Effect of Bushen Huoxue decoction on inhibiting osteogenic differentiation of vascular smooth cells by regulating OPG/RANK/RANKL system in vascular calcification.

Abstract

To investigate the effects of Bushen Huoxue Decoction (BSHXD) and its underlying molecular mechanisms on inhibiting osteogenic differentiation of vascular smooth muscle cells (VSMCs) in vascular calcification via regulating the mRNA expression of osteoprotegerin (OPG) and the receptor activator of the nuclear factor-kappa B ligand (RANKL). VSMCs from the aortas of rats were cultured in vitro. Osteogenic differentiation of VSMCs was induced by high levels of an inorganic phosphate medium (2.4 mM). BSHXD-containing serum was prepared using the serum-pharmacological method. VSMCs were plated using 6-well plates at an approximate density of 4.0×104 cells/mL and cultured for 10 days. This was followed by the application of different concentrations of BSHXD-containing serum. The percentage of concentrations of BSHXD-containing serum in high, middle and low dosage group was 20%, 10% and 5%, respectively. Calcium nodules were evaluated by alizarin red S staining, and alkaline phosphatase (ALP) activity and calcium deposition were both examined as per the instruction of the test kits on the 3rd, 6th, and 10th days. Protein expression level of ALP and α-smooth muscle actin (α-SMA) were detected by Western blot on the 3rd, 6th, and 10th days. The mRNA expression of the OPG and RANKL were also detected by real-time PCR on the 3rd, 6th, and 10th days. Compared with the control group, BSHXD significantly attenuated the calcium nodules that were examined by alizarin red-S staining. Protein expression levels of α-SMA were up-regulated and ALP were down-regulated on the BSHXD group (P<0.05). BSHXD also attenuated the ALP activity and calcium deposition of the VSMCs (P<0.05). These changes were associated with the effect of BSHXD on up-regulating the expression of OPG mRNA and down-regulating the expression of RANKL mRNA in the process of osteogenic differentiation of VSMCs. BSHXD has a beneficial effect on inhibiting osteogenic differentiation of VSMCs induced by high levels of phosphate. The underlying mechanism appears to be related to the modulation of expressions of OPG mRNA and RANKL mRNA in the VSMCs, thereby preventing the phenotypic changes of VSMCs to an osteogenic phenotype.