OBJECTIVETo observe the effect of extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chuanxiong (EXT) on delaying vascular smooth muscle cells (VSMCs) aging in aged rats.

METHODSVSMCs were obtained by the modified tissue explants technique and were shown to be positive for smooth muscle α-actin (SM-α-actin) by immunohistochemistry staining. VSMCs obtained from the young rats were served as the young control group; VSMCs obtained from the old rats were treated with no drug (the old group), with low dose extracts (20 mg/L, the EXT low-concentration group) and high dose extracts (40 mg/L, the EXT high concentration group), and with Probucal (10(-6) mol/L, the Probucal group) as a positive control. All groups were cultured for 24 h in the medium with 10% serum for 24 h followed by another 24 h in the serum-free medium. At the end of the 48-h culture, the following analyses were performed including determination of senescence-associated β-galactosidase (SAβ-Gal) activity, flow cytometry analysis of cell cycle, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses of p16, Cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma (Rb) mRNA expression, and Western blotting analyses of p16, cyclin D1, CDK4 and phosphoretinoblastoma (pRb) protein expressions.

RESULTS(1) In comparison to the younger rats, VSMCs from aged rats had significantly more SAβ-Gal positive cells (P<0.01) and more cells in S phase (P<0.05). VSMCs from the all treated groups showed a significant decrease in both SAβ-Gal positive cells (P<0.05) and S phase (P<0.05) compared to the old rats. (2) Compared with the young group, VSMCs in the old group had a significant decrease in p16 and Rb mRNA expression and a significant increase in Cyclin D1 and CDK4 mRNA expression. Compared with the old group, VSMCs in the treated groups had a significant increase in p16 and Rb mRNA expression and a significant decrease in Cyclin D1 and CDK4 mRNA expression (P<0.05). (3) Compared with the young group, VSMCs in the old group had a significant decrease in p16 protein expression and a significant increase in Cyclin D1, CDK4 and pRb protein expressions (P<0.05). Compared with the old group, VSMCs in the treated groups had a significant increase in p16 protein expression and a significant decrease in cyclinD1, CDK4 and pRb protein expressions (P<0.05).

CONCLUSIONSVSMCs obtained from old rats showed typical signs of cellular senescence and vascular aging. EXT had an effect on delaying senescence of VSMCs in vitro by altering the p16-cyclinD/CDK-Rb pathway.