Ganagliflozin proline attenuates osteogenic dysfunction and inflammatory signaling during exposure to advanced glycation end products in murine osteoblasts

Figure 6 Receptor for advanced glycation end products knockdown and phorbol 12-myristate 13-acetate stimulation support pathway-associated responses during advanced glycation end product-modified bovine serum albumin exposure. MC3T3-E1 cells were assigned to normal-glucose + negative-control small interfering RNA (siNC), advanced glycation end product-modified bovine serum albumin (AGE-BSA) + siNC, AGE-BSA + receptor for advanced glycation end products (RAGE)-targeting small interfering RNA (siRAGE), or AGE-BSA + siRAGE + phorbol 12-myristate 13-acetate (PMA) (100 nmol/L; treatment duration reported in the materials and methods) groups. A: Western blotting, relative-expression heatmap, and densitometric analysis of total p65, phosphorylated p65 (p-p65), and the p-p65/p65 ratio. RAGE knockdown reduced AGE-BSA-associated p65 phosphorylation, whereas PMA increased p65 phosphorylation after RAGE knockdown; B: Quantitative polymerase chain reaction analysis of Rela expression; C: Western blotting, relative-expression heatmap, and densitometric analysis of RAGE and tumor necrosis factor alpha (TNF-α). PMA did not restore RAGE expression but increased TNF-α expression after RAGE knockdown; D: Quantitative polymerase chain reaction analysis of Ager and Tnf expression; E: Western blotting and densitometric analysis of collagen I and osteocalcin expression; F: Quantitative polymerase chain reaction analysis of Col1a1 and Bglap expression; G: Western blotting and densitometric analysis of alkaline phosphatase and runt-related transcription factor 2 expression; H: Quantitative polymerase chain reaction analysis of Alpl and Runx2 expression. RAGE knockdown increased the measured osteogenic markers, whereas PMA partially attenuated their recovery. Data are presented as the mean ± SD from three independent biological experiments (n = 3). Technical replicate measurements were averaged within each experiment. Multiple-group comparisons were performed using one-way analysis of variance followed by Tukey’s multiple-comparisons test. PMA was used as a pleiotropic stimulus capable of engaging nuclear factor kappa B (NF-κB)-associated signaling, not as a selective NF-κB agonist. Nuclear p65 translocation and nuclear/cytoplasmic fractionation were not evaluated. Ganagliflozin proline was absent from this experiment; therefore, these results do not establish that RAGE/NF-κB-associated signaling mediates its protective effects. Uncropped Western blot source images are provided in the Supplementary material. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NF-κB: Nuclear factor kappa B; p-p65: Phosphorylated p65; NG: Normal-glucose; AGE: Advanced glycation end product; siNC: Negative-control small interfering RNA; siRAGE: Receptor for advanced glycation end products-targeting small interfering RNA; RAGE: Receptor for advanced glycation end products; TNF-α: Tumor necrosis factor alpha; mRNA: Messenger RNA; OCN: Osteocalcin; COL1: Collagen I; ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2.