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Introduction
Several natural products have been used for vascular diseases and some traditional herbal prescriptions have been
employed for treatment of atherosclerosis. Ginsenosides
are the most important active constituents identified in
ginseng[1], which can protect against
atherosclerosis[2]. Ginsenoside Rg1, a steroidal saponin of high abundance in ginseng, can relax
the rat aorta and enhance NO
production[2_3]. Gene chip analyses find ginsenoside Rg1 can affect the expression levels of
genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in
TNF-a stimulated endothelial cells[4]. Atherosclerosis is the principal vascular lesion which is initiated by vascular endothelial cell
damage followed by an intimal hyperplasia of VSMC. VSMC change their phenotype from a contractile to a synthetic state
and actively proliferate in the intima of atherosclerotic arteries, secreting excess amounts of extra-celluar matrix components
including collagen and
proteo-glycans[5]. Thus inhibition of VSMC hyperpro-liferation is
one of the key pharmacological strategies for prevention of atherosclerosis.
TNF-a, a cytokine that is secreted by VSMC in the atherosclerotic neointima,
can promote cell growth and movement, both of which are
critical for the initiation and progression of vascular
lesions[6]. An investigation of ginsenoside Rg1-induced alterations in protein expression in
TNF-a-activated VSMC will aid in understanding the molecular mechanism of ginsenoside Rg1.
Proteomics describe all the proteins encoded from a specific
genome[7]. Proteomic analysis involves the qualitative
alterations in proteins along with the quantitative changes in protein expression levels that occur in response to a given set
of conditions[8]. The rapid increase in proteomic-based drug studies stems from the potential benefits associated with the
elucidation of drug mechanisms.
As little is known regarding the vascular molecular mechanism of ginsenoside Rg1 on VSMC and because proteomic
technique has more advantages in molecular identification, we attempted to use proteomic analysis to explain the
molecular mechanism of ginsenoside Rg1 on the proliferation of cultural VSMC.
Materials and methods
Reagents and materials Electrophoresis reagents including acrylamide, methylenebisacrylamide,
N,N,N¡¯,N¡¯,-tetramethylethyl-diamide (TEMED), hydroxymethyl aminomethane (Tris), glycine, sodium dodecyl sulfate (SDS), dithiothreitol
(DTT), 3-[3-(cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate (CHAPS), Immobiline DryStrips, Immobilized pH
gradient (IPG) buffer, IPG coverfluid and low molecular marker were purchased from Amersham Biosciences (Sweden).
Iodoacetamide and trifluoroacetic acid (TFA) were purchased from Acros (Geel, Belgium). Trypsin was obtained from
Boehringer Mannheim (GE, Germany).
TNF-a, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfonyl)
-2H-tetrazolium (MTS) was obtained from Sigma (St Louis, Mo, USA), ginsenoside Rg1 was obtained from Beijing Institute of Biological Products (Beijing, China),
Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) was purchased from Gibco (Los Angeles, USA). Male Sprague-Dawley rats
were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China).
Cell culture Primary VSMC cultures were isolated after removal of the adventitia and scraping of the endothelium.
Arterial strips were then incubated in DMEM with 20% fatal bovine serum (FBS), penicillin 100 U/mL, streptomycin 100
mg/L at 37 ºC in 5% CO2 humidified atmosphere. VSMC were propagated in DMEM with 10% FBS in subsequent passages.
Mean incubation time was 5 d. Cell viability was confirmed by Trypan blue exclusion after each passage and
assay[9]. All assays were conducted with subconfluent cells from passages 3_6.
Proliferation assay VSMC were detached using trypsin and seeded at a density of 5000 cells/well in a 96-well plate and
rendered quiescent. Then VSMC were incubated in DMEM with various concentrations of ginsenoside Rg1 and
TNF-a for 48 h, and 20 µL of MTS was added. The cultures were incubated for a further 1.5 h, after which the absorbance was read in
a multiplate reader. MTS assay is a colorimetric method for determining the number of viable cells in proliferation. It is
composed of a solution of tetrazolium, which is bioreduced by metabolically active cells into a soluble formazan product in
culture medium. Its absorbance can be measured at 490 nm. The quantity of formazan produced is directly proportional to
the number of living cells.
Flow cytometry Aliquots of
5×105 VSMC were centrifuged at
200×g for 5 min, and cell pellets were fixed with 70% ethanol
overnight. The cells were then washed twice with PBS and incubated with 1 g/L RNaseA for 30 min. The cells were then
resuspended in 0.5 mL of solution containing 50 mg/L propidium iodide and stored in the dark for 20 min. Cells were analyzed
on a FACScan Flow Cytometer (FACSCalibur; Becton Dickinson, CA, USA).
Protein extraction and determination of concentrations
Following growth arrest, VSMC were incubated in DMEM with
various concentrations of ginsenoside Rg1 and
TNF-a for 24 h. Cells were then trypsinized and washed with PBS buffer;
1×107 cells were added to 100 µL lysis buffer (8 mol/L urea, 4 % CHAPS, 40 mmol/L Tris base). After a few cycles of quick
freezing and subsequent thawing in liquid nitrogen, 4 µL RNase (10 g/L) was added. After deposition for 20 min, the cells
were centrifuged at 12 000×g for 30 min. The supernatant was transferred and stored. Protein concentrations were
determined using the Bradford assay.
Two-dimensional electrophoresis (2-DE) and image analysis
Isoelectric focusing (IEF) was carried out on gel strips as
described by Ying et al[10]. After IEF separation, the gel strips were immediately equilibrated for two steps in equilibrium
buffer, and the second dimension separation was performed using ETTAN DALT six (Amersham Biosciences). The gels
were stained and scanned with an ImageScanner (Amersham Biosciences) in transmission mode. Spot detection and
matching were performed using ImageMaster 2D Elite 4.01 (Amersham Biosciences). The gel of
TNF-a treated cells was selected as a reference gel. Other gels were matched with the reference gel. Values of spot abundance were normalized and
exported for statistical analysis.
In-gel digestion and MALDI-TOF-MS
identification In-gel digestion of proteins from 2D gels was carried out as
described by Steiner et al[11]. Spots were excised and destained with 25 mmol/L ammonium bicarbonate/50% acetonitrile, then
dried in a vacuum concentrator (Savant, Holbook, NY, USA). The dried gel pieces were rehydrated with 5
mL of 20 mg/L trypsin solution, and digested at 37 ºC for 18_20 h. Tryptic peptides were extracted using 5% TFA and dried in a vacuum
concentrator. MS was performed on a Reflex III MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).
Database searching was performed in the NCBI database and pI were acquired from image analysis as described by Lim
et al[12].
RNA isolation and reverse transcriptase polymerase chain reaction
(RT-PCR) Total RNA was extracted from
cultured cells according to the supplied protocol. Total RNA was reverse transcripted at 42 ºC for 1 h using reverse
transcriptase. The cDNA was amplified using glyseralde-hyde-3-phosphate dehydrogenase(GAPDH) cDNA as an internal
control for the amplification of p21 and protein kinase
C-z (PKC-z) expressed by the cells. Primers for the GAPDH were as
follows: sense: 5¡¯-ACCACAGTCCATGCC-ATCAC-3¡¯; antisense: 5¡¯-TCCACCACCCTGTTGCTGTA-3¡¯. Primer sequences for
p21 were as follows: sense 5¡¯-CCGATA-CAGGTGATGATGATGG-3¡¯, antisense: 5¡¯-GCCAGAAGTG-AAGCCAAGG-3¡¯; Primer
sequences for PKC-z were as follows: sense: 5¡¯-GCCTCCAGTAGATGACAAGAAC-3¡¯, antisense:
5¡¯-GAGTGTAAGCCAACCAGGAAG-3¡¯. The sample was amplified for 28 cycles with the following parameters: 94 ºC 30 s, 58 ºC
20 s, 72 ºC 30 s. The PCR products were subjected to 2% agarose gel electro-phoresis. Quantitative data normalized to
GAPDH were obtained from a densitometer and analyzed with the included Quantity One 4.4.0 software (BIO-RAD, USA).
Statistical analysis Data was reported as mean±SD. One-way ANOVA or Student¡¯s
t-test was used for statistical analysis of the original data, and significance was accepted at
P<0.05.
Results
Effects of ginsenoside Rg1 on proliferation of
TNF-a stimulated VSMC Following growth arrest, VSMC were
stimulated with TNF-a for 24 h.
TNF-a induced VSMC proliferation significantly compared with the control. However, ginsenoside
Rg1 inhibited VSMC growth at doses as low as 20 mg/L (Figure 1). This observation was not a result of cell necrosis as
ginsenoside Rg1-treated cells remained greater than 90% viable after Trypan blue staining. The effects of ginsenoside Rg1
on cell cycle progresssion were determined by flow cytometry (Table 1). Quiescent VSMC was induced to enter S phase by
stimulation with TNF-a. The population of
G0/G1 cells decreased with a significant concomitant rise in S phase cells.
Ginsenoside Rg1 significantly inhibited VSMC from
G1 to S progresssion, as shown by the increase in
G0/G1 cells accompanied by a concurrent decrease in S phase cells. Therefore, ginsenoside Rg1 not only inhibited VSMC proliferation, but also
arrested VSMC in G0/G1.
Protein expression in normal, TNF-a-stimulated and ginsenoside Rg1-treated VSMC
Protein samples extracted from normal, TNF-a-stimulated and ginsenoside Rg1-treated VSMC were loaded on IPGphor gel strips for IEF. Vertical
SDS-PAGE was performed immediately following IEF. This experiment was repeated three times. A representative batch of gels
is shown in Figure 2. Two dimentional gels of normal,
TNF-a-stimulated and ginsenoside Rg1-treated VSMC were analyzed
for the purpose of quantitative spot comparisons. Analyzed results showed 845±22 spots in a normal cell map, 892±242
spots in a TNF-a-stimulated cell map, and 879±21 spots in a ginsenoside Rg1-treated cell map. The gels of
TNF-a stimulated cells were selected as reference gels, the gels of normal and ginsenoside Rg1 treated cells were matched to reference gels.
Differential analysis showed 24 protein spots were different in intensity, including 17 spots that were increased and 7 spots
that were decreased after TNF-a stimulation. Ginsenoside Rg1 treatment could prevent this change or reverse it to some
degree (Figure 2, Table 2).
Identification of proteins associated with different
expressions in normal and ginsenoside Rg1-treated VSMC compared with
TNF-a-stimulated VSMC by PMF The differently expressed spots were excised from stained Coomassie Brilliant R-250 gels and submitted to in-gel tryptic digestion.
The resulting peptide mixtures were analyzed by MALDI-TOF-MS. The resulting peptide mass maps were used to search
against a comprehensive protein sequence database, typical peptide mass maps were listed in Figure 3. Table 3 lists the
proteins expressed differently in normal and ginsenoside Rg1-treated VSMC compared with
TNF-a-stimulated VSMC. We identified 8 proteins by database searching according to identified
standards[12]. Other proteins need to be investigated
further because of their poor score. The expression of G-protein coupled receptor kinase,
PKC-z,
N-ras protein were increased, while cycle related protein p21, was decreased by
TNF-a compared with normal VSMC. Ginsenoside Rg1 could restore the expression levels of these proteins, at least in part, to basic levels of untreated cells.
Effects of ginsenoside Rg1 on mRNA expression in VSMC
TNF-a down-regulated more than 60% of p21 mRNA
expression compared with normal VSMC, while ginsenoside Rg1 upregulated more than 50% of p21 mRNA expression
compared with TNF-a stimulated VSMC. The effects of ginsenoside Rg1 on
PKC-z mRNA expression in VSMC had contrary changes.
TNF-a upregulated PKC-z to more than 40% of mRNA expression compared with normal VSMC, while ginsenoside
Rg1 down-regulated PKC-z to more than 17% of mRNA expression compared with
TNF-a stimulated VSMC (Figure 4).
Discussion
VSMC in healthy vessel walls have low mitogenic
activity. During the early stages of arterial wall injury or
athero-sclerosis, VSMC may undergo a phenotypic
transformation[13]. They become activated and then proliferate and migrate to the
intimal layer where they accumulate lipids and participate in plaque formation
atherosclerosis[14]. The inhibition of this
process is considered to be of great benefit in the maintenance of vascular homeostasis and in the prevention and
development of atherosclerosis. Both ischemic and
direct vascular injury result in the elaboration of proinflam-matory substances, including
TNF-a, which regulate VSMC proliferation and promote vessel
stenosis[15]. Other investigators have suggested that
TNF-a induces VSMC proliferation[16]. In our present study, we found that
TNF-a stimulated VSMC proliferation. Ginsenoside Rg1 is a major ingredient used
in many traditional Chinese medicines to cure atherosclerosis. It has biphasic regulation and little adverse effect. Our results
suggest that ginsenoside Rg1 inhibits the proliferation of VSMC in a dose-dependent manner and this action is not due to
a cell-toxicity effect.
There have been no previous studies to report that ginsenoside Rg1 has the action to inhibit proliferation of VSMC.
Cell-cycle analysis using flow cytometry confirmed the results of MTS methods. Flow cytometry results showed that
ginsenoside Rg1 arrested the VSMC cell cycle at
G1 in growing-phase cells and inhibited the cell cycle progression from
G0/G1 to S phase in
G0-arrested cells. These results suggest that the inhibitory effect of ginsenoside Rg1 is exerted at a point in
the G1 phase. It has been reported that cyclin dependent kinase (CDK) promote
G1/S transition and that p21 is an intrinsic
inhibitor of the kinase activity of
CDK[17]. At 40 mg/L, ginsenoside Rg1 increased the p21 protein level while producing
G1 arrest. RT-PCR results suggested ginsenoside Rg1 increased p21 mRNA levels. These results indicate that the induction of
p21 by ginsenoside Rg1 is due to an induced increase in its mRNA level. It has recently been reported that the migration of
VSMC can be inhibited by an overproduction of p21 in cultured
VSMC[18], thus the induction of p21 by ginsenoside Rg1 may
be one of the mechanisms by which ginsenoside Rg1 exerts its action on migration activity.
PKC could mediate activation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK)
in VSMC. The proliferation of VSMC is mediated by the MAPK
pathway[19]. The down-regulation of
PKC-z by ginsenoside Rg1 may be one of the mechanisms that inhibit proliferation.
PKC-z, an atypical PKC that are Ca2+-independent and do not
require diacylglycerol for activation, can phosphorylate and decrease the half-life of
p21[20]. PKC also play a role in p21 mRNA
stability[21], which hints that activation of
PKC-z results in a decrease of the p21 level. The induction of p21 and
inhibition of PKC-z may be the mechanism by which ginsenoside Rg1 inhibits VSMC proliferation.
Because upregulation of N-ras activated MAPK pathway and induced
inflammation[22_23], ginsenoside Rg1 could
downregulate the N-ras level, which suggests it exerts anti-inflammation and anti-proliferation effects by the N-ras pathway.
The present results suggest that ginsenoside Rg1 inhibits the proliferation of VSMC through an induction of p21 and
inhibition of PKC-z. The above results suggest that ginsenoside Rg1 is proved to be a valuable drug to cure atherosclerosis.
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