β-Casein phosphopeptides (β-CPP), a class of bioactive peptides derived from β-casein, possess excellent calcium-binding capacity and demonstrate potential applications in promoting bone health. In this study, β-CPP was prepared through enzymatic hydrolysis to investigate its effects on the proliferation, differentiation, and mineralization of osteoblasts (MC3T3-E1), with its mechanism of action being analyzed through transcriptomics. The results indicated that when the concentration of β-CPP was 50 μg/mL, the proliferation rate of MC3T3-E1 cells was significantly increased to 128.43% after 72 h of treatment (P < 0.05). After 7 days of treatment, alkaline phosphatase activity was 2.17 times higher than that of the control group, and after 21 days, the mineralization rate was 2.15 times higher than that of the control group, demonstrating that β-CPP significantly enhanced alkaline phosphatase activity and extracellular matrix mineralization in MC3T3-E1 cells (P < 0.05). RNA-seq results revealed 476 differentially expressed genes in β-CPP-treated MC3T3-E1 cells, among which 258 genes were upregulated and 218 genes were downregulated. Gene Ontology (GO) analysis indicated that the differentially expressed genes were primarily involved in the regulation of biological processes. KEGG pathway enrichment analysis showed that signaling pathways such as TGF-β and PI3K-Akt were involved in the differentiation of MC3T3-E1 cells. This study revealed the promoting effects of β-CPP on the proliferation, differentiation, and mineralization of MC3T3-E1 cells, providing a theoretical basis for the application of β-CPP in bone health and functional foods.
