Abstract: BACKGROUND: The diseased osteogenic differentiation function of bone marrow mesenchymal stem cells is a key pathological link in the occurrence and development of osteoporosis. Curcumin can promote osteogenic differentiation and treat osteoporosis by regulating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, but the specific mechanism of action remains unclear.
OBJECTIVE: To investigate the mechanism by which curcumin regulates the phosphatidylinositol 3-kinase/protein kinase B signaling pathway to promote osteogenic differentiation for the treatment of osteoporosis. 
METHODS: Network pharmacology was used to screen the drug action targets of curcumin and target genes related to osteogenic differentiation and osteoporosis. The intersection targets of the three were used to construct a protein-protein interaction network diagram and perform Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses and molecular docking. Bioinformatics was used to screen potential signature genes in the protein-protein interaction network and construct a clinical prediction model. Single-cell transcriptomics technology was used to analyze the expression patterns of signature genes in osteoblast lineage subpopulations. Finally, cell and animal experiments were conducted for verification.
RESULTS AND CONCLUSION: (1) A total of 366 drug targets of curcumin were screened, and 134 intersection targets were identified among curcumin, osteogenic differentiation, and osteoporosis. (2) Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the intersection targets were involved in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway. (3) Molecular docking showed that the main active components of curcumin could be well docked with the targets related to the phosphatidylinositol 3-kinase/protein kinase B signaling pathway. (4) Osteoporosis-related gene sets were downloaded from the Gene Expression Omnibus database. The 134 target genes in the protein-protein interaction network were screened by 113 combinations of machine learning algorithms to obtain 14 potential signature genes, including target genes in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway: PIK3R1, PIK3CG, GSK3β, and AKT1. These 14 genes may play important roles in curcumin promoting osteogenic differentiation for the treatment of osteoporosis. (5) Single-cell transcriptomics analysis showed that there was a cell subpopulation with high GSK3β expression in the osteoblast lineage of osteoporosis patients, and this cell subpopulation might be closely related to the occurrence and development of osteoporosis. (6) Pseudotime analysis found that during the development of osteoporosis, the expression level of GSK3β gradually increased, while the expression levels of PIK3R1, PIK3CG, and AKT1 gradually decreased. (7) Cell experiments showed that after GSK3β knockout, the osteogenic differentiation of mouse MC3T3-E1 subclone 14 was promoted, as evidenced by deeper staining in alkaline phosphatase staining and Alizarin Red S staining, and significantly increased alkaline phosphatase activity and calcium deposition (P < 0.05). The mRNA and protein expression levels of β-catenin were significantly increased (P < 0.05). Cell counting kit-8 and EdU results showed that 5 and 10 μmol/L curcumin had no obvious toxicity and could significantly promote the proliferation of MC3T3-E1 subclone 14 cells. In addition, intervention with 5 and 10 μmol/L curcumin significantly promoted the osteogenic differentiation of MC3T3-E1 subclone 14. (8) Animal experiments showed that curcumin improved osteoporosis in the femoral tissue of ovariectomized rats by increasing the number of trabecular bones and ameliorating the disorder of trabecular bone arrangement. After curcumin intervention, the expression and phosphorylation levels of phosphatidylinositol 3-kinase and protein kinase B1 in the distal femoral bone tissue of osteoporotic rats were significantly increased (P < 0.05), and the phosphorylation level of GSK3β was significantly decreased (P < 0.05). In addition, the expression levels of c-MYC, Cyclin D1, bone morphogenetic protein 2, osteocalcin, osteopontin, and Osterix were significantly increased (P < 0.05). In conclusion, the increased expression of GSK3β in the phosphatidylinositol 3-kinase/protein kinase B signaling pathway may be a key factor leading to the decline of osteogenic differentiation function and the occurrence and development of osteoporosis. Curcumin treats osteoporosis by activating the phosphatidylinositol 3-kinase/protein kinase B signaling pathway to promote osteogenic differentiation.

Key words: curcumin, osteogenic differentiation, osteoporosis, single-cell transcriptomics, phosphatidylinositol 3-kinase, protein kinase B, signaling pathway