Inhibitory Effect of Ginsenoside Rg1 on Vascular Smooth Muscle Cell Proliferation Induced by PDGF-BB Is Involved in Nitric Oxide Formation

Ginsenoside Rg1 (Rg1) has been reported to suppress the proliferation of vascular smooth muscle cells (VSMCs). This study aimed 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 (20 mg·L−1, 60 mg·L−1, and 180 mg·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 180 mg·L−1 did 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/G1 phase cell percent in the cell cycle, accompanied with an upregulating c-fos mRNA expression (P < 0.01), which was reversed by additions of Rg1(20 mg·L−1, 60 mg·L−1, and 180 mg·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+]i induced by PDGF-BB. It was concluded that Rg1 could inhibit the VSMC proliferation induced by PDGF-BB through restricting the G0/G1 phase 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.


Introduction
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 [1] and the left ventricular hypertrophy induced by abdominal aorta coarctation [2] in rats and protect rat cardiomyocyte from hypoxia/reoxygenation oxidative injury [3]. 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 [6], leukocyte chemotaxis [7], and endothelial cell apoptosis [8]. 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][10][11]. The inhibitory effect of S-nitroso-N-acetylpenicillamine and sodium nitroprusside, the NO donors, on VSMC proliferation was thought via cGMP-dependent [12] and cGMP-independent [13][14][15] mechanisms. On the other hand, Rg1 has been reported to cause endothelial-dependent relaxation in the rat aorta [16], enhance endogenous NO production in human umbilical vein endothelial cells [17], rat kidney [18], and in porcine coronary arteries [19]. Interestingly, some pharmacological effects of Rg1 have been attributed to endogenous NO production, such as its improving effect on male mice copulatory behavior [20] and its protection against left ventricular hypertrophy induced by abdominal aorta coarctation in rats [21]. 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.
Sprague-Dawley rats (150∼200 g) were purchased from the Animal Center of Institute of Surgery Research of the Third Military Medical University (Chongqing, China).

Cell Culture and Experimental
Design. Rat VSMCs were isolated from rat thoracic aorta using the explanting technique [22]. Briefly, the aortic pieces (about 1 mm 3 ) removed the endothelium, and adventitia were cultured in Dulbecco's modified Eagle's medium (Invitrogen Gibco, USA) containing 20% bovine serum, penicillin 100 U/mL, streptomycin 100 μg/mL at 37 • C in a humidified atmosphere of 95% air and 5% CO 2 . 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: Rg1 20 mg · L −1 , 60 mg · L −1 , 180 mg · L −1 were added, respectively, to the cells treated with 25 μg · L −1 PDGF-BB. The above concentrations of PDGF-BB or Rg1 used in each group were final concentration.

Assessment of VSMC Proliferation by MTT Assay. The rat
VSMCs were harvested by trypsinization and plated in a 96well plate at a density of 1 × 10 5 cells/mL. PDGF-BB and Rg1 were added to each well as the mention in experimental design (PDGF-BB was added at 30 min after the addition of Rg1). Briefly, MTT assays were carried out as follow: VSMCs were grown in 100 μL medium at 37 • C under 5% CO 2 for 24 h in 96-well plates, followed by a incubation with 10 μL MTT (5 g·L −1 ) for 4 h. Then 100 μL (dimethyl sulfoxide) DMSO were added to each well, and an absorbance at 490 nm was read on a Microplate reader.

Cell-Cycle Analysis Using Flow Cytometry (FCM).
VSMCs were seeded in six-well plastic culture dishes at a density of 5 × 10 5 cells/mL, PDGF-BB and Rg1 were added as MTT assay after cell synchronization and cultured for 24 h. 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 2 mL 70% ice-ethanol overnight. After centrifugalization, the VSMCs were washed with PBS twice again. Finally, the DNA contents of VSMCs were determined by PI staining and FCM analysis.

Real-Time RT-PCR Analysis of c-Fos, and eNOS mRNA.
VSMCs were seeded in 25 mL culture flask at a density of 1 × 10 5 cells/flask, total RNA was isolated by using the TRIzol (MRC Co., Cincinnati, USA) method after 24 h of PDGF-BB and Rg1 action. Two-step reverse-transcription polymerase chain reaction was used to detect the expression by iCycle iQ Real-Time PCR Detection System (BIO-RAD Co., California, USA), with SYBR Green PCR Master Mix (ABI Co., Foster, USA). The Primers were designed with Express Software and synthesized by TaKaRa

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 according to the manufacturer's instructions in the assay kits. VSMC culture cell synchronization and experimental design were in accordance with MTT assay. 2+ ] i ) in VSMCs. The cultured VSMCs were centrifuged at 150 g for 5 min at room temperature.The supernatant was discarded, and the cells were washed twice with Hanks solution (in mmol/L : NaCl 137.0, CaCl 2 1.3, KCl 5.4, MgSO 4 0.8, NaHPO 4 0.38, KH 2 PO 4 0.44, NaHCO 3 4.2, sucrose 5.6, BSA 0.2%; pH 7.4), then the cells were incubated in Hanks solution containing 3 μmol/L Fura-2/AM for 40 min at 37 • C, followed by washing twiced to remove the extracellular dye. Finally, the cell number was adjusted to 1 × 10 −6 cells/mL for measurement of [Ca 2+ )] i .

Measurement of the Intracellular Free Calcium Concentration ([Ca
The fluorescence value from 1 mL cell suspension was measured by a RF-5000 dual-wavelength spectrofluorometer (Shimadzu Company, Japan) with excitation wavelengths at 340/380 nm and emission wavelength at 480 nm. The ratio (R) of the fluorescence signals at 340/380 (nm) was calculated automatically, and the maximal R value (R max ) and minimal R value (R min ) were determined by the addition of 10 μL 10% TritonX-100 (final concentration 0.1%) and 10 μL 500 mmol/L EGTA (final concentration 5 mmol/L), respectively. The [Ca 2+ ] i was calculated by the formula described before [22] where FD and FS are the fluorescence proportionality coefficients obtained at 380 nm under R min and R max conditions, respectively. The number 224 is the K D value of Fura-2/AM. To observe the possible influence of Rg1 on the elevated [Ca 2+ )] i induced by PDGF-BB, Rg1 was added before the addition of this growth factor, and the fluorescence intensity was detected at 5 min after PDGF-BB addition.

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.

Effect of Rg1 on MTT Assay in Rat VSMCs.
In normal cells without any treatment of growth factor, Rg1 180 mg · 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 20 mg · L −1 (P > 0.05), and was remarkably inhibited by Rg1 60 mg · L −1 and 180 mg · L −1 (P < 0.05 and P < 0.01), suggesting the antiproliferating effect of Rg1 on VSMCs (Figure 1).

Effect of Rg1 on the Cell Cycle Induced by PDGF-BB in Rat
VSMCs. The results shown in Figure 2  induced by PDGF-BB through restricting the G 0 /G 1 -phase to S-phase progression in cell cycle.

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). 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 (20 mg · L −1 ) tended to increase the NO content (P > 0.05), and the higher concentrations of Rg1 (60 mg · L −1 , 180 mg · 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)). 2+ ] i in VSMCs by Rg1 treatment. The [Ca 2+ ] i levels of VSMCs were detected at 3 minutes after the addition of PDGF-BB or at 33 minutes after Rg1administration. In our experimental conditions, the basal [Ca 2+ ] i was about 120 nmol/L in normal VSMCs; it tended to increase by Rg1 180 mg · L −1 (P > 0.05), and was elevated to 182 nmol/L by the addition of PDGF-BB 25 μg · 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 [Ca 2+ ] i induced by this growth factor in a dosedependent manner ( Figure 5).

Discussion
VSMC proliferation has been known to be an important 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 [4], and PDGF-BB is known to be a mitogen involved in the development of vascular proliferative lesions observed in atherosclerosis and in restenosis after angioplasty [25,26]. In the present study, we further certified the antiproliferative effect of Rg1 on the VSMCs from ratthoracic aorta, using PDGF-BB to induce cell proliferation. Our study also certified that Rg1 could cause G 0 /G 1 cell cycle arrest in rat VSMCs, which was very consistent with the report [4]. 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][28][29]. The final result of these pathways activation is transcription and activation of the early response genes cfos 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 system, including inhibition of VSMC proliferation [9], and Rg1 has been known to promote the endogenous NO production in many tissues [16][17][18][19][20][21], in the present study, we have emphatically observed the influences of Rg1 on the NO content in supernatant, cGMP level and [Ca 2+ ] i in VSMCs, as well as the expression of eNOS in the cells, to clarify the relationship between the antiproliferative effect of Rg1 and NO formation. 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 [12] and cGMP-independent [13][14][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 formation, 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 [32]. 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 [Ca 2+ ] i [33], and Rg1 has been reported to inhibit Ca 2+ influx in the neurons suffered from hypoxic-ischemic injury [34]. Because vasoconstrictor agonists and growth factors generally cause an increase in cytosolic Ca 2+ within seconds to minutes [33], we detected the [Ca 2+ ] i level 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 [Ca 2+ ] i induced by PDGF-BB. Obviously, the increased [Ca 2+ ] i could not be attributed to NO formation, and might be the direct action of Rg1 to Ca 2+ influx or Ca 2+ release from the endoplasmic reticulum. We conjectured that the NO formation-promoting effect of Rg1 might be not strong enough to reduce the [Ca 2+ ] i level of VSMCs at the Ca 2+ -detection time(3 minutes after PDGF-BB addition), and the antiproliferative effect of Rg1 via promoting NO-formation perhaps occurred when larger amount of eNOS expression had been restored. On the other hand, some investigators reported that the Ca 2+ -increasing agents including Ca 2+ ionophore A23187, ionomycin, cyclopiazonic acid, and di-tert-butylhydroquinone had little effect on proliferation of VSMCs, and they thought that an increase in [Ca 2+ ] i per se does not appear to be important in VSMC replication [35]. What is the relationship between the Ca 2+ -increasing effect of Rg1 and its VSMC proliferationinhibiting effect remained to be studied.
It is concluded that one of the mechanisms for Rg1 inhibition on VSMC proliferation is related to the upregulation of eNOS mRNA and increases the formation of NO and cGMP.