Evodiamine inhibits PDGF‑BB‑induced proliferation of rat vascular smooth muscle cells through the suppression of cell cycle progression and oxidative stress

Abstract

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development of in‑stent restenosis. Evodiamine is an indole alkaloid extracted from the Chinese medicine, evodia, and has been shown to inhibit tumor cell proliferation and protect the cardiovascular system. However, whether evodiamine affects VSMC proliferation remains to be elucidated. Therefore, the present study examined the effects and the mechanisms of action of evodiamine on the proliferation of rat VSMCs. The cells were treated with evodiamine alone or in combination with platelet‑derived growth factor‑BB (PDGF‑BB) stimulation. It was found that evodiamine inhibited PDGF‑BB‑induced VSMC proliferation in a dose‑dependent manner, without inducing cell death. Evodiamine also retarded cell cycle progression, evidenced by the suppression of the expression of cell cycle‑promoting cyclin proteins and cyclin‑dependent kinases. In addition, evodiamine attenuated the PDGF‑BB‑induced phosphorylation of mitogen‑activated protein kinases p38 and extracellular signal‑regulated kinases 1/2, however, it had no effect on the phosphorylation of Akt. Evodiamine also inhibited the increase of reactive oxygen species generation and upregulated the mRNA expression levels of genes encoding antioxidant enzymes. These findings provide important insights into the mechanisms underlying the vasoprotective actions of evodiamine and suggest that it may be a useful therapeutic agent for the treatment of vascular occlusive disease.

Figure 1.

Evodiamine inhibits PDGF-BB-induced VSMC proliferation. (A) Chemical structure of evodiamine. To measure cell toxicity, (B) VSMCs were treated with 0.1, 0.5, 1, 2 or 4 µM evodiamine for 30 h, followed by a CCK-8 analysis. To measure cell proliferation, VSMCs were pretreated with 0.1 or 0.5 µM evodiamine for 6 h and then stimulated with 10 ng/ml PDGF-BB for 24 h. Cell proliferation was determined using a (C) CCK-8 assay, (D) direct cell counting and an (E and F) EdU incorporation assay. Magnification, ×200. Data are presented as the mean + standard deviation of three independently prepared samples, each with six measurements. *P<0.05 and **P<0.01, compared with the control group; ^##P<0.01, compared with the PDGF-BB-stimulated group. Evo, evodiamine; PDGF-BB, platelet-derived growth factor-BB; VSMCs, vascular smooth muscle cells; OD, optical density.

Figure 2.

Evodiamine inhibits cell cycle progression. Vascular smooth muscle cells were pretreated with 0.5 µM evodiamine for 6 h and then stimulated with 10 ng/ml PDGF-BB for 24 h. (A) Cell cycle phase distributions were assessed using flow cytometry. (B) Results of the statistical analysis. Data are presented as the mean + standard deviation of three independently prepared samples, each with five measurements. **P<0.01, compared with the control group; ^##P<0.01, compared with the PDGF-BB-stimulated group. Evo, evodiamine; PDGF-BB, platelet-derived growth factor-BB.

Figure 3.

Evodiamine affects the protein expression levels of key regulators involved in cell cycle progression. Vascular smooth muscle cells were pretreated with 0.5 µM evodiamine for 6 h and then stimulated with 10 ng/ml PDGF-BB for 24 h. (A) Representative image from three separate experiments. GAPDH was used as an internal control. (B-I) Graphs of the results of statistical analyses of protein expression levels. Data are presented as the mean + standard deviation of three independent experiments. *P<0.05 and **P<0.01, compared with the control group; ^#P<0.05 and ^##P<0.01, compared with the PDGF-BB-stimulated group. Evo, evodiamine; PDGF-BB, platelet-derived growth factor-BB; PCNA, proliferating cell nuclear antigen; CDK cyclin-dependent kinase.

Figure 4.

Evodiamine inhibits PDGF-BB-induced kinase activation. Vascular smooth muscle cells were pretreated with 0.1 or 0.5 µM evodiamine for 24 h and then stimulated with 10 ng/ml PDGF-BB for 15 min. The protein expression levels of phosphorylated and total kinases were examined using qestern blot analysis. (A) Representative image from three separate experiments. GAPDH was used as an internal control. (B-D) Signal ratios of phosphorylated kinases to total kinases were calculated. Data are presented as the mean + standard deviation of three independent experiments. **P<0.01 compared with the control group; ^#P<0.05 and ^##P<0.01, compared with the PDGF-BB-stimulated group. Evo, evodiamine; PDGF-BB, platelet-derived growth factor-BB; ERK, extracellular signal-regulated kinase; p-, phosphorylated.

Figure 5.

Evodiamine ameliorates PDGF-BB-induced oxidative stress in VSMCs. VSMCs were pretreated with 0.1 or 0.5 µM evodiamine for 24 h, and then stimulated with 10 ng/ml PDGF-BB for 1 h. (A) ROS detection using 2′ 7′ dichlorofluorescin diacetate staining. Magnification, ×200. (B) Quantitative data of five independent experiments, expressed as the fold increase, compared with the control. (C-F) mRNA expression levels of antioxidant genes were assessed using reverse transcription-quantitative polymerase chain reaction analysis. Data are presented as the mean+ standard deviation of three independent experiments. *P<0.05 and **P<0.01, compared with the control group; ^##P<0.01, compared with the PDGF-BB-stimulated group. Evo, evodiamine; PDGF-BB, platelet-derived growth factor-BB; VSMCs, vascular smooth muscle cells; ROS, reactive oxygen species; HO-1, heme oxygenase-1; GPx-1, glutathione peroxidase 1; SOD, superoxide dismutase.