[Effects and mechanism of morroniside on osteogenic differentiation and proliferation of mouse MC3T3-E1 cells]

Objective: To study the effects of morroniside (MOR) on the proliferation and osteogenic differentiation of mouse MC3T3-E1 cells.

Methods: The 4th generation MC3T3-E1 cells were randomly divided into 6 groups: control group (group A), MOR low dose group (10 μmol/L, group B), MOR medium-low dose group (20 μmol/L, group C), MOR medium dose group (40 μmol/L, group D), MOR medium-high dose group (80 μmol/L, group E), and MOR high dose group (100 μmol/L, group F). The proliferation activity of each group was detected by cell counting kit 8 (CCK-8) assay; the bone differentiation and mineralized nodule formation of each group were detected by alizarin red staining; real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect cyclin-dependent kinase inhibitor 1A (P21), recombinant Cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP), collagen type Ⅰ (COL-1), bone morphogenetic protein 2 (BMP-2), and adenosine A2A receptor (A2AR) mRNA expressions; Western blot was used to detecte the expressions of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and adenosine A2AR protein.

Results: The CCK-8 assay showed that the absorbance ( A) values of groups B to F were significantly higher than that of group A at 24 hours of culture, with group C significantly higher than the rest of the groups ( P<0.05). The MOR concentration (20 μmol/L) of group C was selected for the subsequent CCK-8 assay; the results showed that the A values of group C were significantly higher than those of group A at 24, 48, and 72 hours of culture ( P<0.05). Alizarin red staining showed that orange-red mineralized nodules were visible in all groups and the number of mineralized nodules was significantly higher in groups B and C than in group A ( P<0.05). RT-qPCR showed that the relative expressions of P21, CCND1, and PCNA mRNAs were significantly higher in group C than in group A ( P<0.05). The expressions of ALP, BMP-2, COL-1, and adenosine A2AR mRNAs in groups B to E were significantly higher than those in group A, with the expressions of ALP, BMP-2, COL-1 mRNAs in group C significantly higher than the rest of the groups ( P<0.05). Compared with group A, the expressions of OPN and RUNX2 proteins in groups B and C were significantly increased, while those in group D and E were significantly inhibited ( P<0.05). There was no significant difference between groups B and C and between groups D and E ( P>0.05). The relative expression of adenosine A2AR protein in groups B to E was significantly higher than that in group A, with group C significantly higher than the rest of the groups ( P<0.05).

Conclusion: MOR can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells; the mechanism of MOR may be achieved by interacting with adenosine A2AR.