Baicalin Inhibits The Proliferation Of Vascular Smooth Muscle Cell Induced By PDGF

The proliferation of vascular smooth muscle cells (VSMCs) is the important pathological basis of some vascular proliferative diseases, such as hypertension, atherosclerosis and restenosis. Therefore, it is essential to improve and reverse the vascular remodeling induced by proliferation of VSMCs for treatment of cardiovascular diseases. Baicalin, one of the major flavonoids extracted from the roots of Scutellaria baicalensis Georgi, has been showed that it has the effects on cleaning oxygen free radicals, improving microcirculation and relaxing the blood vessel. However, its exact mechanism of cardiovascular protection and anti-atherosclerosis remains unclear. In the present study, we designed to elucidate the effect of baicalin on the proliferation of in VSMCs induced by PDGF and the related mechanism.Methods:1 VSMC culture5-week-old male SD rats were selected. The VSMCs from rat aorta were isolated and were cultured in DMEM medium containing 10 % FBS. The cells used for the experiment were passage 3 ~ 6. The cells grown into 60 % ~ 70 % confluences were serum-starved, and then the VSMCs were treated with PDGF for 24 h following preincubation by baicalin (5, 10, 20, 40μmol/L) for 24 h.2 Wound healing assaysFor evaluation of the effects of baicalin on VSMC migration, VSMCs were plated in a six-well plate. When the cells grew to 100 % confluent, the monolayer was scratched using a pipette tip. Medium and suspension cells were aspirated, and new medium containing various concentrations of baicalin was added before treated with PDGF for 24 h. Cells were observed under the microscope and the activity of migration was assessed.3 Immunocytochemistry assaysVSMCs were treated by Triton X-100 after being fixed by 4 % paraformaldehyde. The apecific antibodies were added and were then stained by 3,3-diaminobenzidine (DAB). The positive cells were counted under microscope.4 Cell cycle analysis by flow cytometryThe VSMCs were harvested and fixed with 70 % ethanol. Following two more washes with PBS, the cells were stained with 50μg/ml of propidium iodide (PI). Cell cycle distribution was then analyzed by flow cytometry.5 Western blot analysisEqual amounts of protein extracts from VSMCs were separated on 10 % SDS-PAGE, and then blotted onto PVDF membrane. The membrane was immunologically stained with specific antibodies. The results were analyzed by digital imaging system. Results:1 Baicalin inhibits VSMC proliferation induced by PDGF The results of cell counting assays showed that baicalin (5, 10, 20, 40μmol/L) treatment resulted in a significant reduction of VSMC proliferation in a dose-dependent manner.Immunocytochemistry and Western blot analysis showed that expression of PCNA was lower in control group. PDGF (10 ng/ml) stimulation resulted in significant increase in PCNA expression in VSMCs. However, the amount of PCNA was significantly reduced in the baicalin-treated VSMCs (P<0.05), consistent with cell counting results. The results suggested that baicalin inhibited the proliferation of VSMCs induced by PDGF.2 Baicalin inhibits PDGF-induced VSMC migration Wound healing assays demonstrated that VSMC migration was significantly decreased after incubation with baicalin (20μmol/L) compared with the PDGF-treated group. Immunocytochemistry and Western blot analysis showed that expression of ICAM-1, VCAM-1, MMP-2 and OPN induced by PDGF was markedly reduced in baicalin-treated group in a does-dependent manner (P<0.05). The results indicated that baicalin inhibits PDGF-induced VSMC migration by modulating adhesion molecules expression.3 Effect of baicalin on cell cycle in VSMCsThe effect of baicalin on cell cycle progression was determined by flow cytometry. In response to PDGF stimulation, the number of VSMCs distributed in G0/G1 phase was decreased, while the cells in S phase increased dramatically. Treatment with baicalin (5, 10, 20, 40μmol/L), the cell population of G0/G1 phase increased significantly by 5 %, 19 %, 25 % and 29 %, respectively compared with PDGF-group (P<0.05). The results suggest that baicalin arrests the cell cycle of VSMCs stimulated by PDGF in the G0/G1 phase in a does-dependent manner.4 Baicalin inhibits the expression and interaction of cyclinE and CDK2Western blot analysis showed that cyclinE and CDK2 protein levels in VSMCs were down-regulated in response to baicalin preincubation, in a does-dependent manner. Howerer, CDK inhibitor p27 was up-regulated under the same conditions. Furthermore, co-immunoprecipitation analysis showed that baicalin could inhibited interaction between cyclinE and CDK2 in a does-dependent manner.Conclusions:1 Baicalin inhibits the proliferation of VSMCs induced by PDGF.2 Baicalin inhibits VSMC migration induced by PDGF via inhibiting adhesion molecules expression.3 Baicalin blocks the cell cycle of VSMCs stimulated by PDGF in the G0/G1 phase through suppressing cyclinE and CDK2 expression and their interaction between each other.