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

Figure 5 Ganagliflozin proline improves osteogenic and mineralization-associated readouts during advanced glycation end product-modified bovine serum albumin exposure. A: Western blotting, relative-expression heatmap, and densitometric analysis of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) in advanced glycation end product-modified bovine serum albumin (AGE-BSA)-exposed MC3T3-E1 cells. Glyceraldehyde-3-phosphate dehydrogenase was the loading control. Compared with AGE-BSA alone, ganagliflozin proline (Gana Pro) increased ALP, RUNX2, and OCN expression; B: Quantitative polymerase chain reaction analysis of Alpl, Runx2, and Bglap expression. No superiority of Gana Pro over canagliflozin (Cana) is inferred unless supported by a multiplicity-adjusted direct comparison; C: Representative RUNX2 immunofluorescence images and fluorescence quantification in the normal-glucose (NG), AGE-BSA, and AGE-BSA + Gana Pro groups. RUNX2 is shown in red, and 4’,6-diamidino-2-phenylindole-stained nuclei are shown in blue; D: Representative ALP staining, ALP-positive area, and ALP activity in the NG, AGE-BSA, AGE-BSA + Cana, and AGE-BSA + Gana Pro groups. ALP-positive staining was expressed as a percentage of the analyzed field area, and ALP activity in cell lysates was expressed as U/L; E: Representative Alizarin Red S staining, positive-area quantification, and calcium content. Alizarin Red S-positive area was expressed as a percentage of the analyzed field area, whereas calcium content was reported as μg/well. Gana Pro increased both mineralization-associated readouts compared with AGE-BSA alone. Because calcium content was not normalized to protein, DNA, or viable-cell number, a contribution from differences in cell abundance cannot be excluded. Data are presented as the mean ± SD from three independent biological experiments (n = 3). Technical wells and image fields were averaged within each biological experiment and were not treated as independent observations. Multiple-group comparisons were performed using one-way analysis of variance followed by Tukey’s multiple-comparisons test. Uncropped Western blot source images are provided in the Supplementary material. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; OCN: Osteocalcin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NG: Normal-glucose; Cana: Canagliflozin; Gana Pro: Ganagliflozin proline; DAPI: 4’,6-diamidino-2-phenylindole; AGE: Advanced glycation end product; mRNA: Messenger RNA.