Figure 4.

Inhibition of Gpx1 expression by siRNA abrogated the protective effect of Na₂SeO₃ on the osteogenic potential and mitochondrial antioxidant functions of OP-BMMSCs. OP-BMMSCs were pre-treated with siRNA to inhibit Gpx1 expression and then exposed to 100 nM Na₂SeO₃. (A) RT-PCR revealed that transfection with Gpx1-siRNA inhibited the gene expression of Gpx1, n = 4. (B) Western blot confirmed that the protein level of GPx1 in siRNA-treated OP-BMMSCs was significantly down-regulated, n = 3. (C) The enzyme activity of total GPx was significantly decreased after siRNA transfection, n = 4. (D) The intracellular ROS has increased in siRNA-transfected cells even exposure to Na₂SeO₃, n = 3. (E) Inhibition of Gpx1 increased mitochondrial superoxide production, n = 3. (F) Transfection with Gpx1-siRNA reduced the ATP content in Na₂SeO₃-treated cells, n = 3. (G) Transfection with Gpx1-siRNA decreased the MMP in Na₂SeO₃-treated cells, n = 3. (H) Inhibition of Gpx1 significantly down-regulated the gene expressions of Nd4, sdha, Cox4 and Atp5a, n = 4. (I) Western blot confirmed that treatment with Gpx1-siRNA decreased the protein levels of ND4, SDHA, COX4 and ATP5A, n = 3. (J) Inhibition of Gpx1 suppressed the matrix mineralization of Na₂SeO₃-treated cells. Scale bar = 200 μm, n = 4. (K) Quantification of the stained mineral layers, n = 4. (L) Inhibition of Gpx1 down-regulated the gene expressions of osteoblast-specific markers in Na₂SeO₃-treated cells, n = 4. Values represent mean ± SEM. Statistically significant differences are indicated by * where P < 0.05 or ** where P < 0.01 between the indicated groups.