Osteogenic differentiation of rat bone marrow stem cells induced by acanthopanax senticosus glycoside

Objective 　To investigate the effect of acanthopanax on inducing osteogenesis of rat bone marrow stem cells (BMSCs). Methods 　BMSCs were extracted from SD rats, and the surface antigens CD45, CD29 and CD90 were identified by flow cytometry at the third generation. Different concentrations of acanthopanax were added to the classical osteogenic medium to make it being 9 groups: A(1×10–4mol/L), B(1×10–5mol/L), C(1×10–6mol/L), D(1×10–7mol/L), E(1×10–8mol/L), F(1×10–9mol/L), G(1×10–10mol/L), H(classical osteogenic group), and I(negative control group). The cell counting kit CCK-8 was used to detect cell proliferation, RT-qPCR was performed to detect the mRNA expression of bone morphogenetic protein (BMP), and then the optimal concentration of acanthopanax was selected and used to the later experiments. On the 12th day of culturing with optimal concentration, RT-qPCR was performed to detect osteogenic differentiation-related gene expression: RUNX, OSX, BSP and OCN. Western blotting was used to detect the levels of transmembrane receptor protein 1 (Notch1) and hairy enhancer of split 1 (Hes1). On the 21th day of culturing, the mineralized calcium nodules were stained with alizarin red. Results 　The surface antigens of the third generation BMSCs were consistent with the stem cell identification criteria. CCK-8 results indicated that the proliferation of BMSCs was inhibited in group A and group B 120h after cultivation, so the two groups were discarded in the later culture. RT-qPCR results showed that among groups C-G with acanthopanax, the expression of BMP in group E (1×10–8mol/L) was the highest (4.91±0.46), so 1×10–8mol/L was selected as the optimal concentration of acanthopanax to finish the later experiments. The results of RT-qPCR showed that the expression of OSX was significantly higher in group E (30.72±1.96) than in group I (1.02±0.27) and group H (9.99±0.59, P<0.05); the expression of BSP (8.15±0.47) was higher than in group I (1.09±0.31) and group H (6.03±0.8, P<0.05); and the expression of OCN (5.91±0.68) was higher than in group I (1.18±2.91) and group H (3.05±0.53, P<0.05). However, the expression of RUNX was higher in group E (1.99±0.09) than in group I (1.02±0.19, P<0.05), but was lower than in group H (2.51±0.06, P<0.05). Western blotting suggested that the Notch1 in group E (4608±103) was higher than in group I (2638±308), but lower than in group H (5218±182, P<0.05); Hes1 expression in group E (8885±17) was higher than in group I ( 6241±461), but lower than in H group (12289±629, P<0.05). The alizarin red staining indicated that the number of mineralized calcium nodules was higher in group E than in group H, suggesting that the osteogenic effect in group E (with acanthopanax concentration of 10–8mol/L) is better than in group H. Conclusion 　Acanthopanax may cooperate with dexamethasone to promote the osteogenesis of BMSCs, which may be related to the Notch signaling pathway. DOI: 10.11855/j.issn.0577-7402.2019.03.05