Abstract: BACKGROUND: Asperosaponin VI has good osteogenic effects, but its ability to promote cellular osteogenesis under hypoxia environment is not yet clear.
OBJECTIVE: To investigate the effect and potential mechanism of Asperosaponin VI on osteogenic differentiation of MC3T3-E1 cells under hypoxia environment.
METHODS: MC3T3-E1 cells were divided into three groups. Cells in the control group were cultured in a complete medium under normoxic conditions (volume fraction of 21% O2); cells in the hypoxia group were cultured in the complete medium under hypoxia conditions (volume fraction of 0.5% O2); and cells in the Asperosaponin VI group were cultured in the complete medium containing Asperosaponin VI under hypoxia conditions (volume fraction of 0.5% oxygen). After 24 hours of culture, cell counting kit-8 method and EdU staining were used to detect cell proliferation activity, TUNEL staining and western blot assay were performed to detect cell apoptosis, and flow cytometry was used to detect intracellular reactive oxygen species levels and cell cycle distribution. Each group of cells was cultured in an osteogenic induction medium containing 1×10-5, 1×10-6, and 1×10-7 mol/L Asperosaponin VI under hypoxia conditions for 7 days. The optimal concentration of Asperosaponin VI for intervention was identified using alkaline phosphatase staining under optical microscopy. Western blot was used to detect the expression of bone morphogenetic protein 2, osteopontin, and PI3K/AKT signaling axis-related proteins.
RESULTS AND CONCLUSION: (1) Compared with the control group, the proliferation ability of MC3T3-E1 cells decreased under hypoxia conditions. 1×10-6 mol/L 
Asperosaponin VI could significantly improve the cell proliferation ability under hypoxia conditions and reduce cell apoptosis. (2) Compared with the hypoxia group, the Asperosaponin VI group showed a decrease in intracellular reactive oxygen species and a significant increase in the proportion of cells in the S phase. (3) Compared with the hypoxia group, the cell morphology in the 1×10-6 mol/L Asperosaponin VI group was elongated, with more protrusions and darker alkaline phosphatase staining. (4) Compared with the hypoxia group, the expression of bone morphogenetic protein 2 and osteopontin increased in the Asperosaponin VI group, while the expression of p-PI3K and p-AKT proteins decreased. These findings indicate that under hypoxia conditions, Asperosaponin VI can promote osteogenic differentiation of MC3T3-E1 cells, possibly by regulating the PI3K/AKT pathway.

Key words: MC3T3-E1 cell, hypoxia, Asperosaponin VI, PI3K, AKT, pathway, cell differentiation, osteogenesis