Decoding Geniposide's Mechanism: From miR-208a-3p/SMAD5 Modulation To Osteoblast Proliferation and Differentiation.

Geniposide (GEN), primarily extracted from the Gardenia jasminoides Ellis, was investigated in this study for its molecular mechanisms in regulating osteoblast proliferation and differentiation and in promoting tibial fracture (TF) healing. A TF mouse model was established and fracture healing was assessed by biomechanical examination and hematoxylin-eosin (H&E) staining. The effects of GEN on MC3T3-E1 cell proliferation, apoptosis, differentiation and its regulation of miR-208a-3p/SMAD5 axis were analyzed. The targeting relationship between miR-208a-3p and SMAD5 was verified by RT-qPCR, Western blot and dual luciferase reporter gene assay. It was found that GEN significantly promoted fracture healing in TF mice, as evidenced by enhanced bone formation and improved biomechanical parameters (maximum load, elastic modulus and elastic deflection). In MC3T3-E1 cells, GEN increased cell proliferation and differentiation while inhibiting apoptosis. Mechanistically, GEN upregulated SMAD5 expression by downregulating miR-208a-3p, and miR-208a-3p was confirmed to directly targeted the 3’UTR of SMAD5. Both in vivo and in vitro experiments demonstrated that elevating miR-208a-3p levels or reducing SMAD5 expression attenuated the fracture healing benefits of GEN. In summary, this study proposes for the first time that GEN promotes TF healing by regulating the miR-208a-3p/SMAD5 axis to enhance osteoblast proliferation and differentiation.