Panaxquin quefolium diolsaponins dose-dependently inhibits the proliferation of vascular smooth muscle cells by downregulating proto-oncogene expression.

OBJECTIVES: Panax quinquefolium saponins (PQS) potentially prevent atherosclerosis in vivo. The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in coronary heart disease and restenosis after percutaneous coronary intervention. Here, we investigated the potential effect of Panax quinquefolium diolsaponins (PQDS), a subtype of PQS, on angiotensin II (AngII)-induced VSMC proliferation.
MATERIALS AND METHODS: Isolated rat VSMCs were identified by immunocytochemical analysis. Cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell cycle and proliferation index were analyzed using flow cytometry. The messenger ribonucleic acid (mRNA) expression of proto-oncogenes was evaluated using reverse transcription polymerase chain reaction.
RESULTS: Over 98% of cultured VSMCs were immunopositive for anti-α-smooth muscle actin. AngII promoted cell proliferation, whereas PQDS significantly suppressed VSMC growth in a dose-dependent manner. Moreover, PQDS suppressed AngII-induced proliferation of VSMCs by arresting the Gap 0/Gap 1 phase. Down-regulated mRNA expressions of proto-oncogenes occurred after PQDS application.
CONCLUSIONS: Our study demonstrates that PQDS may reduce AngII-stimulated VSMC proliferation by suppressing the expression of proto-oncogenes. These results may provide insights for the development of novel traditional Chinese medicines to prevent atherosclerosis.

Introduction

Atherosclerosis, which potentially results in severe coronary heart disease and myocardial infarction, has emerged as a major life-threatening complication and cause of death in developed countries. Percutaneous coronary intervention (PCI) is recognized as an effective and safe form of treatment for single and multivessel coronary atherosclerotic disease. However, following coronary intervention, restenosis (i.e., a reoccurrence in the narrowing of blood vessels) remains an unsolved, yet important, clinical problem.[1] It has been generally accepted that the proliferation of abnormal vascular smooth muscle cells (VSMCs) is one of the most prominent features in the development of atherosclerosis and may contribute to restenosis after PCI. Therefore, after coronary intervention, the inhibition of VSMC proliferation may provide an effective alternative therapy.

Angiotensin II (AngII) is an important component of the renin-angiotensin system, which has been demonstrated to regulate the growth of VSMCs.[2] It has been reported that AngII is able to stimulate VSMC proliferation by promoting the incorporation of 3H-thymidine at concentrations ranging from 10−9 mol/L to 10−6 mol/L.[3] Moreover, growth has been observed to be significantly stimulated when 10−8-10−6 mol/L of AngII is applied. These observations have been confirmed in subsequent studies,[4] resulting in AngII being widely administered to induce VSMC proliferation. AngII stimulates the growth and migration of VSMCs in vitro, through the induction of autocrine growth factors. In addition, the continuous administration of AngII promotes the proliferation of VSMC in the arterial wall of injured rats in vivo.[2] Cell proliferation is controlled by the cell cycle, which comprises four phases: Gap 1 (G1), S (synthesis), Gap 2 (G2) and M (mitosis). In comparison, cells in the Gap 0 (G0) phase are in a quiescent state and do not divide.[5] Moreover, pro-oncogenes, such as c-myc and c-fos, have been shown to regulate the growth of VSMCs.[6] The activation of proto-oncogenes may contribute to AngII-induced VSMC proliferation as the exposure of rat VSMCs to AngII has been found to result in the sequential activation of c-myc and platelet-derived growth factor A-chain messenger ribonucleic acid (mRNA) expression.

A range of pharmacological therapies have been used to suppress the migration and proliferation of VSMCs, including calcium channel antagonists, β-blockers angiotensin converting enzyme inhibitor, rapamycin, taxol and probucol.[78] However, there remains a paucity of information about the safety of these drugs as studies on their effects have primarily conducted in vitro.[78] Traditional Chinese medicine has been demonstrated to be safe and effective at treating various human diseases, possibly due to the use of multiple ingredients that act on multiple-target sites.[91011] However, the effect of traditional Chinese medicine on the suppression of VSMC growth remains unclear. Panax quinquefolium saponins (PQS), which is extracted from the roots, stems and leaves of the North American variety of ginseng (Panax quinquefolium), has been proposed to protect low density lipoproteins from oxidation and may have a potential role in preventing atherosclerosis in vivo.[12] PQS mainly consists of Panax quinquefolium diolsaponins (PQDS) and Pana xquinquefolium tritolsaponins. It has been reported that PQDS has an anti-myocardial ischemic effect on mice. This effect is achieved by decreasing crown arterial resistance and cardiac oxygen consumption and promoting the 86Rb utilization rate. As a result, the blood flow capacity of the cardiac muscle increases, the oxygen consumption capacity of the cardiac muscle reduces, myocardial infarction size diminishes and the activity of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) in blood serum decreases.[131415] However, the effect of PQDS on VSMC proliferation requires investigation.

In the present study, we evaluate the effect of PQDS on VSMC proliferation induced by stimulating AngII.[3] Within this context, diltiazem (Dil), a calcium channel antagonist, which has been reported to inhibit VSMC proliferation,[16] was used as a standard drug. Cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The cell cycle and proliferation index (PI) were analyzed using flow cytometry. The mRNA expression of proto-oncogenes (c-myc, c-fos and c-jun) was assessed using a reverse transcription polymerase chain reaction (RT-PCR). Based on our observations, we determined whether PQDS reduces AngII-induced VSMC proliferation by suppressing the expression of proto-oncogenes (c-myc, c-fos and c-jun). The results are intended to provide a theoretical basis for the clinical application of PQDS in preventing atherosclerosis following coronary intervention.

Materials and Methods

Reagents

RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from Gibco Co., USA. Rabbit anti-α-smooth muscle actin (α-SMA) antibody was obtained from Lab Vision Corp., USA. An anti-rabbit streptavidin-peroxidase kit and a DAB kit were obtained from Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China. All other reagents and chemicals were purchased from Sigma (St. Louis, MO, USA).

Cell Culture and Drug Treatment

Cell culture was conducted following previously described methods.[1718] Briefly, primary VSMCs were obtained from the aortic media of male Sprague-Dawley rats (120-150 g) using the tissue explant technique. Tissue blocks were maintained in the culture medium until VSMCs migrated and became approximately 80% confluent. VSMCs were subsequently transferred to a new culture dish and maintained in RPMI 1,640 medium supplemented with 10% FBS in a humidified 37°C, 5% CO2 incubator. Subcultures in passages 3-6 were used for identification by using immunocytochemical analysis with a rabbit anti-α-SMA antibody. Experiments were conducted using VSMCs in passages 4 and 10. To stimulate the proliferation of VSMCs, 10−7 mol/L AngII was added to the culture medium. For PQDS treatment, various concentrations (25, 50 or 100 mg/L) of PQDS or 0.1 μmol/L Dil were applied, accompanied with 10−7 mol/L Ang II.[19] This study was approved by the ethics committee of the Bethune First Hospital of Jilin University.

Determination of Cell Proliferation

To evaluate cell proliferation, a MTT assay was performed as described previously.[1820] VSMCs were seeded onto a 96-well plate at a density of 103-104 cells/well, 24 h prior to drug treatment. At 48 h after drug incubation, 20 μL of MTT (5 mg/mL) was added to each well. After a further 4 h of incubation at 37°C, the medium was removed and 150 mL dimethyl sulfoxide was added to each well to resuspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 490 nm (A490) using a scanning microplate spectrophotometer (DG-3022A, Huadong Electron Tube Factory, Shanghai, China).

Flow Cytometry Analysis

Cell cycle and proliferation was analyzed using flow cytometry analysis[1821] using FACS Aria (BD Bioscience, San Jose, CA, USA). Briefly, cells were arrested in the G0 phase through 24 h of serum-free medium incubation. After 48 h of drug treatment, cells were collected by centrifugation and fixed in pre-cooled, 70% ethanol for a further 18 h. Subsequently, the cells were adjusted to a density of 1 cells/mL × 106 cells/mL and stained with propidiumiodide at 37°C. At 30 min after incubation, PI-labeled cells were analyzed using flow cytometry. Cell cycle analysis was performed using ModFit Lt3.0 (BD Becton, Dickinson and Company, New Jersey, USA) software. The PI was calculated using the formula: PI = (S + G2/M)/(G0/G1+ S + G2/M).

RT-PCR

The mRNA level of the proto-oncogenes (c-myc, c-fos and c-jun) was examined using RT-PCR on a TC-312 thermal cycler (Techne, Duxford, Cambridge, United Kingdom). Total RNA was extracted using conventional technology and RNA purification was determined by using a spectrophotometer. Subsequently, total RNA was retrotranscribed into double-chained complementary deoxyribonucleic acid (DNA) and used for PCR amplification with the specific primers for the indicated genes [Table 1]. The thermocycling conditions were 95°C for 15 min, followed by 30 cycles at 94°C for 45 s, 55°C for 30 s, 72°C for 60 s and a 10 min final extension step at 72 °C. The amplified products were stored at 4°C until use. The PCR products were confirmed by 2% agarose gel electrophoresis and a gel imaging system was used for gray-scale analysis. The integrated optical density of the target gene/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was recorded.

Table 1

The primers and PCR conditions

Statistical Analysis

Statistical package for the Social Sciences 10 (Statistical package for the Social Sciences company, Chicago, USA) software and expressed as ± s. Statistical significance was assessed using one-way analysis of variance followed by Student-Newman-Keuls test. The significance level was set at P < 0.05.

Results

Primary Culture and Identification of VSMCs

At 3 and 5 d following in vitro culture, the initial migration of VSMCs in the tissue sections was observed. Excessive proliferation occurred with prolonged culture time. As examined by the inverted phase contrast microscope, these cells exhibited a typical, spindle-shaped morphology and a multilayered hill-and-valley growth pattern. The longitudinal axis of the cells ran in a direction that was perpendicular to the tissue margins. Bipolar cells were commonly observed to have a diffuse cytoplasm and round or mitotic nuclei. After 10 d of in vitro culture, a proportion of the cells were aligned in parallel to one another, with an overlapping growth pattern being detected in some regions. Immunostaining for α-SMA identified over 98% of cells as VSMCs. In addition, enhanced immunoactivity of α-SMA was predominately observed in the cytoplasm of the VSMCs with limited nuclear labeling [Figure 1].

Figure 1

Identification of VSMCs using immunocytochemical analysis. Over 98% of cells were α-SMA-immunopositive, confirming the high purification of cultured VSMCs

PQDS Inhibited AngII-induced Cell Proliferation

AngII has been widely used to stimulate the proliferation of VSMCs, both in vitro and in vivo.[2] Cell viability and proliferation was determined using MTT assays. Consistent with previous reports, 48 h of 10−7 mol/L AngII incubation promoted the remarkable growth of VSMCs [Figure 2, P < 0.05 compared to the control]. The standard drug Dil (0.1 μM) caused a major decrease in the growth rate of AngII-stimulated VSMCs (P < 0.05 compared to the AngII treatment group). In addition, the application of 50 or 100 mg/L of PQDS significantly reduced the growth rate of VSMCs stimulated by AngII (P < 0.05 compared to the AngII treatment group). The low PQDS treatment dose (25 mg/L) induced a slight reduction in cell proliferation, but no significant difference was observed (P > 0.05 compared to the AngII treatment group). No significant difference was observed between the Dil and PQDS treatment groups (P > 0.05). These results indicate that PQDS is able to suppress AngII-induced VSMC proliferation in a dose-dependent manner.

Figure 2

Cell proliferation after a 48 h incubation period using MTT assays. VSMCs were incubated with 10-7 mol/L AngII, with or without the application of PQDS (25, 50, and 100 mg/L). The x-axis represents PQDS dose (mg/L); the y-axis represents MTT optical density (OD). A concentration of 0.1 μM Diltiazem (Dil) was used was used as the standard drug. #P < 0.05 compared to the control group; *P < 0.05 compared to the AngII treatment group

Effect of PQDS on the Cell Cycle and PI of VSMCs

Flow cytometric analysis was performed to explore whether the PQDS inhibits cell proliferation by arresting the G0/G1 phase in VSMCs. As shown in Figure 3a-f, the number of cells in the G0/G1 phase decreased following treatment with 10−7 mol/L AngII (67.11 ± 2.56% vs. control 77.57 ± 1.75%, P < 0.05). Meanwhile, AngII elevated the number of cells and PI in the S and G2/M phases [Figure 3g and h]. This result indicates that AngII promotes the transition from the G0/G1 phase to the S phase during the cell cycle progression in VSMCs. In addition, the administration of different PQDS concentrations noticeably elevated the number of cells in the G0/G1 phase (P < 0.05 compared to the AngII group). The application of 50 and 100 mg/L AngII significantly reduced the percentage of cells in the G2/M phase (P < 0.05 compared to the AngII group). In contrast, the application of 25 mg/L AngII slightly decreased the number of cells in the G2/M phase (P > 0.05). Consistent with the MTT results, the effect of PQDS on G0/G1 arrest appeared to be dose-dependent as higher concentrations of PQDS (50 or 100 mg/L) more strongly inhibited VSMC proliferation. In addition, 0.1 μmol/L Dil elevated the number of cells in the G0/G1 phase (P < 0.05) and reduced the percentage of cells in the G2/M phase (P < 0.05), indicating that Dil inhibited growth. Different concentrations of both Dil and PQDS suppressed the AngII-stimulated PI Figure 3h.

Figure 3

Effect of PQDS on the cell cycle and proliferation index of VSMCs. (a-f) are the representative data of the cell cycle analysis for (a) the control, (b) Ang II, (c) Ang II+PQDS (25 mg/L), (d) Ang II+PQDS (50 mg/L), (e) Ang II+PQDS (100 mg/L), and (f) Ang II+diltiazem (0.1 μM), determined by flow cytometry. The number of cells in the G0/G1 phase, the S phase (DNA synthesis phase), and the G2/M-phase (mitosis) are shown. (g) The percentage of cells in each phase using flow cytometry. The x-axis represents the doses of drugs. (h) The proliferation index of cells after PQDS or diltiazem treatment. The x-axis represents the drug doses used. #P< 0.05 compared to the control group; *P < 0.05 compared to the AngII treatment group; ^ P < 0.05 compared to the diltiazem treatment group

Effect of PQDS on the mRNA Level of Proto-oncogenes (c-myc, c-fos and c-jun)

Proto-oncogenes c-myc, c-fos and c-jun may be involved in the mechanism by which PQDS modulates the reduction of AngII-induced cell proliferation in VSMCs. To test this hypothesis, RT-PCR was carried out with specific primers for c-myc, c-fos, c-jun and the GAPDH gene. Enhanced mRNA expression of each proto-oncogene was detected after 48 h of 10−7 mol/L AngII treatment [Figure 4]. A 0.1 μM concentration of Dil caused a noticeable decrease in the levels of these proto-oncogenes (P < 0.05 compared to the AngII group). Treatment with 25, 50 and 100 mg/L PQDS noticeably reduced c-myc and c-jun levels (P < 0.05 compared to the AngII group). Treatment with 25 mg/L PQDS caused a slight decrease in c-fos expression (P > 0.05 compared to the AngII group), while 50 and 100 mg/L PQDS significantly reduced c-fos levels (P < 0.05 compared to the AngII group). Hence, PQDS caused the mRNA expression of c-myc, c-fos and c-jun to be down-regulated in a dose-dependent manner. The results imply that PQDS may reduce AngII-induced VSMC proliferation by inactivating proto-oncogenes.

Figure 4

PQDS reduced the mRNA level of proto-oncogenes. (a) The expression of proto-oncogenes (c-myc, c-fos, and c-jun) was evaluated by RT-PCR. Total RNA was extracted from cultured VSMCs at 48 h after treatment (for PCR amplification primers see Table 1). (b) The mRNA level of proto-oncogenes was quantified relative to the GAPDH level. The x-axis represents the drug doses used. Data was quantified from three independent experiments. #P< 0.05 compared to the control group; *P< 0.05 compared to the AngII treatment group; ^P< 0.05 compared to the diltiazem treatment group

Discussion

The abnormal proliferation and migration of VSMCs is a key event in the development of atherosclerosis and may contribute to restenosis following PCI. Consequently, the inhibition of VSMCs proliferation represents an important therapeutic strategy for preventing these diseases. Endothelial cells exert a large influence on VSMCs by releasing both contracting and relaxing factors, in addition to their ability to synthesize a large number of molecules that influence the growth of VSMCs. Hence, the progression of disease may be suppressed by understanding the underlying mechanism of VSMC proliferation, facilitating the development of drugs to prevent VSMC growth. Existing studies show that traditional Chinese medicines have limited adverse and/or toxic effects in the therapy of atherosclerosis, indicating advantages for their development.

In the present study, AngII was used to stimulate the proliferation of VSMCs. Cell viability and proliferation was determined using MTT assays, from which it was found that the dose-dependent application of PQDS reduced the growth rate of VSMCs stimulated by AngII [Figure 2]. Moreover, PQDS was found to inhibit VSMC proliferation, arresting development in the G0/G1 phase. In contrast, the administration of different concentrations of PQDS noticeably elevated the number of cells in the G0/ G1 phase in a dose-dependent manner [Figure 3]. In general, abnormal VSMC function may induce myocardial ischemia.[22] A previous study showed that PQDS has an anti-myocardial ischemic effect, with the ability to decrease crown arterial resistance, increase cardiac muscle blood current capacity, reduce the oxygen consumption capacity of cardiac muscle, diminish myocardial infarction size and decrease the activities of CPK and LDH in blood serum.[13] Therefore, PQDS may suppress VSMC proliferation, protect against the impairment of coronary circulation and consequently, prevent myocardial ischemia.

It has been demonstrated that extracellular stimuli may initiate the rapid transcriptional activation of various genes, which have been classed together under the rubric of immediate early response genes (IEGs). IEGs mediate a complex cascade of mechanisms, linking membrane stimulation to long-term alterations in cellular phenotype. Accumulating evidence shows that some of these genes, termed proto-oncogenes, serve as key control switches in the regulation of cell growth. For example, c-myc is involved in the G0/G1 transition from quiescence to proliferation and is required for continuous cellular mitogenesis. The nuclear protein fos, which specifically binds to chromosomal DNA, is involved in regulating DNA replication and transcription. The c-jun gene is activated early in response to growth-promoting agents in a wide variety of cell types. Existing research has also detected the up-regulation of mRNA levels for c-myc and c-fos in VSMCs after AngII stimulation. In the current study, the mRNA expression of c-myc, c-fos and c-jun was promoted after 48 h of 10−7 mol/L AngII treatment [Figure 4]. Of note, PQDS caused the downregulation of mRNA expression for proto-oncogenes in a dose-dependent manner, implying that PQDS may reduce AngII-induced VSMC proliferation by suppressing the activities of proto-oncogenes. In this study, we observed that PQDS efficiently suppressed AngII-induced VSMC proliferation after 48 h incubation. Calcium has been reported to mediate cell proliferation and the generation of cell matrices, which represent a common pathway involved in bioactive peptide-regulated cell proliferation.[2324] Intracellular calcium becomes elevated during the mitogenetic process, following induction by growth factors. In addition, a voltage-gated calcium channel contributes to growth factor-induced cell proliferation.[25] Previous studies have indicated that certain panaxa diolsaponins (i.e., Rb1, Rb2, Rb3, Rc and Rd) may block the calcium channel.[2627] As Rb1, Rb2, Rb3 and Rd are the main components of PQDS,[28] it is possible that PQDS may act as a calcium channel inhibitor, suppressing VSMC proliferation by blocking calcium influx after AngII stimulation. By using a whole-cell patch clamp, Zhang et al. found that panaxa diolsaponin Rb1 reduces the L-type calcium channel current in ischemic cardiomyocytes, without influencing the maximum activated voltage and reverse potential.[29] This experiment indicates that Rb1 may decrease the concentration of calcium in the cytoplasm by inhibiting Ca2+ influx. In addition, PQS has been observed to suppress high K+ levels induced by elevated [Ca2+] I; hence, PQS may inhibit the entry of calcium into cells by a voltage-dependent Ca2+ channel.[30]

We found that PQDS could suppress the proliferation of VSMC induced by AngII at 48 h after incubation. However, the proliferation inhibition of PQDS after 36 h, 72 h or longer-term incubation has not yet been determined. Moreover, the proliferation inhibition of PQDS at the dose of 100 mg/L was better than the dose of 50 mg/L and 25 mg/L. Future study will be continued to evaluate the best dose and incubation time of PQDS treatment. In addition, the involvement of apoptosis-related genes (such as bax, p53 and fas) in the suppression of PQDS-mediated growth requires further clarification. In conclusion, this study provides a theoretical basis for the clinical application of PQDS in preventing atherosclerosis.