Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 314395, 7 pages
InhibitoryEffectof GinsenosideRg1 on
PDGF-BB IsInvolved inNitricOxideFormation
Jing Huang,1,2Li-Sheng Li,1Dan-LiYang,1Qi-HaiGong,1
1Department of Pharmacology of Zunyi Medical College and the Key Laboratory of Basic Pharmacology of Guizhou,
Zunyi 563003, China
2Department of Pharmacology, Mindong Medical School, 65 Manchun Road, Fujian, Fuan, 355017, China
Correspondence should be addressed to Xie-Nan Huang, firstname.lastname@example.org
Received 12 October 2011; Accepted 17 December 2011
Academic Editor: Youn Chul Kim
Copyright © 2012 Jing Huang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
to observe the role of nitric oxide (NO) in Rg1-antiproliferative effect. VSMCs from the thoracic aorta of SD rats were cultured
by tissue explant method , and the effect of Rg1 (20mg·L−1, 60mg·L−1, and 180mg·L−1) on platelet-derived growth factor-BB
(PDGF-BB)-induced proliferation was evaluated by MTT assay. The cell cycle was analyzed by flow cytometry. For probing the
mechanisms, the content of NO in supernatant and cGMP level in VSMCs was measured by nitric oxide kit and cGMP radio-
immunity kit, respectively; the expressions of protooncogene c-fos and endothelial NO synthase (eNOS) mRNA in the VSMCs
were detected by real-time RT-PCR; the intracellular free calcium concentration ([Ca2+]i) was detected with Fura-2/AM-loaded
VSMCs. Comparing with that in normal group, Rg1 180mg·L−1did not change the absorbance of MTT and cell percent of
G0/G1, G2/M, and S phase in normal cells (P > 0.05). Contrarily, PDGF-BB could increase the absorbance of MTT (P < 0.01)
and the percent of the S phase cells but decrease the G0/G1phase cell percent in the cell cycle, accompanied with an upregulating
c-fos mRNA expression (P < 0.01), which was reversed by additions of Rg1(20mg·L−1, 60mg·L−1, and 180mg·L−1). Rg1
administration could also significantly increase the NO content in supernatant and the cGMP level in VSMCs, as well as the
eNOS mRNA expression in the cells, in comparison of that in the group treated with PDGF-BB alone (P < 0.01). Furthermore,
Rg1 caused a further increase in the elevated [Ca2+]iinduced by PDGF-BB. It was concluded that Rg1 could inhibit the VSMC
proliferation induced by PDGF-BB through restricting the G0/G1phase to S-phase progression in cell cycle. The mechanisms may
be related to the upregulation of eNOS mRNA and the increase of the formation of NO and cGMP.
Ginsenoside Rg1 (Rg1) is believed to be one of the main
active principles in ginseng (Panax ginseng C. A. Meyer),
a traditional Chinese medicine used to enhance stamina
and capacity to cope with fatigue as well as physical stress.
It has been reported that Rg1 has many beneficial effects
on several systems. For example, in cardiovascular system,
Rg1 can ameliorate the ventricular remodeling induced by
myocardial infarction  and the left ventricular hyper-
trophy induced by abdominal aorta coarctation  in rats
and protect rat cardiomyocyte from hypoxia/reoxygenation
oxidative injury . Also, Rg1 has been reported to inhibit
proliferation of vascular smooth muscle cells (VSMCs)
induced by tumor necrosis factor-α and block the cell cycle
in the G1-phase via depressing the signaling pathways of
ERK, PI3K/PKB, and PKC [4, 5]. However, cell proliferation
is modulated by many factors, more studies should be done
to elucidate the action mechanisms of the antiproliferative
effect of Rg1.
Nitric oxide (NO) has been known to exert many
different vasoprotective effects, such as inhibitions of platelet
2 Evidence-Based Complementary and Alternative Medicine
aggregation , leukocyte chemotaxis , and endothelial
cell apoptosis . For vasculature, mounting evidence has
indicated that NO can inhibit VSMC proliferation in vitro
and prevent the development of intimal hyperplasia after
vascular injury in vivo [9–11]. The inhibitory effect of
S-nitroso-N-acetylpenicillamine and sodium nitroprusside,
the NO donors, on VSMC proliferation was thought via
cGMP-dependent  and cGMP-independent [13–15]
mechanisms. On the other hand, Rg1 has been reported to
cause endothelial-dependent relaxation in the rat aorta ,
enhance endogenous NO production in human umbilical
vein endothelial cells , rat kidney , and in porcine
coronary arteries . Interestingly, some pharmacological
effects of Rg1 have been attributed to endogenous NO pro-
behavior  and its protection against left ventricular
hypertrophy induced by abdominal aorta coarctation in rats
. However, whether the antiproliferative effect of Rg1 on
VSMC involves in endogenous NO production is unknown.
This study aimed to investigate the possible influence of
Rg1 on the cell-cycle progression induced by platelet-derived
growth factor-BB (PDGF-BB) and observe the role of NO in
Rg1 antiproliferative effect on VSMC.
from Beijing Naturally Occurring Drugs Research Insti-
tute, China. Other reagents and their sources were mainly
as follows: platelet-derived growth factor-BB (PDGF-BB)
(Sigma-Aldrich Co. USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-
dye, and Rnase A (Beijing Solarbio Science Technology Co.
Beijing, China); Nitric oxide kit (Jiangsu Beyotime institute
of Biotechnology. Jiangsu, China); cGMP radio-immunity
kit (Beijing Puerweiye Biological Engineering Co. Beijing,
China); β-actin primer endothelial nitric oxide synthase
(eNOS) primer, c-fos primer and Cyclin D1 primer (Dalian
TakaRa Biological Engineering Co. Dalian, China).
Sprague-Dawley rats (150∼200g) were purchased from
the Animal Center of Institute of Surgery Research of the
Third Military Medical University (Chongqing, China).
2.2. Cell Culture and Experimental Design. Rat VSMCs were
isolated from rat thoracic aorta using the explanting tech-
nique . Briefly, the aortic pieces (about 1mm3) removed
the endothelium, and adventitia were cultured in Dulbecco’s
modified Eagle’s medium (Invitrogen Gibco, USA) contain-
ing 20% bovine serum, penicillin 100 U/mL, streptomycin
100μg/mL at 37◦C in a humidified atmosphere of 95% air
and 5% CO2. After about 10 days, the cells were removed
by trypsinization and successively subcultured. Cells from
passages 3 to 10 were used for the experiments.
The VSMCs were randomly divided into six groups:
Normal group (Normal): no drug was added; N + Rg1 group
(N + Rg1): Rg1 180mg·L−1was added in normal cells;
PDGF-BB model group (Model): PDGF-BB 25μg·L−1was
added in normal grown cells; Rg1 low-dose group (Rg1-L),
middle-dose group (Rg1-M), and high-dose group (Rg1-H):
Rg1 20mg·L−1, 60mg·L−1, 180mg·L−1were added,re-
spectively, to the cells treated with 25μg·L−1PDGF-BB. The
were final concentration.
2.3. Assessment of VSMC Proliferation by MTT Assay. The rat
VSMCs were harvested by trypsinization and plated in a 96-
well plate at a density of 1 × 105cells/mL. PDGF-BB and
Rg1 were added to each well as the mention in experimental
design (PDGF-BB was added at 30min after the addition of
Rg1). Briefly, MTT assays were carried out as follow: VSMCs
were grown in 100μL medium at 37◦C under 5% CO2for
24h in 96-well plates, followed by a incubation with 10μL
MTT (5g·L−1) for 4h. Then 100μL (dimethyl sulfoxide)
was read on a Microplate reader.
2.4. Cell-Cycle Analysis Using Flow Cytometry (FCM).
VSMCs were seeded in six-well plastic culture dishes at a
density of 5×105cells/mL, PDGF-BB and Rg1 were added as
MTT assay after cell synchronization and cultured for 24h.
Thereafter, VSMCs were harvested by trypsinization and
collected from the six-well plate, placed in Eppendorf tubes
and washed with 4◦C PBS twice, then fixed with 2mL 70%
ice-ethanol overnight. After centrifugalization, the VSMCs
of VSMCs were determined by PI staining and FCM analysis.
2.5. Real-Time RT-PCR Analysis of c-Fos, and eNOS mRNA.
VSMCs were seeded in 25mL culture flask at a density of
1×105cells/flask, total RNA was isolated by using the TRIzol
(MRC Co., Cincinnati, USA) method after 24h of PDGF-BB
and Rg1 action. Two-step reverse-transcription polymerase
chain reaction was used to detect the expression by iCycle iQ
USA), with SYBR Green PCR Master Mix (ABI Co., Foster,
USA). The Primers were designed with Express Software
and synthesized by TaKaRa Biological Engineering Company
(Dalian, China). The following Primers were used: c-fos
CCC GGC ATC CTT ATT CAA TTA TCA, reverse primer
(5?–3?) GTG TTA TCC CAC AGC ATG TCA ACA G; eNOS
(GenBank accession no. NM 021838): forward primer (5?–
3?) AGC TGG ATG AAG CCG GTG AC, reverse primer (5?–
3?) CCT CGT GGT AGC GTT GCT GA; β-actin (GenBank
accession no. NM 031144): forward primer (5?–3?)GGC
CAA CCG TGA AAA GAT GA, reverse primer (5?–3?) CAG
CCT GGA TGG CTA CGT ACA. The reaction conditions
were 95◦C 10min 1 cycle; 95◦C 15s, annealing temperature
1min, 40 cycles. The threshold cycle (Ct) values of target
genes were normalized with β-actin of the same sample, and
expressed as were relative to controls.
2.6. Measurements of NO Content in Supernatant and cGMP
Level in VSMCs. The measurements of NO content in
supernatant and cGMP level in VSMCs were undertaken
VSMC culture cell synchronization and experimental design
were in accordance with MTT assay.
Evidence-Based Complementary and Alternative Medicine3
2.7. Measurement of the Intracellular Free Calcium Con-
centration ([Ca2+]i) in VSMCs. The cultured VSMCs were
centrifuged at 150g for 5min at room temperature.The
supernatant was discarded, and the cells were washed twice
with Hanks solution (in mmol/L:NaCl 137.0, CaCl2 1.3,
KCl 5.4, MgSO40.8, NaHPO40.38, KH2PO40.44, NaHCO3
4.2, sucrose 5.6, BSA 0.2%; pH 7.4), then the cells were
incubated in Hanks solution containing 3μmol/L Fura-
2/AM for 40min at 37◦C, followed by washing twiced to
remove the extracellular dye. Finally, the cell number was
adjusted to 1 ×10−6cells/mL for measurement of [Ca2+)]i.
The fluorescence value from 1mL cell suspension was
measured by a RF-5000 dual-wavelength spectrofluorometer
(Shimadzu Company, Japan) with excitation wavelengths
at 340/380nm and emission wavelength at 480nm. The
ratio (R) of the fluorescence signals at 340/380 (nm) was
calculated automatically, and the maximal R value (Rmax)
and minimal R value (Rmin) were determined by the addition
of 10μL 10% TritonX-100 (final concentration 0.1%) and
10μL 500mmol/L EGTA (final concentration 5mmol/L),
respectively. The [Ca2+]i was calculated by the formula
described before  [Ca2+)]i= 224 × [(R − Rmin)/(Rmax−
R)] × FD/FS, where FD and FS are the fluorescence
proportionality coefficients obtained at 380nm under Rmin
and Rmax conditions, respectively. The number 224 is the
KD value of Fura-2/AM. To observe the possible influence
of Rg1 on the elevated [Ca2+)]iinduced by PDGF-BB, Rg1
was added before the addition of this growth factor, and the
fluorescence intensity was detected at 5min after PDGF-BB
2.8. Statistical Analysis. All data are presented as mean ±
S.E.M. and were analyzed by the one-way ANOVA followed
by Student’s t-test (two-tails) with the SPSS 13.0 software
(SPSS Inc, Chicago, Illinois, USA), and significance was set
at P < 0.05.
3.1. Effect of Rg1 on MTT Assay in Rat VSMCs. In normal
cells without any treatment of growth factor, Rg1 180mg·L−1
did not change the MTT absorbance, while the addition
of PDGF-BB could significantly increase the absorbance in
MTT assay (P < 0.01), which tended to be inhibited by an
addition of Rg1 20mg·L−1(P > 0.05), and was remarkably
inhibited by Rg1 60mg·L−1and 180mg·L−1(P < 0.05 and
P < 0.01), suggesting the antiproliferating effect of Rg1 on
VSMCs (Figure 1).
3.2. Effect of Rg1 on the Cell Cycle Induced by PDGF-BB in Rat
VSMCs. The results shown in Figure 2 demonstrated that
Rg1 180mg·L−1had no any effect on the growth of normal
cells, while PDGF-BB could significantly increase the percent
of S-phase cells and degrade the G0/G1-phase cell percent in
the cell cycle (P < 0.01) administration of Rg1 (20mg·L−1,
could markedly decrease the S-phase cell percent and
upgrade the G0/G1phase percent in the cell cycle. The data
suggested that Rg1 could inhibit the VSMC proliferation
Normal N + Rg1Model Rg1-LRg1-M Rg1-H
Figure 1: Effect of Rg1 on PDGF-BB-induced VSMC proliferation
(MTT assay, x ± s, n
24h. Normal: vehicle; N+Rg1: Rg1 180mg·L−1; model: PDGF-
BB 25μg·L−1; Rg1-L: Rg1 20mg·L−1+ PDGF-BB 25μg·L−1;
Rg1-M: Rg1 60mg·L−1+PDGF-BB 25μg·L−1; Rg1-H: Rg1
180mg·L−1+ PDGF-BB 25μg·L−1. Data were mean ± S.E.M.
##SignificantdifferencefromnormalcontrolatP < 0.01∗significant
difference from model control at P < 0.05, and
difference from model control at P < 0.01.
= 7). The VSMCs were cultured for
induced by PDGF-BB through restricting the G0/G1-phase
to S-phase progression in cell cycle.
3.3. Effect of Rg1 on the Expression of c-Fos and eNOS mRNA.
The results from real-time RT-PCR assay indicated that
PDGF-BB could elevate the c-fos mRNA expression by about
10 times of that in the normal control, and Rg1 could
significantly blunt the c-fos expression (P < 0.01). On the
other hand, addition of PDGF-BB decreased the expression
of eNOS mRNA by about 67%, and Rg1 could reverse the
decreasing eNOS expression in a concentration-dependent
manner (P < 0.01) (Figure 3).
3.4. Changes of NO Content in Supernatant and cGMP
Level in VSMCs. As shown in Figure 4(a), PDGF-BB could
significantly degrade the content of NO in the supernatant
of the cultured VSMCs (P < 0.01), the addition of the low
concentration of Rg1 (20mg·L−1) tended to increase the
NO content (P > 0.05), and the higher concentrations of
Rg1 (60mg·L−1, 180mg·L−1) could significantly elevate
the content of NO (P < 0.05 or P < 0.01). Consistent with
the changes of NO content in supernatant, the cGMP level in
VSMCs was also decreased by PDGF-BB (P < 0.01) and was
markedly increased by additions of all three concentrations
of Rg1 (P < 0.05 or P < 0.01) (Figure 4(b)).
3.5. Change of [Ca2+]i in VSMCs by Rg1 treatment. The
[Ca2+]i levels of VSMCs were detected at 3 minutes after
the addition of PDGF-BB or at 33 minutes after Rg1-
administration. In our experimental conditions, the basal
[Ca2+]iwas about 120nmol/L in normal VSMCs; it tended
to increase by Rg1 180mg·L−1(P > 0.05), and was elevated
to 182nmol/L by the addition of PDGF-BB 25μg·L−1(P <
0.05). When Rg1 was administered at 30 minutes before
PDGF-BB addition, it could cause a further increase in the
elevated [Ca2+]i induced by this growth factor in a dose-
dependent manner (Figure 5).
4 Evidence-Based Complementary and Alternative Medicine
0 50 100
0 50 100150200
N + Rg1
050 100150 200
0 50100 150 200
0 50 100 150200
0 50 100 150200
N + Rg1
∗ ∗∗ ∗
∗ ∗ ∗ ∗
Figure 2: Effect of Rg1 on the VSMC cell cycle in the presence of PDGF-BB (x ± s, n = 4 ∼ 6,%). A: representative results of flow
cytometry measurements to determine the cell-cycle stages of VSMCs; B:percentage of cells in each phase of the cell cycle. Normal: vehicle;
N+Rg1: Rg1 180mg·L−1; model:PDGF-BB 25μg·L−1; Rg1-L:Rg1 20mg·L−1+ PDGF-BB 25μg·L−1; Rg1-M:Rg1 60mg·L−1+PDGF-
BB 25μg·L−1; Rg1-H:Rg1 180mg·L−1+PDGF-BB 25μg·L−1.Data were mean ± S.E.M.##Significant difference from normal control at
P < 0.01;∗∗significant difference from model control at P < 0.01.
Evidence-Based Complementary and Alternative Medicine5
eNOS mRNA c-fos mRNA
∗ ∗ ∗ ∗
∗ ∗ ∗ ∗
∗ ∗∗ ∗
∗ ∗ ∗ ∗
∗ ∗ ∗ ∗
∗ ∗∗ ∗
Figure 3: Effects of Rg1 on the changes of expressions of c-fos and
eNOS mRNA induced by PDGF-BB in VSMC (x ± s,n = 3); real-
time RT-PCR analysis of c-fos, and eNOS mRNA were performed
model:PDGF-BB 25μg·L−1; Rg1-L:Rg1 20mg·L−1+ PDGF-BB
25μg·L−1; Rg1-M:Rg1 60mg·L−1+PDGF-BB 25μg·L−1; Rg1-
H:Rg1 180mg·L−1+PDGF-BB 25μg·L−1. Data were mean ±
S.E.M.##Significant difference from normal control at P < 0.01;∗∗
significant difference from model control at P < 0.01.
VSMC proliferation has been known to be an impor-
tant component of vessel wall remodelling in response to
injury, such as after angioplasty and during atherosclerosis
formation [23, 24]. The development of highly effective
antiproliferative drugs is necessary for the prevention and
treatment of hypertrophic vascular diseases. Rg1 has been
reported to inhibit TNF-α-induced human arterial smooth
muscle-cell proliferation and cause cell-cycle arrest in G1
phase , and PDGF-BB is known to be a mitogen
involved in the development of vascular proliferative lesions
[25, 26]. In the present study, we further certified the
antiproliferative effect of Rg1 on the VSMCs from rat-
thoracic aorta, using PDGF-BB to induce cell proliferation.
Our study also certified that Rg1 could cause G0/G1 cell
cycle arrest in rat VSMCs, which was very consistent with
the report . Furthermore, VSMC proliferation has been
known to be promoted by the concerted action of several
distinct signal transduction pathways, such as phospholipase
C isoforms and mitogen-activated protein kinase (MAPK)
cascade [27–29]. The final result of these pathways activation
is transcription and activation of the early response genes c-
fos and c-jun. Rg1 has been reported to inhibit the PKC and
MAPK signalings in TNF-α-treated human arterial smooth
muscle cells [4, 5]; in this study, we found that Rg1 could
also significantly inhibit the elevated c-fos mRNA expression
induced by PDGF-BB, which might be the result of the
transduction pathway inhibitions.
Because NO has been shown to play diverse roles in
the physiology and pathophysiology of the cardiovascular
NormalModel Rg1-LRg1-M Rg1-H
The content of NO (µM)
NormalModel Rg1-L Rg1-MRg1-H
Figure 4: Effects of Rg1 on the NO content in supernatant (a) and
cGMP level in cultured VSMCs (b) in the presence of PDGF-BB.
VSMCs were cultured for 24h, the NO content in supernatant and
cGMP level in the cells were determined with the corre- sponding
Kits. Normal: vehicle; model: PDGF-BB 25μg·L−1; Rg1-L:Rg1
20mg·L−1+ PDGF-BB 25μg·L−1; Rg1-M:Rg1 60mg·L−1+
PDGF-BB 25μg·L−1; Rg1-H:Rg1 180mg·L−1+ PDGF-BB
25μg·L−1. Data were mean ± S.E.M.##Significant difference from
normal control at P < 0.01;∗∗significant difference from model
control at P < 0.01.
Normal N + Rg1 ModelRg1-L Rg1-M Rg1-H
Figure 5: Effect of Rg1 on the intracellular free Ca2+concen-
tration ([Ca2+]i) of VSMCs. The [Ca2+]i was detected after 3
minutes of PDGF-BB addition with Fura-2/AM loaded VSMCs,
and Rg1 administrations were performed before 30 minutes
of the addition of PDGF-BB. Normal:vehicle; model:PDGF-
BB 25μg·L−1; Rg1-L:Rg1 20mg·L−1+ PDGF-BB 25μg·L−1;
Rg1-M:Rg1 60mg·L−1+ PDGF-BB 25μg·L−1; Rg1-H:Rg1
180mg·L−1+ PDGF-BB 25μg·L−1. Data were mean ± S.E.M.
#SignificantdifferencefromnormalcontrolatP < 0.05;∗significant
difference from model control at P < 0.05,∗∗Significant difference
from model control at P < 0.01.
6 Evidence-Based Complementary and Alternative Medicine
system, including inhibition of VSMC proliferation ,
and Rg1 has been known to promote the endogenous NO
production in many tissues [16–21], in the present study, we
have emphatically observed the influences of Rg1 on the NO
content in supernatant, cGMP level and [Ca2+]iin VSMCs,
as well as the expression of eNOS in the cells, to clarify the
relationship between the antiproliferative effect of Rg1 and
It is known that NO stimulates cytosolic soluble guanylyl
cyclase to increase cGMP formation, which accompany the
activation of cGMP-dependent protein kinase [30, 31]and it
has also been known that the NO donors, such as S-nitroso-
N-acetylpenicillamine (SNAP) and sodium nitroprusside,
inhibit VSMC proliferation by cGMP-dependent  and
cGMP-independent [13–15] mechanisms. In our study, we
found that both NO content in supernatant and cGMP level
in VSMCs were degraded by addition of PDGF-BB and
elevated by Rg1-treatment. The results seemed to suggest
that the increase in cGMP level was from the NO formation
after Rg1 addition. Consistent with above results, we also
found that Rg1 could significantly upregulate the expression
of eNOS in VSMCs in a concentration-dependent manner.
Taking the above results together, it appears to be possible
that Rg1 promotes the NO formation through upregulating
the NOS (at least for eNOS) expression, and to stimulate
cytosolic soluble guanylyl cyclase to increase cGMP forma-
tion, then the NO itself and cGMP participate the inhibition
on VSMC proliferation. It has been reported that in human
umbilical vein endothelial cells, Rg1 can downregulate miR-
214 (a microRNA related closely to eNOS) expression,
leading to an increase in eNOS expression . Whether
miR-214 downregulation is involved in the up-regulation of
eNOS in this model used in our work remains to be studied.
Notably, the preceding studies indicate that NO inhibits
VSMC proliferation by preventing an increase in cell [Ca2+]i
, and Rg1 has been reported to inhibit Ca2+influx in the
vasoconstrictor agonists and growth factors generally cause
an increase in cytosolic Ca2+within seconds to minutes
, we detected the [Ca2+]ilevel of VSMCs at 3 minutes
after addition of PDGF-BB. Surprisingly, the result showed
that in our experimental conditions, Rg1 (administered
at 30 minutes before PDGF-BB addition) could cause a
further increase in the elevated [Ca2+]iinduced by PDGF-
BB. Obviously, the increased [Ca2+]icould not be attributed
to NO formation, and might be the direct action of Rg1 to
Ca2+influx or Ca2+release from the endoplasmic reticulum.
We conjectured that the NO formation-promoting effect of
addition), and the antiproliferative effect of Rg1 via promot-
ing NO-formation perhaps occurred when larger amount
of eNOS expression had been restored. On the other hand,
some investigators reported that the Ca2+-increasing agents
acid, and di-tert-butylhydroquinone had little effect on
proliferation of VSMCs, and they thought that an increase
in [Ca2+]i per se does not appear to be important in
VSMC replication . What is the relationship between the
Ca2+-increasing effect of Rg1 and its VSMC proliferation-
inhibiting effect remained to be studied.
It is concluded that one of the mechanisms for Rg1
inhibition on VSMC proliferation is related to the up-
regulation of eNOS mRNA and increases the formation of
NO and cGMP.
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