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

Figure 4 Ganagliflozin proline restores collagen I expression and reduces inflammatory readouts during advanced glycation end product-modified bovine serum albumin exposure. A: Western blotting, relative-expression heatmap, and densitometric analysis of collagen I (COL1), advanced glycation end product (AGE)-modified protein immunoreactivity, and the receptor for advanced glycation end products (RAGE). MC3T3-E1 cells were cultured under normal-glucose, high-glucose, AGE-bovine serum albumin (BSA), AGE-BSA + canagliflozin (10 μmol/L), or AGE-BSA + ganagliflozin proline (Gana Pro) (5 μmol/L) conditions. Glyceraldehyde-3-phosphate dehydrogenase was the loading control. Compared with AGE-BSA alone, Gana Pro increased COL1 expression and reduced anti-AGE immunoreactivity and RAGE expression. The anti-AGE signal represents overall AGE-modified protein immunoreactivity and does not measure COL1-specific glycation or crosslinking; B: Quantitative polymerase chain reaction analysis of Col1a1 and Ager expression. Gana Pro increased Col1a1 expression and reduced Ager expression compared with AGE-BSA alone; C: Western blotting, heatmap, and densitometric analysis of nuclear factor kappa B (NF-κB) p65, phosphorylated p65 (p-p65), the p-p65/p65 ratio, and tumor necrosis factor alpha (TNF-α). Compared with AGE-BSA alone, Gana Pro reduced p-p65, the p-p65/p65 ratio, and TNF-α expression; D: Quantitative polymerase chain reaction analysis of Rela and Tnf expression. The high-glucose comparison lacked a mannitol or L-glucose osmotic control. The observed reductions in inflammatory readouts do not establish that RAGE/NF-κB signaling mediates the effects of Gana Pro. Uncropped Western blot source images are provided in the Supplementary material. 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. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. COL1: Collagen I; AGE: Advanced glycation end product; RAGE: Receptor for advanced glycation end products; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NG: Normal-glucose; HG: High-glucose; Cana: Canagliflozin; Gana Pro: Ganagliflozin proline; NF-κB: Nuclear factor kappa B; p-p65: Phosphorylated p65; TNF-α: Tumor necrosis factor alpha; mRNA: Messenger RNA.