Background: Osteoporosis, characterized by bone loss, usually occurs with the increased bone resorption and decreased bone formation. H₂O₂-induced MC3T3-E1 cells are commonly used for the study of osteoblastic activities, which play a crucial role in bone formation.

Objective: This study aimed to investigate the effects of Phosphocreatine (PCr) on the osteoblastic activities in H₂O₂-induced MC3T3-E1 cells and elaborate on the possible molecular mechanism.

Methods: The Osteoprotegerin (OPG)/Receptor Activator of NF-κB Ligand (RANKL) ratio and osteogenic markers were detected to investigate the effects of PCr on osteoblastic activities, and the osteoblastic apoptosis was detected using Hochest staining. Moreover, oxidative stress, Adenosine Triphosphate (ATP) generation and the expression of Sirtuin 1 (SIRT1), Forkhead Box O 1 (FOXO1) and Peroxisome Proliferator-Activated Receptor Γ Coactivator-1α (PGC-1α) were also examined to uncover the possible molecular mechanism in H₂O₂-induced MC3T3-E1 cells.

Result: The results showed that PCr promoted the osteoblastic differentiation by increasing the expression levels of osteogenic markers of Alkaline Phosphatase (ALP) and Runt-related transcription factor 2 (Runx2), as well as increased the OPG/RANKL ratio and suppressed the osteoblastic apoptosis in H₂O₂-induced MC3T3-E1 cells. Moreover, treatment with PCr suppressed reactive oxygen species (ROS) over-generation and promoted the ATP production as well as increased the PGC-1α, FOXO1 and SIRT1 protein expression levels in H₂O₂-induced MC3T3-E1 cells.

Conclusion: PCr treatment could promote osteoblastic activities via suppressing oxidative stress and increasing the ATP generation in H₂O₂-induced MC3T3-E1 cells. In addition, the positive effects of PCr on osteoblasts might be regulated by SIRT1/FOXO1/ PGC-1α signaling pathway.