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

Figure 3 Concentration screening and proliferative responses to ganagliflozin proline during advanced glycation end product-modified bovine serum albumin exposure. A-C: Cell counting kit-8 metabolic-activity measurements after 12 hours, 24 hours, and 48 hours, respectively. MC3T3-E1 cells were exposed to commercial advanced glycation end product-modified bovine serum albumin (AGE-BSA) (25-400 μg/mL total protein), canagliflozin (1-40 μmol/L), or ganagliflozin proline (Gana Pro) (1-40 μmol/L). Additional groups received 200 μg/mL AGE-BSA with 1 μmol/L, 5 μmol/L, or 10 μmol/L Gana Pro. The single-agent drug series evaluated concentration-associated effects on metabolic activity and did not constitute a comprehensive safety assessment. Based on the combined concentration-screening results, 5 μmol/L Gana Pro was selected as the working concentration for subsequent experiments; it was not defined as universally optimal; D: Representative 5-ethynyl-2’-deoxyuridine (EdU) images and quantification in the normal-glucose, AGE-BSA, AGE-BSA + 1 μmol/L Gana Pro, AGE-BSA + 5 μmol/L Gana Pro, and AGE-BSA + 10 μmol/L Gana Pro groups. EdU-positive nuclei are red, and Hoechst 33342-stained nuclei are blue. AGE-BSA exposure was associated with reduced EdU incorporation, whereas 5 μmol/L Gana Pro increased the EdU-positive proportion; E: Representative propidium iodide DNA-content histograms for the corresponding groups; F: Percentages of cells in the G0/G1, S, and G2/M phases. AGE-BSA exposure was associated with increased G0/G1-phase accumulation and reduced S-phase representation. Treatment with 5 μmol/L Gana Pro partially restored S-phase representation. Data are presented as the mean ± SD from three independent biological experiments (n = 3). Technical replicate wells were averaged within each 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. The AGE-BSA preparation lacked a matched unmodified BSA control, and its endotoxin content was not analyzed. Gana Pro-only groups were included in the metabolic-activity screen but not in the downstream molecular and osteogenic assays. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. For panels A-C, statistical comparisons were made against the corresponding normal-glucose group within each experimental series at the same time point. NG: Normal-glucose; HG: High-glucose; AGE: Advanced glycation end product; Cana: Canagliflozin; Gana Pro: Ganagliflozin proline; EdU: 5-ethynyl-2’-deoxyuridine.