Background: Osteoporosis is a common systemic metabolic disease, leading to increased bone fragility and risk of fractures. Research has shown that Adenosine triphosphate (ATP) synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5A1), a crucial component in ATP production, is inhibited in dexamethasone (DEX)-induced osteoblasts. Therefore, this study aimed to investigate the molecular mechanism underlying the inhibitory impact of DEX on osteogenic differentiation in rat bone marrow mesenchymal stem cells (BMSCs).

Methods: Rat BMSCs were treated with varying concentrations of DEX for 14 days, followed by subsequent analyses. The expression levels of calpastatin (CAST), calpain 1 (CAPN1), and ATP5A1 were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting analyses. Furthermore, osteogenic marker proteins and ATP activity were evaluated employing Western blotting analysis and enzyme-linked immunosorbent assay (ELISA). Moreover, to determine the regulatory role of DEX on the CAST-CAPN1 axis, overexpression plasmids for CAST (oe-CAST) and CAPN1 (oe-CAPN1) were constructed. Additionally, osteogenic differentiation and ATP activity in BMSCs were analyzed using qRT-PCR, Western blotting, Alizarin Red S staining, and ELISA.

Results: With increasing concentrations of DEX, the expression of the CAST-CAPN1-ATP5A1 axis in BMSCs was significantly altered (p < 0.05). DEX downregulated the levels of osteogenic markers, including Runt-Related Transcription Factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN), while reducing ATP activity (p < 0.05). However, oe-CAST partially mitigated the inhibitory effects of DEX on osteogenic differentiation and ATP activity (p < 0.05). In contrast, oe-CAPN1 exacerbated the effects of DEX and reversed the regulatory impact of CAST (p < 0.05).

Conclusion: DEX inhibits osteogenic differentiation and reduces ATP activity in BMSCs by modulating the CAST-CAPN1 axis.