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

Figure 7 Evidence-bounded working model of ganagliflozin proline-associated protection and receptor for advanced glycation end products/nuclear factor kappa B-associated signaling in MC3T3-E1 cells. Commercial advanced glycation end product-modified bovine serum albumin (AGE-BSA) exposure was associated with increased AGE-modified protein immunoreactivity, receptor for advanced glycation end products (RAGE) expression, p65 phosphorylation, and tumor necrosis factor alpha (TNF-α) expression. These changes occurred together with reduced proliferation, osteogenic-marker expression, alkaline phosphatase (ALP) activity, and mineralization-associated readouts. Ganagliflozin proline (Gana Pro) increased 5-ethynyl-2’-deoxyuridine incorporation, collagen I (COL1) and osteogenic-marker expression, ALP activity, calcium content, and matrix mineralization while reducing RAGE, p65 phosphorylation, and TNF-α readouts. In a separate experiment without Gana Pro, RAGE knockdown followed by phorbol 12-myristate 13-acetate (PMA) stimulation supported the functional involvement of RAGE/nuclear factor kappa B-associated signaling in AGE-BSA-associated injury. Solid connections represent relationships measured directly within the corresponding experimental module. The dashed connection between reduced pathway readouts and Gana Pro-associated functional recovery denotes a proposed contribution requiring direct Gana Pro plus PMA rescue testing. The model does not imply that Gana Pro directly inhibits COL1 glycation or crosslinking. Because matched unmodified bovine serum albumin and endotoxin-controlled conditions were absent, the initiating injury cannot be attributed exclusively to AGE modifications. The model is restricted to MC3T3-E1 cells and does not imply validation in primary, human, or in vivo osteogenic systems. AGE: Advanced glycation end product; OCN: Osteocalcin; COL1: Collagen I; ALP: Alkaline phosphatase; RUNX2: Runt-related transcription factor 2; TNF-α: Tumor necrosis factor alpha; NF-κB: Nuclear factor kappa B; RAGE: Receptor for advanced glycation end products; p-p65: Phosphorylated p65; Gana Pro: Ganagliflozin proline.