Wang, H. X.; LUO, Z.; ZHAO, C.; CHEN, D.; YAN, X.; GUAN, D.

Background: Osteoporosis (OP) manifests primarily in middle-aged and elderly individuals, representing an age-related condition characterised by diminished bone mass and alterations in bone tissue structure, potentially resulting in fractures and compromising the patient's quality of life.The potential of kaempferol to modulate osteogenic differentiation, enhance bone metabolism, and potentially offer therapeutic benefits in OP cases is of particular interest. This study, employing a combination of network pharmacology and experimental verification, investigated the underlying mechanisms by which kaempferol stimulates the proliferation and osteogenic differentiation of mouse embryonic osteoblast precursor cells MC3T3-E1 subclone 14 through the PI3K/AKT signalling pathway. The findings provide a rational foundation for the potential of kaempferol to promote osteogenesis and enhance the treatment of OP. Methods: The present study identified target genes regulated by kaempferol during osteogenesis and differentiation using network pharmacology. To this end, a protein-protein interaction (PPI) network was constructed, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and molecular docking were performed.The cytotoxicity of kaempferol was assessed using the CCK-8 method and the cell clone method in the cell detection section.The macroscopic regulatory mechanism of kaempferol in osteogenic differentiation was studied using alkaline phosphatase staining, activity assay, Alizarin Red staining, and calcium quantification. Furthermore, real-time quantitative polymerase chain reaction (RT qPCR) and Western blot methods were employed to detect the microscopic expression differences of mRNA and protein related to the PI3K/AKT signalling pathway.The present study identified target genes regulated by kaempferol during osteogenesis and differentiation using network pharmacology. To this end, a protein-protein interaction (PPI) network was constructed, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses and molecular docking were performed. Results: A total of 203 target genes regulated by kaempferol were identified during osteogenic differentiation, with the majority of these genes being associated with biological processes related to cell proliferation and regulation.Four of these target genes act on the PI3K/AKT signalling pathway and show good interactions with kaempferol. Furthermore, kaempferol (5 M, 10 M) has been shown to enhance the vitality and proliferation of MC3T3-E1 Subclone 14 cells, as well as to increase alkaline phosphatase activity and calcium deposition. Furthermore, kaempferol (5M, 10 M) has been observed to upregulate the mRNA expression of phosphoinositide 3-kinase (Pi3k), {beta}-catenin, Myc proto-oncogene protein (c-Myc), and cyclin D1 in MC3T3-E1 Subclone. 14 cells, and promotes the phosphorylation of PI3K, serine/threonine protein kinase AKT (AKT1) and glycogen synthase kinase, as well as the phosphorylation of glycogen synthase kinase-3{beta} (GSK3{beta}) (p < 0.05), thereby upregulating the expression of {beta}-catenin, C-MYC and CYCLIN D1 proteins and increasing the levels of p-PI3K/PI3K, p-AKT1/AKT1 and p-GSK3{beta}/GSK3{beta} levels, thereby promoting osteogenic differentiation of MC3T3-E1 Subclone 14. Conclusion: Kaempferol has been demonstrated to have the capacity to significantly promote the osteogenic differentiation of MC3T3-E1 Subclone 14. This process is thought to be achieved by regulating the PI3K/AKT signalling pathway and affecting the expression of osteogenic-related genes. It has been shown to have a preventive and therapeutic effect on the occurrence and development of osteoporosis.