Table 1.
Primers of AKT, Bax, P53, P85, BCL-2, Cytochrome C and β-actin.
Fig 1.
Potential Cytotoxicity of Cyasterone on BMSCs.
The chemical structure of Cyasterone (A). The cytotoxic effects of Cyasterone on BMSCs were determined by increasing concentrations (0, 1, 5, 10 and 20μM) for 24 hours using a CCK8 assay (B, C). Cyasterone was not cytotoxic to BMSCs at concentrations of 1 to 10μM at 24h. Cyasterone ameliorate DXM-induced cell death in a concentration-dependent manner, especially when the concentration of Cyasterone at a level of 10μM. (Values represent the averages±S.D. Significant differences between different groups are indicated as *P < 0.05, **P < 0.01, vs DXM alone treatment group, n = 3).
Fig 2.
The apoptosis rate of BMSCs in each group.
The apoptosis rate of DXM group and DXM+Cyasterone group was higher than that of the Control group, while the apoptosis rate of DXM+Cyasterone group was lower than that of the DXM group. It was showed that Cyasterone reduced the DXM-induced apoptosis compared with DXM group. (*P<0.05, vs Control, #P<0.05, DXM+Cyasterone vs DXM, n = 3).
Fig 3.
Results of immunofluorescence detection of the expression of Caspase-3 and Caspase-9 in cells.
The bar graph showed both caspase-3 and caspase-9 were punctually overexpressed in the cytoplasm. (400×, the nucleus is stained in blue, and the target gene protein is stained in red, Cya is short for Cyasterone, P<0.05, DXM+Cya vs DXM, n = 3).
Fig 4.
Results of PCR of the mRNA expression of AKT, BAX, P53, P85, Bcl-2, Cytochrome C in cells.
The mRNA expression of BAX, P53, Bcl-2 and Cytochrome C decreased in DXM group compared with the Control group, but the mRNA expression of BAX, P53 and P85 increased in DXM+Cyasterone group compared with that DXM group. (*P<0.05, vs Control; #P<0.05, DXM+Cyasterone vs DXM, n = 3).
Fig 5.
Results of WB of the protein expression of AKT, BAX, P53, P85, Bcl-2, Cytochrome C in cells.
The protein expression level of BAX, Bcl-2 and P53 decreased in DXM group compared with Control group, while the protein expression level of AKT, Bcl-2 decreased in DXM+Cyasterone group compared with DXM group. (*P<0.05, vs Control; #P<0.05, DXM+Cyasterone vs DXM, n = 3).
Fig 6.
Representative images of Micro-CT and HE staining, and the structural parameters of Micro-CT.
Fig 6 (A, B) showed the HE staining and Micro-CT reconstruction images of the three groups of rats; Fig 6A showed the results of Micro-CT after sampling of the femoral head of the three groups of rats; Fig 6B showed that the trabecular bone in the model group was obviously sparser and partially broken than in the Control group (Scale bar: 400 μm, the red arrow represents the necrotic area while the white arrow shows the normal area); The Fig 6C showed that the values of BV/TV, Conn.D and Tb.N increased in the Cyasterone group and the value of Tb.Sp decreased in the Cyasterone group. (The samples conformed to normal distribution and the variance was homogeneous, and one-way analysis of variance was used for comparison among multiple groups. *P<0.05, **P<0.01, Model group compared with Control group; #P<0.05, N = 5, Cyasterone group compared with model group).