Abstract: BACKGROUND: Alpinetin has anti-inflammatory, antitumor and antibacterial effects and has been proven to alleviate osteoporosis, but the effect and mechanism of alpinetin on osteoporotic fractures are still unclear.
OBJECTIVE: To investigate the improvement effect of alpinetin in rates with osteoporotic fracture by regulating the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signaling pathway. 
METHODS: A rat model of osteoporotic fracture was constructed using bilateral ovariectomy. The successfully modeled rats were randomly separated into model group, low-, medium-, and high-dose alpinetin group, and inhibitor group, with 12 rats per group. Another 12 rats were regarded as the sham operation group. On the day of fracture, rats in the low-, medium-, and high-dose alpinetin groups were gavaged with 7.5, 15, and 30 mg/kg alpinetin + intraperitoneally injected with an equal amount of normal saline, respectively; the inhibitor group was gavaged with 30 mg/kg alpinetin + intraperitoneally injected with 5 mg/kg 
H-89 (a pathway inhibitor); and the model and sham operation groups were given (gavaged + intraperitoneally injected) an equal amount of normal saline, once a day for 8 consecutive weeks. Radiological examination was applied to evaluate fracture healing and assess healing scores; bone mineral density scanners were used to measure bone mineral density at the fracture site; three-point bending experiment and compression experiment were conducted to evaluate the biomechanical status of the femur; pathological damage at the fracture site in rats was observed by hematoxylin-eosin staining; enzyme linked immunosorbent assay (ELISA) was used to detect changes in serum alkaline phosphatase, osteocalcin, type I collagen cross-linked C-terminal peptide, and cAMP levels; western blot was applied to detect the expression of bone morphogenetic protein 2 and cAMP/PKA/CREB pathway proteins in femoral tissue. 
RESULTS AND CONCLUSION: Compared with the sham operation group, the fracture healing score, bone mineral density, maximum load, maximum stress, alkaline phosphatase, osteocalcin, cAMP levels, bone morphogenetic protein 2, p-PKA/PKA, p-CREB/CREB expression in the model group were decreased, the level of type I collagen cross-linked C-terminal peptide was increased (P < 0.05). The above indicators showed opposite changes in all the alpinetin groups compared with the model group (P < 0.05). Compared with the high-dose alpinetin group, the changes in the above indicators were reversed in the inhibitor group (P < 0.05). To conclude, alpinetin may accelerate fracture healing in osteoporotic fracture rats, by activating the cAMP/PKA/CREB signaling pathway.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: alpinetin, cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-responsive element binding protein, osteoporosis, fracture healing, rat