To investigate the effect of β-sitosterol on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by regulating the transforming growth factor β1 (TGF-β1)/mothers against decapentaplegic drosophila homolog 3 (Smad3) pathway.

BMSCs were randomly assigned into control group, low-dose β-sitosterol (β-sitosterol-L) group, medium-dose β-sitosterol group (β-sitosterol-M), and high-dose β-sitosterol group (β-sitosterol-H). Control group was cultured normally and β-sitosterol-L, -M and -H group was intervened by 1.5、3.0、6.0 μg·mL^-1 β-sitosterol. BMSCs were randomly divided into control group (normal culture), β-sitosterol group (6 μg·mL^-1 β-sitosterol), SIS3 group (5 μmol·L^-1 SIS3), and β-sitosterol+SIS3 group (6 μg·mL^-1 β-sitosterol+5 μmol·L^-1 SIS3). Enzyme-linked immunosorbent assay (ELISA) was used to detect the alkaline phosphatase (ALP) activity was measured in each group. Alizarin red staining was used to determine the calcium deposition rate of each group, and then the osteogenic differentiation of BMSCs in each group was detected. Western blot was used to detect the osteogenic differentiation of BMSCs and the expression of TGF-β1/Smad3 pathway related proteins in each group.

The ALP activities of the β-sitosterol-L, β-sitosterol-M, β-sitosterol-H and the control groups were (1.24±0.10), (1.53±0.08), (1.84±0.11) and (0.95±0.09) U·mg prot^-1, respectively; the calcium deposition rates were (196.21±13.67)%, (301.73±16.90)%, (423.60±20.25)% and (100.00±0)%, respectively; the relative expression levels of bone morphogenetic protein 2 (BMP-2) protein were 0.49±0.05, 0.89±0.07, 1.30±0.09 and 0.13±0.02, respectively; the relative protein expression of TGF-β 1 were 0.47±0.04, 0.85±0.07, 1.26±0.09 and 0.10±0.01, respectively; the p-Smad3/Smad3 values were 0.32±0.03, 0.59±0.06, 0.94±0.05 and 0.08±0.02, respectively. Compared with the control group, the above indicators of the β-sitosterol-L, β-sitosterol-M and β-sitosterol-H groups showed an increase in a dose-dependent manner, and the differences were statistically significant (all P<0.05). The ALP activities of the β-sitosterol group, SIS3 group, β-sitosterol+SIS3 group and the control group were (1.78±0.14), (0.43±0.08), (0.97±0.13) and (0.91±0.10) U·mg prot^-1, respectively; the calcium deposition rates were (410.35±19.72)%, (45.62±13.91)%, (109.14±16.53)% and (100.00±0)%, respectively; the relative expression levels of BMP-2 protein were 1.79±0.16, 0.27±0.07, 0.87±0.10 and 0.85±0.11, respectively; the relative protein expression levels of TGF-β1 were 1.47±0.10, 0.11±0.03, 0.56±0.09 and 0.55±0.06, respectively; The p-Smad3/Smad3 values were 0.94±0.06, 0.09±0.02, 0.45±0.05 and 0.43±0.04, respectively. Compared with the control group, the above indicators were all increased in the β-sitosterol group, while they were all decreased in the SIS3 group, and the differences were statistically significant (all P<0.05). Compared with the β-sitostero group, the above indicators were all decreased in the β-sitosterol+SIS3 group, and the differences were statistically significant (all P<0.05).

β-sitosterol can promote osteogenic differentiation of BMSCs by activating the TGF-β1/Smad3 pathway.