Runbei Dong1, Yutao Jia2, Houzhi Yang1, Gan Luo1, Yuqiao Li1, Tianwei Sun2. 1. Graduate School of Tianjin Medical University, Tianjin, 300070, P. R. China. 2. Department of Spinal Surgery, Tianjin Union Medical Centre, Tianjin, 300121, P. R. China.
Abstract
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.
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.