhiza plants cultivated in a local farm were
prepared in the Herbal Source Biotechnology Company
(Tainan County, Taiwan). MLB was purified according to
the protocol described by Tanaka et
al[3]. The dried roots (8.8 kg) were extracted with 50 L methanol under reflux for
8 h and concentrated to a brown syrup. The syrup was
suspended in H2O and partitioned with chloroform. MLB
was harvested after repeated column chromatography of the
H2O extract using Sephadex LH-20 and
H2O as an eluent.
Preparation of plasma membrane from rat brains and
heart Male Sprague-Dawley (Narll: SD) rats (3 months old)
were purchased from the National Laboratory Animal Center
(Nankang, Taipei) and raised under specific pathogen-free
conditions. The rats, fed with rat chow (Rodent Laboratory
Chow 5001, Purina, MO, USA) and tap water ad
libitum, received humane care throughout the studies in accordance
with the guidelines of a guidebook for the care and use
of laboratory animals. The animals were sacrificed
by decapitation, and the brain and the heart organs were
removed immediately after complete exsanguination.
The plasma membrane was isolated from the rat brain
and heart at 4 °C according to the methods described by Lin
and Way[13,16]. Briefly, the brain cortex or heart was
homogenized with 10_20 volumes of 0.32 mmol/L sucrose solution
containing 5.0 mmol/L
4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES) and 1.0 mmol/L EDTA (pH
7.5). The brain or heart homogenate was centrifuged at
1000×g for 10 min, and the resultant supernatant was further
centrifuged at 17 000×g for 30 min to precipitate the crude
plasma membrane fraction. The latter was washed twice and
suspended in 0.32 mol/L sucrose HEPES-buffer and was
subjected to centrifugation at 63
000×g for 1 h with a discontinuous sucrose density gradient consisting of successive
layers of 0.3, 0.8, and 1.0 mmol/L. The plasma membrane
collected at the interface between 0.8 and 1.0 mmol/L sucrose
was suspended in 0.32 mol/L sucrose solution and used for
enzyme assays within 2 h.
Western blot analysis For SDS-PAGE,
Na+,K+-ATPase samples were mixed with sample buffer containing 62.5
mmol/L Tris-HCl (pH 6.8), 2% SDS, 0.02% bromophenol blue,
10% glycerol, and 5% β-mercaptoethanol according to the
Bio-Rad (Hercules, CA, USA) instruction manual. The
samples were boiled for 5 min and stored at 4
°C prior to electrophoresis. For the Western blot analysis, the proteins
resolved in SDS-PAGE were transferred to a PVDF
(polyvinyli-dene difluoride) membrane (PerkinElmer, Boston, MA, USA)
in a Bio-Rad Trans-Blot system (USA) according to
the manufacturer's instructions. The membrane was subjected
to immunodetection using an antibody against the
b-subunit of Na+,K+-ATPase raised in rabbits (Sigma, St Louis, MO, USA)
and anti-rabbit IgG raised in goats and conjugated with
alkaline phosphatase (Jackson ImmunoResearch, West
Grove, PA, USA) as the primary and secondary antibodies,
respectively.
Measurement of Na+,K+-ATPase activity
The activity of Na+,K+-ATPase was determined by measuring the amount of
inorganic phosphate (Pi) liberated from ATP according to
Muszbek et al[17]. A commercial
Na+,K+-ATPase from porcine cerebral cortex (Sigma, USA, 0.3 units/mg) or plasma
membrane fraction purified from rats was incorporated into a
reaction mixture of 1 mL containing 3 mmol/L ATP, 5 mmol/L
MgCl2, 80 mmol/L NaCl, 20 mmol/L KCl, and 40 mmol/L
Tris-HCl (pH 7.4); the enzymatic reaction was terminated by 200
µL of 30% (w/v) trichloroacetic acid after 15 min. After
centrifugation at 3300×g for 15 min, 500 µL supernatant was
taken to measure the inorganic phosphate by the
spectrophotometric method described by Goldberg and
Fernander[18]. Activity was expressed as mmol Pi liberated from ATP by 1
mg of Na+,K+-ATPase during 1 h. Protein
content was quantified with a Bradford protein assay kit (Sigma, USA). To
observe the potency of the inhibitors, commercial
Na+,K+-ATPase or the purified plasma membrane fraction was
incubated with ouabain or MLB of various concentrations at
37 °C for 10 min prior to incorporation into the reaction mixture.
Cerebral ischemia of gerbils Eighteen male gerbils
(65_80 g) were randomly divided into 3 groups: the control group
and 2 MLB groups, and fed regular meals (20
mg·kg-1·d-1). In the present study, the focal cerebral ischemia was performed
by occlusion of the right common carotid artery (CCA) and
the right middle cerebral artery (MCA) as modified from the
method of Yang et al[19]. All gerbils were anesthetized with
chloral hydrate (360 mg/kg, ip) and allowed to breathe
spontaneously. A thermostatically-regulated heating pad
(CMA/150, Carnegie Medicine, Stockholm, Sweden) was
used to maintain body temperature at 37 °C during
ischemia/reperfusion. The gerbils' heads were placed in a stereotaxic
frame (Stoelting, Wood Dale, IL, USA) and the right CCA,
exposed through a ventral midline incision in the neck, was
carefully separated from the vago-sympathetic trunks for
later occlusion by a mini-aneurysm clip. Following a midline
incision, the skull was craniectomized to expose the right
MCA. An 8_0 suture was positioned so that it encircled the
right MCA for later occlusion. A focal ischemic/reperfusion
lesion was made by simultaneous occlusion of the right CCA
and the ipsilateral MCA for 60 min. The suture and the
mini-aneurysm clip were released, and followed by 23 h
reperfu-sion. Either distilled water (0.6 mL) or MLB (0.6 or 6 mg/kg in
0.6 mL) were fed via an intragastric route 1 h after reperfusion
according to the research protocol.
Visualization of infarct size in gerbil brains
For the determination of infarct sizes, the gerbils were sacrificed and
the brains were removed and sliced. Slices 2 mm were
immersed in a 2% solution of 2,3,5-triphenyltetrazolium
chloride (TTC) stain as described by Bederson et
al[20]. After 20 min, the slices were placed in 10% buffered formalin in the
dark and refrigerated until photographed. The slices were
projected and traced. The infarct size was quantified by
cutting out and weighing the traced normal and infarct area
using the Optimal computer software.
Statistical analysis The data are expressed as mean±
SEM and analyzed by ANOVA and Student's t-test.
Differences were considered statistically significant at
P<0.05.
Results
Structural comparison between ouabain and MLB
Having a steroid backbone, ouabain, as well as other cardiac
glycosides, possesses a rigid structure (Figure 1). MLB, a
derivative of caffeic acid tetramer, also has a relatively rigid
structure due to the formation of salt bridges between
Mg2+ and the 4 oxygen atoms of carboxyl groups originating from
the 4 caffeic acid fragments. The molecular organization and
configuration of ouabain and MLB in 3-D structures are
somewhat similar from a particular viewpoint (lower portions of
the 2 3-D structures in Figure 1), although they are totally
different compounds with distinct molecular weights (584.65
and 740.67 for ouabain and MLB, respectively).
Inhibition of porcine Na+,
K+-ATPase by ouabain and MLB To test if MLB could lead to a similar therapeutic effect
via the same mechanism triggered by ouabain, that is,
accentuating the force of myocardial contraction by elevating
calcium concentration via the inhibition of
Na+,K+-ATPase, a commercial
Na+, K+-ATPase from porcine cerebral cortex was
used to evaluate the inhibitory potency of MLB using
ouabain as a control. The results showed that both ouabain and
MLB could inhibit the commercial
Na+,K+-ATPase in a dose-dependent manner (Figure 2). In our assay condition, the
inhibition potency on Na+,K+-ATPase, equivalent to that for
ouabain, was observed for MLB of approximately half
dosage by weight.
Inhibition of Na+,K+-ATPase in the purified plasma
membrane from rat tissues The presence of
Na+,K+-ATPase in the plasma membrane purified from rat brains was confirmed
by Western blot analysis using the commercial porcine
Na+,K+-ATPase as a positive control (Figure 3). Under the same
assay conditions, the relative potency of ouabain and MLB
was also observed for their inhibition on
Na+,K+-ATPase activity of the plasma membrane purified from rat brains,
although these 2 inhibitors exhibited lower competence in
comparison with their inhibition on commercial
Na+,K+-ATPase (Figure 4). No apparent difference was detected for MLB
inhibition on Na+,K+-ATPase activity of the plasma
membranes purified from rat heart and brain tissues, respectively
(data not shown).
Neuroprotection of MLB on cerebral ischemic damage
in gerbils Recent observations of the neuroprotection of
cardiac glycosides against ischemic
stroke[14] provoked an inquiry as to whether MLB could also act as anti-ischemic
agents in vivo. To examine this possibility, gerbils were
administered with 2 different concentrations of MLB after a
focal cerebral ischemia. All the treated animals were found
to have infarction in the cortex and caudate-putamen. The
mean total infarct sizes, visualized by TTC staining (Figure
5), in the 3 groups (control, 6 mg MLB, and 0.6 mg MLB) were
17.35%±0.51%, 7.42%±1.40%, and 7.67%±2.35%, respectively
(Figure 6). These results indicate that post-treatment with 6
mg or 0.6 mg MLB significantly reduced the infarct sizes of
gerbil brains in cerebral ischemia by 57.3% and 55.8%,
respectively (P<0.01).
Discussion
Cardiac glycosides are drugs clinically used to relieve
the symptoms of congestive heart
failure[21]. Although these compounds unquestionably improve the conditions of
patients, safe administration of these drugs has been
regarded as a difficult task due to their narrow safety margin
and severe side effects. Extensive efforts have been made to
develop novel cardiotonic agents, such as new digitalis-like
molecules through chemical synthesis and
modification[21_25]. As these derivatives possess the same or similar steroid
backbone, side-effects are unlikely to be eliminated. In this
study, MLB extracted from Danshen, presumably partially
mimicking the steroid structure of cardiac glycosides on the
basis of its relatively rigid structure stabilized by the salt
bridges formed between Mg2+ and carboxyl groups, was
demonstrated to inhibit
Na+,K+-ATPase more potently than
ouabain in vitro. In contrast with the side effects of cardiac
glycosides, MLB has been considered an antioxidant
without significant adverse effects[2]. Therefore, we believe that
MLB is of great potential to replace cardiac glycosides, after
clinical trials, for the treatment of congestive heart failure.
It has been shown that cardiac glycosides inhibit
Na+,K+-ATPase by binding to its
α-subunit from the extracellular
side[26]; however, the detailed binding site has not been
illustrated. Based on molecular modeling and docking, 2
different locations in the α-subunit of
Na+,K+-ATPase were proposed for ouabain binding. Cerri
et al[27] proposed that ouabain penetrated into and bound with transmembrane
regions, that is, both the extracellular loops of the
α-subunit and the interprotodimeric cleft constituted by the
transmembrane helices of both subunits. In contrast, Qiu
et al[28] predicted that ouabain lay on the surface of the
transmembrane ion channel without penetration, that is, ouabain was
mainly located in the area surrounded by some extracellular
loops of α-subunit and a few amino acid residues of the
transmembrane helices exposed to the extracellular space.
Based on the current study, we propose that MLB may
trigger the same molecular mechanism responsible for the
therapeutic effect of cardiac glycosides via the reversible
inhibition on Na+,K+-ATPase. Further studies are indispensable
to see if MLB and cardiac glycosides bind to the same or
different region of Na+,K+-ATPase. Although the inhibition
of MLB on Na+,K+-ATPase is possibly responsible for the
cardiac therapeutic effect of Danshen, the molecular
mechanism triggered by this inhibitory activity is unlikely
responsible for other known medicinal effects of MLB, such as
vasodilating, antihypertensive, antioxidative, and free
radical scavenging activities[5_10].
In a recent study using a cortical brain slice-based
compound screening platform, cardiac glycosides were
demonstrated to provide neuroprotection against ischemic
stroke[14]. In that work, neuroprotective activity and delayed
therapeutic potential were observed for neriifolin as well as other
cardiac glycosides in this brain slice assay model. The same
phenomenon was observed when we examined the
neuro-protective effect of MLB against ischemic stroke in a similar
brain slice assay model (Figures 5, 6). Furthermore, the
protective effects against cerebral ischemia-reperfusion injury
and attenuation of the infarction area in a middle cerebral
artery occlusion animal model by a mixture of total salvianolic
acids extracted from Danshen were also reported
previously[29]. Recent studies indicate that
Mg2+ possesses a neuroprotec-tive effect on brain trauma in
rats[30] and is capable of modulating the L-type calcium channel in the
myocardium[31]. Since MLB is composed of 2 portions, an organic moiety of caffeic
acid tetramer and the cation Mg2+, it is also possible that the
Mg2+ salt-bridged against MLB may partly play a role to
protect the brain against ischemic injury. It remains to be
investigated whether cardiac glycosides and MLB exert
neuro-protection against ischemic stroke via the same mechanism
triggered by the inhibition of
Na+,K+-ATPase.
Although blocking excitotoxicity-induced calcium
overload of neurons after ischemic injury has been the focus of
many therapeutic efforts, abnormally low levels of cytosolic
calcium may cause the death of neuronal cells, as proposed
in the "calcium set-point
hypothesis"[32]. As suggested by Lee
et al[33], calcium starvation and apoptosis may be the
predominant causes of cell death in the penumbra,
particularly at later time intervals. Therefore, it is possible that the
neuroprotection of MLB against ischemic stroke may partly
be a result of an increasing intracellular calcium level through
the inhibition of Na+,K+-ATPase (A diagram was shown on
the cover of this issue).
It is known that the biological mechanisms underlying
brain damage during reperfusion subsequent to ischemia are
presumably attributed to energy failure, free radical damage,
and intracerebral synthesis of the platelet activating factor.
It is possible that the neuroprotection of MLB observed in
this study may have partly resulted from the reduction of
ATP consumption via the inhibition of
Na+,K+-ATPase, consistent with the concept of a defense strategy against
hypoxia proposed by Hochachka[34]. Edema is one of the
major complications of cerebral ischemia, being at the time
an aggravating factor. MLB may limit the formation of
cerebral edema and suppress its neurological changes. In brief,
our results in the present study support the notion that MLB
provides anti-ischemic neuroprotection in gerbils subjected
to focal ischemia and reperfusion. Of course, identification
of the signal transduction pathway downstream from the
inhibition of Na+,K+-ATPase by MLB and assessment of other
stimulatory effects of this potential drug in brain are also
interesting and challenging tasks.
Acknowledgments
We thank Professor Chih-ning SUN for critical reading of
the manuscript and Mr Chi-ming WU for assistance in
preparation of the rat plasma membrane.
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