Abstract
OBJECTIVE: To explore the effect of oridonin (ORI) for suppressing thioacetamide (TAA)-induced osteoclast differentiation of RAW264.7 cells and antagonizing the inhibitory effect of TAA on osteogenic differentiation of bone mesenchymal stem cells (BMSCs). METHODS: The effects of TAA and ORI on the proliferation of RAW264.7 cells and SD rat BMSCs were examined using CCK-8 assay. TRAP staining and immunofluorescence staining were used to observe the effects of TAA and ORI on osteoclast differentiation in RAW264.7 cells. The expressions of osteoclast-specific proteins in the treated cells were detected using Western blotting, and p65 nuclear translocation and reactive oxygen species (ROS) production in the cells were assessed with immunofluorescence assay and flow cytometry. Alkaline phosphatase (ALP) staining and alizarin red staining were used to examine the effects of TAA and ORI on osteogenic differentiation of BMSCs, and the expressions of osteogenic and apoptosis-related proteins in the cells were detected with Western blotting. RESULTS: Compared with RAW264.7 cells treated with TAA alone, the cells with the combined treatment with TAA and ORI showed decreased osteoclast differentiation (P < 0.01) and significant inhibition of the MAPK/NF-κB pathway (P < 0.01) with reduced p65 nuclear translocation and intracellular ROS production (P < 0.01). In rat BMSCs, treatment with TAA alone significantly inhibited ALP activity and formation of calcified nodules (P < 0.01) and induced obvious cell apoptosis. Compared with TAA-treated BMSCs, the cells treated with both TAA and ORI showed upregulated expressions of the BMP-2/RUNX2 pathway with enhanced ALP activity (P < 0.01) and calcium deposition (P < 0.01) and a lowered cell apoptosis level. CONCLUSION: ORI inhibits TAA-induced osteoclast differentiation via regulating the MAPK/NF-κB pathway and antagonizes TAA-induced inhibition of bone formation by regulating the BMP-2/RUNX2 pathway.