Fig. 4. RSV inhibits PDGF-BB-induced VSMCs proliferation and migration through Akt and mTOR-mediated autophagy activation.
(A) Western blot analysis for Akt and mTOR phosphorylation. The cells were incubated with rapamycin (100 nM, mTOR inhibitor) for 4 h, and added RSV (40 μM) and PDGF-BB (25 ng/ml) for 48 h. (B) Measurement of LC3-I/II expression using specific signal inhibitors in PDGF-BB-stimulated VSMCs. The cells were incubated with RSV (40 μM), U0126 (10 μM, ERK1/2 inhibitor), MK-2206 (10 μM, Akt inhibitor), and added PDGF-BB for 48 h. (C) MTT assay for RSV, MK-2206, and PDGF-BB-stimulated VSMCs proliferation. The cells were incubated with RSV (40 μM), MK-2206 (10 μM, Akt inhibitor), and PDGF-BB (25 ng/ml) for 48 h. ****p < 0.0001 vs. Con group, ####p < 0.0001 vs. PDGF-BB-treated group. (D) Representative images for PDGF-BB-treated VSMCs migration. The cells were incubated with RSV (40 μM), MK-2206 (10 μM, Akt inhibitor) and PDGF-BB (25 ng/ml) for 48 h. ****p < 0.0001 vs. Con group, ####p < 0.0001 vs. PDGF-BB-treated group. Scale bar: 100 μm. (E) Western blot analysis for MMP-2 and PCNA expression. The cells were incubated with RSV (40 μM), MK-2206, and PDGF-BB (25 ng/ml) for 48 h. Data were analyzed using mean ± SEM. All experimental techniques are described in the Methods section. n = 3 per group. RSV, rosuvastatin; PDGF, platelet-derived growth factor; VSMCs, vascular smooth muscle cells; mTOR, mammalian target of rapamycin; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCNA, proliferating cell nuclear antigen.
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