The incidence of cardiovascular diseases has increased remarkably over the past decade; atherosclerosis, which leads to decreased arterial lumen size, is a major cause of morbidity and mortality [1]. Great efforts have been made to develop efficient therapies to overcome stenosis and occlusion of coronary arteries and to restore normal blood flow. Percutaneous transluminal coronary angioplasty, which has advanced over the past decades, can restore arterial blood flow to the heart.

The most important problem associated with percutaneous coronary intervention (PCI) has been restenosis, which is the major factor hampering the long-term success of this procedure [2]. Drug-eluting stents significantly reduced the restenosis rate to less than 10 % [3, 4]. Nevertheless, drug-eluting stents may be associated with the potential drawback of impairing re-endothelialization and increasing the risk of late thrombosis [5, 6]. Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature in post-PCI restenosis; thus, to explore the regulatory mechanism and to develop a method for inhibiting the proliferation of VSMCs might be a feasible therapeutic strategy for preventing restenosis. Platelet-derived growth factor (PDGF) can be synthesized by VSMCs and vascular endothelial cells after vascular injury. PDGF induces the proliferation of VSMCs and has it been proposed as a key mediator in the progression of atherosclerosis and restenosis after PCI. Therefore, inhibition of PDGF-stimulated VSMC proliferation may represent an effective means of blocking restenosis after PCI.

Magnolol (6,6′,7,12-tetramethoxy-2,2′-dimethyl-1-beta-berbaman) is a compound isolated from the root and bark of Magnolia officinalis and has been reported to have a series of pharmacological properties, e.g., anti-inflammatory, antibacterial, and antitumoral effects [7, 8, 9]. Magnolol has different bioactivities related to VSMCs. Magnolol promotes the release of endothelium-derived relaxing factor and inhibits calcium influx to relax VSMCs [10]. Magnolol has been reported to block the proliferation of VSMCs induced by tumor necrosis factor-α, which was associated with the inhibition of ERK1/2 phosphorylation and reduced expression of cyclin-dependent kinases (CDKs) and cyclins [11, 12]. Magnolol could inhibit the activation of mitogen-activated protein kinase families, including ERK1/2, p38, and JNK [13], which play an important role in the regulation of VSMC proliferation. However, the effects of magnolol on the proliferation of VSMCs induced by PDGF-BB and the related molecular mechanisms remain unknown. Therefore, in this study, we investigated the effect of magnolol on the proliferation of VSMCs induced by PDGF-BB and elucidated the underlying molecular mechanisms. We demonstrate that magnolol inhibits the proliferation and induces cell cycle arrest at the G0/G1 phase by modulating the expression of cell cycle regulators via blocking of ROS production and Ras-MEK1/2-ERK1/2 pathway activation.

Materials and methods

Materials

Recombinant human PDGF-BB was obtained from R&D Systems, Inc. (Minneapolis, Minn., USA). Cell Counting Kit-8 (CCK-8) and Cell Cycle and Apoptosis Analysis kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 5-Bromo-2′-deoxyuridine (BrdU) assays and 2′,7′-dichlorodihydrofluorescein diacetate were purchased from Sigma Chemical (St. Louis, Mo., USA). Total-ERK1/2 and phospho-ERK1/2 antibodies were from Cell Signaling Technology (Danvers, Mass., USA). Glyceraldehyde 3-phosphate dehydrogenase, cyclin D1, cyclin E, and CDKs 2/4 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). For the in vitro study, magnolol was dissolved in dimethyl sulfoxide medium and the final concentration of dimethyl sulfoxide did not exceed 0.1 %.

Cell culture

VSMCs were isolated from the thoracic aorta of Sprague Dawley rats (150–200 g) and digested with 0.2 % collagenase I. Cells were cultured in DMEM, supplemented with 10 % fetal bovine serum and 1 % penicillin-streptomycin, and maintained at 37 °C in a humidified 5 % CO2 incubator. The purity of the VSMC cultures was determined by immunocytochemical localization of α-smooth muscle actin. When the cultures reached confluence, cells were subcultured using 0.25 % trypsin. Cells between passages 3 and 10 were used in this study. VSMCs were grown to 70 and 80 % confluency and serum-deprived for 24 h. VSMCs were treated with different concentrations of magnolol for 1 h prior to stimulation with 20 ng/ml PDGF-BB.

Measurement of cell proliferation, DNA synthesis, and cell viability

The CCK-8 and BrdU assays were performed according to the manufacturer’s protocols. VSMCs were seeded in 96-well plates at a density of 5 × 103/well. Two days later, at 60–70 % confluency, cells were serum-deprived for 24 h, then preincubated for 1 h with various concentrations of magnolol, and stimulated with PDGF-BB (20 ng/ml) for 24 and 48 h. Proliferation and viability were determined using the CCK-8 assay. At 4 h after adding the CCK-8 reagent, the absorbance at 450 nm was measured on a microplate reader. DNA synthesis was determined by measuring the incorporation of BrdU, which was added for the last 2 h of treatment.

Cell cycle progression assay

The proportions of cells in various phases of the cell cycle were measured by flow cytometry. Serum-starved VSMCs were treated with 20 μM magnolol for 1 h prior to stimulation with 20 ng/ml PDGF-BB for 24 h. The cells were digested, washed twice with phosphate-buffered saline (PBS), and then fixed with 70 % ethanol (ethanol + PBS) overnight at 4 °C. Cells were centrifuged at 1,500 rpm for 5 min and then re-suspended in PBS. The Cell Cycle and Apoptosis Analysis Kit was used to determine cell cycle progression.

Real-time reverse transcription polymerase chain reaction

Serum-starved VSMCs were preincubated with magnolol for 1 h and stimulated with PDGF-BB for an additional 24 h. Total RNA was extracted from VSMCs using TRIZol (Invitrogen). Expression levels of the desired genes were quantified by using the ABI Sybr Green PCR Master Mix and an ABI StepOnePlus Real-Time PCR System. The expression levels of the target genes were normalized to that of glyceraldehyde-3-phosphate dehydrogenase.

Western blotting

To assess the protein levels of cyclin D1, cyclin E, CDK2, and CDK4, serum-starved VSMCs were treated for 1 h with different concentrations of magnolol, stimulated with PDGF-BB for an additional 24 h, and then harvested. To determine the protein levels of Ras, Ras-GTP, T-MEK, P-MEK, T-ERK1/2, and P-ERK1/2 (cell signaling), VSMCs were treated for 1 h with different concentrations of magnolol, stimulated with PDGF-BB for 15 min, and then harvested.

Assessment of intracellular reactive oxygen species

Intracellular production of reactive oxygen species (ROS) was measured on a fluorescence microplate reader. After serum starvation for 24 h, VSMCs were treated for 1 h with various concentrations of magnolol, followed by treatment with 20 ng/ml PDGF-BB at 37 °C for 15, 30, or 60 min. After a brief wash, cells were stained with 10 μM of 2′,7′-dichlorodihydrofluorescein diacetate at 37 °C for 30 min, then digested with 0.25 % trypsin, and re-suspended in PBS. Cells were immediately analyzed using a fluorescence microplate reader at excitation and emission wavelengths of 485 and 525 nm.

Statistical analysis

The results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests. A value of p < 0.05 was considered significant.

Results

Magnolol inhibits PDGF-BB-stimulated VSMC proliferation and DNA synthesis

We observed an effect of various concentrations of magnolol (5–20 μM) on the proliferation of VSMCs at 24 and 48 h in the CCK-8 cell proliferation assay. VSMC growth was significantly increased after treatment with 20 ng/ml PDGF-BB for 24 and 48 h compared with the serum-free group, and magnolol inhibited PDGF-BB-induced VSMC proliferation in both a concentration- and time-dependent manner (Fig. 1 a). The BrdU incorporation assay revealed an inhibitory effect of magnolol on VSMC DNA synthesis that was concentration- and time-dependent (Fig. 1 b). To confirm that the test results were not due to magnolol-induced toxicity or damage to the cells, VSMCs were treated with various concentrations of magnolol for 48 h and cell viability was determined using the CCK-8 assay (Fig. 1 c).

Fig. 1
figure 1

Effects of magnolol on proliferation, DNA synthesis, and viability of vascular smooth muscle cells (VSMCs). Serum-starved VSMCs were treated with various concentrations of magnolol followed by stimulation with platelet-derived growth factor (PDGF-BB) for 24 and 48 h. Cell proliferation was measured using the CCK-8 assay (a), and DNA synthesis was determined with the BrdU incorporation assay (b). Cell viability was measured using the CCK-8 assay (c). Data represent the mean ± SD. #p < 0.05 versus control (untreated group); *p < 0.05 versus cells exposed to PDGF-BB; n = 6

Full size image

Magnolol induces G0/G1 phase arrest in VSMCs

Serum-starved VSMCs were treated for 1 h with various concentrations of magnolol before stimulation with 20 ng/ml PDGF-BB for an additional 24 h. Cell cycle analysis of VSMCs treated with PDGF-BB showed an increase in the number of cells in the S and G2 phase. Magnolol arrested PDGF-BB-stimulated cells in the G1 phase. This result indicates that treatment with magnolol results in an appreciable increase in VSMCs in the G1 phase with a concomitant decrease in the proportion of cells in the S phase (Fig. 2).

Fig. 2
figure 2

Effect of magnolol on cell cycle progression. Serum-starved vascular smooth muscle cells were treated with various concentrations of magnolol and then stimulated with platelet-derived growth factor (PDGF)-BB for 24 h. Cell cycle progression was assessed by flow cytometric analysis of the DNA content. Results represent the mean ± SD (n = 3)

Full size image

Magnolol inhibits expression of cyclin D1, cyclin E, and CDK2/4 in PDGF-BB-exposed VSMCs

To elucidate the inhibitory mechanisms of magnolol in the G1-to-S phase arrest, VSMCs were treated for 1 h with various concentrations of magnolol, stimulated with PDGF-BB for 24 h, and then harvested for RT-PCR and Western blotting. PDGF induced significant increases in the mRNA and protein expression levels of cyclin D1, cyclin E, CDK2, and CDK4. Magnolol markedly blocked the mRNA and protein expression of cyclin D1, cyclin E, CDK2, and CDK4 (Fig. 3 a, b).

Fig. 3
figure 3

Effects of magnolol on the mRNA and protein expression of cyclin D1, cyclin E, and cyclin-dependent kinases 2/4 (CDK2/4) in vascular smooth muscle cells (VSMCs). Serum-starved VSMCs were treated for 1 h with various doses of magnolol, stimulated with platelet-derived growth factor (PDGF-BB) for an additional 24 h, and then harvested for reverse transcription-polymerase chain reaction and Western blotting to determine the mRNA (a) and protein (b) levels of cyclin D1, cyclin E, and CDKs 2/4. #p < 0.05 versus control (untreated group); *p < 0.05 versus cells exposed to PDGF-BB; n = 3

Full size image

Magnolol inhibits activation of the Ras-MEK1/2-ERK1/2 pathways

Because the ERK1/2 pathway appears to mediate PDGF-BB-induced cell proliferation of VSMCs, we investigated the effects of magnolol on the ERK1/2 pathway. PDGF-BB induced a significant increase in the activation of Ras, MEK, and ERK1/2 (Fig. 4). Magnolol treatment reduced the expression of Ras-GTP and the level of phosphorylated MEK and ERK1/2 stimulated by PDGF-BB significantly and in a dose-dependent manner (Fig. 4).

Fig. 4
figure 4

Effect of magnolol on the Ras-MEK-ERK1/2 pathways. Serum-starved vascular smooth muscle cells were cultured with various doses of magnolol in the absence or presence of platelet-derived growth factor (PDGF-BB) for 15 min. Protein levels of Ras, Ras-GTP, phospho-MEK, total-MEK, phospho-ERK1/2, and total-ERK1/2 were determined by Western blotting (n = 3)

Full size image

Magnolol inhibits intracellular ROS production

ROS play an important role in cell proliferation and can activate the ERK1/2 pathway. Therefore, we tested whether magnolol could affect PDGF-BB-induced intracellular ROS production. PDGF-BB treatment significantly induced intracellular ROS production in a time-dependent manner (Fig. 5). Magnolol treatment significantly inhibited intracellular ROS production induced by PDGF-BB in a time-and dose-dependent manner (Fig. 5).

Fig. 5
figure 5

Effect of magnolol on platelet-derived growth factor (PDGF)-BB-induced reactive oxygen species (ROS) production. Serum-starved vascular smooth muscle cells were treated for 1 h with various doses of magnolol, followed by culturing in the presence of PDGF-BB for an additional 15, 30, and 60 min. After staining of the cells with 10 μM of 2′, 7′-dichlorodihydrofluorescein diacetate, intracellular ROS production was measured using a fluorescence microplate reader. #p < 0.05 versus control (untreated group); *p < 0.05 versus cells exposed to PDGF-BB; n = 6

Full size image

Discussion

The mechanism by which magnolol inhibits PDGF-BB-induced VSMC proliferation remains poorly understood. In this study, for the first time, we showed that magnolol inhibits proliferation and DNA synthesis induced by PDGF-BB without causing cell cytotoxicity. Flow cytometric analysis showed that magnolol inhibited S-phase entry of VSMCs. We demonstrated that the underlying mechanism by which magnolol inhibits S-phase entry is the inhibition of the mRNA and protein expression of cyclin D1, cyclin E, CDK2, and CDK4. Further analysis showed that magnolol modulates the expression of cell cycle regulators by blocking ROS/Ras/MEK/ERK1/2 signaling pathways. These observations suggest that magnolol could have beneficial protective effects against restenosis after PCI.

Abnormal proliferation of VSMCs plays a critical role in the development of restenosis after PCI. A recent study demonstrated that magnolol inhibits cellular proliferation in various cancer cell lines, including PC-3 (prostate cancer cell line), HL-60 (human promyelocytic leukemia cell line), and MOLT-4 (human acute lymphoblastic leukemia cell line) [14]. Our study demonstrated that magnolol inhibits proliferation and DNA synthesis induced by PDGF-BB in a dose- and time-dependent manner without causing cell cytotoxicity. The cell cycle plays a crucial role in the regulation of cell proliferation. Magnolol has been demonstrated to cause cell cycle arrest [15, 16]. For this reason, we investigated the effect of magnolol on cell cycle progression in VSMCs stimulated by PDGF-BB. Our results demonstrated that magnolol treatment for 24 h resulted in a significant increase in the proportion of cells in the G0/G1 phase and a decrease in the proportion of cells in the S phase in a dose-dependent manner. This finding is consistent with some studies that have shown that magnolol could cause cell cycle arrest in the G0/G1 phase in cancer cells. However, other studies showed that magnolol could cause cell cycle arrest in the G2/M phase. The observed differences may be due to the different cell types used. Next, we investigated whether cell cycle arrest in the G0/G1 phase by magnolol was related to the expression of cell cycle regulators such as cyclin D1 and cyclin E, which form complexes with CDK4 and CDK2 and are essential for progression from the G0/G1 phase to the S phase of the cell cycle. These complexes can promote DNA synthesis through phosphorylation of the retinoblastoma protein [17, 18, 19, 20]. Our experiments indicated that treatment of VSMCs with magnolol resulted in a significant down-regulation of the mRNA and protein expression of cyclin D1, cyclin E, CDK2, and CDK4 in a dose-dependent manner.

Although the mechanism by which magnolol inhibits VSMC proliferation remains unclear, studies showed that activation of ERK1/2 is critical in the PDGF-mediated VSMC proliferation process [21]. Therefore, we investigated the effect of magnolol on the Ras-MEK-ERK1/2 signaling pathway. We found that magnolol inhibited the activation of Ras-MEK-ERK1/2 induced by PDGF-BB. Cyclin D1 is a key molecule in the regulation of the G1/S phase; cyclin D1 transcription is dependent on the activation of ERK [22]. The MEK inhibitor PD98059 can inhibit the expression of cyclin D1 [23]. Furthermore, the ERK1/2 inhibitor U0126 can block the expression of cyclin D1, cyclin E, CDK2, and CDK4 in VSMCs [11]. Magnolol reduced the proliferation of VSMCs and induced cell cycle arrest in the G0/G1 phase by decreasing the expression of cell cycle regulators via blocking of the activation of the Ras-MEK-ERK1/2 signaling pathway.

ROS are intracellular signaling molecules that can activate downstream signals, which are related to cell proliferation and differentiation, e.g., ERK1/2. In addition, they can stimulate the release of transcription factors involved in the regulation of the cell cycle and of proliferation, which is important in the pathogenesis of intimal thickening in atherosclerosis and restenosis. Studies have demonstrated that PDGF can induce intracellular oxidative stress, increase intracellular ROS production, and activate ERK1/2, which can be inhibited by the antioxidant N-acetylcysteine [24, 25]. In this study, we demonstrated that magnolol treatment resulted in a significant decrease in the production of intracellular ROS after PDGF-BB stimulation. Therefore, the inhibitory effects of magnolol on VSMC proliferation are a possible result of its ability to inhibit the activation of ERK1/2 by reducing ROS production induced by PDGF-BB.

Conclusion

In summary, we demonstrated for the first time that magnolol decreases the proliferation of VSMCs induced by PDGF-BB. Moreover, we demonstrated that magnolol inhibited VSMC proliferation by modulating the expression of cell cycle regulators via blocking of ROS production and Ras-MEK-ERK1/2 pathway activation. These findings suggest the potential efficacy of magnolol in the treatment and prevention of atherosclerosis and restenosis.