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Objective To clarify the underlying mechanism of metformin in improvement of glucocorticoid induced osteoporosis (GIOP). Methods Dexamethasone (DEX, 10 μmol/L) alone or combined with metformin (1 mmol/L) was used to treat murine long bone osteocytes, Y4 (MLO-Y4), and the cells without any treatment served as blank control. Oxidant-sensing probe 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the production of reactive oxygen species (ROS), Western blotting was employed to detect the protein levels of AKT and Caspase-3, CCK-8 assay and TUNEL assay were conducted to study the cell viability and apoptosis, and real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to test the mRNA levels of SOD2 and Cat. After the establishment of GIOP model in Sprague Dawley (SD) rats, micro-CT scanning was carried out to evaluate the effect of metformin on bone metabolism. Results CCK-8 assay demonstrated that DEX significantly reduced the proliferation of MLO-Y4 cells when compared with the blank control cells (P < 0.05), while no obvious difference was seen between the DEX+metformin and control cells. DEX group had significantly higher ROS level than the DEX+metformin group and control group, but there was no difference between the latter 2 groups. RT-PCR results showed that the mRNA levels of SOD2 and Cat were notably decreased in the DEX group (P < 0.05), which indicating defects in enzymatic antioxidant defense system. TUNEL assay suggested the apoptosis was increased in the DEX group, but no difference was seen between the metformin group and control group. The results of Western blotting indicated that the DEX group had higher level of Caspase-3 than the other 2 groups, which suggested that metformin inhibits cell apoptosis induced by DEX, while, the AKT level was higher in the metformin group than the DEX group, showing that metformin activates the AKT signaling pathway. Compared with the blank control group, the microstructural parameters, including trabecular thickness (TB. TH), bone and tissue volume ratio (BV/TV), and trabecular number (TB. N) were obviously decreased (P < 0.05), while trabecular spacing (TB.SP) was increased (P < 0.05). But there were no significant differences in these parameters between the DEX+metformin group and control group. Conclusion Metformin reduces the increase of ROS levels induced by glucocorticoids, activates AKT signaling pathway, inhibits the activity of proteolytic enzyme Caspase-3, regulates proliferation and apoptosis of osteocytes, and thus improve bone metabolism of glucocorticoid-induced osteoporosis. |
