bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.04.02.646873

Potential Mechanisms Underlying Kaempferol-Promoted Osteoblast Proliferation and Osteogenic Differentiation: A Network Pharmacology and Experimental Validation Study

Wang, H. X.·LUO, Z.·ZHAO, C.·CHEN, D.·YAN, X.·GUAN, D.
View original source ↗Export metadata ↓

Abstract

BackgroundOsteoporosis (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 patients 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. MethodsThe 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{o} 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. ResultsA 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 (5 M, 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. ConclusionKaempferol 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.

Source connections

Explore related subjects

cell biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Wang, H. X., LUO, Z., ZHAO, C., CHEN, D., YAN, X., GUAN, D.. 2025-04-07. Potential Mechanisms Underlying Kaempferol-Promoted Osteoblast Proliferation and Osteogenic Differentiation: A Network Pharmacology and Experimental Validation Study. https://doi.org/10.1101/2025.04.02.646873

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Sequence context and partner availability specify divergent cell fates from a single transcription factor

Transcription factors drive the gene regulatory networks that control developmental transitions, yet which of their many targets a cell uses remains unresolved. Expressing Cebp, Cebp{beta} or Cebp{delta} identically, alone or with Oct4, Sox2, Klf4 and cMyc, gave four outcomes: adipocyte from fibroblasts and neural stem cells, trophectoderm from embryonic stem cells, and myeloid from B cells. Raising Cebp levels changed how many sites it reached, not which sequence it read: clustered motifs maintained somatic enhancers, while spillover onto low-copy sites created de novo enhancers carrying composites of Cebp and cell-type-restricted partners. In fibroblasts, Cebp also redistributed Oct4, Sox2 and Klf4 from early targets, blocking iPSC formation. Supplying the predicted partners rewrote the output: Spi1 redirected Cebp into parallel adipogenic and myeloid programs, via a transient PpargCD45 state using separate enhancers for each; TFAP2C abolished adipogenic conversion. Motif density and partner composites therefore assemble a factor's instructive output in the receiving cell.

cell biology↗
bioRxiv2026

High-content screening identifies FDA-approved activators of mitophagy

Mitochondrial diseases are highly heterogeneous disorders caused by defects in oxidative phosphorylation, for which only limited therapeutic options are available. The elimination of dysfunctional mitochondria via mitophagy activation is an attractive strategy to treat several mitochondrial diseases, irrespective of the underlying genetic defect. To identify pharmacological activators of this pathway, we performed a high-content screening of 1,971 FDA-approved compounds using cellular reporters of TFEB nuclear translocation and mitophagy. This screening identified 294 compounds that promoted TFEB translocation, nine of which increased the number of mitolysosomes without compromising cell viability or damaging mitochondria. Among the selected compounds, Meptazinol and Ponatinib emerged as the top-ranked mitophagy inducers in fibroblasts from patients with complex I and IV deficiency, respectively, and were prioritised for further characterisation. However, they did not rescue the bioenergetic defect, suggesting that the stimulation of mitophagy per se is not sufficient to ameliorate the OXPHOS defect in mitochondrial diseases. Overall, our findings establish a scalable platform for the identification of pharmacological activators of TFEB and identify candidate compounds for therapeutic modulation of mitophagy.

cell biology↗
bioRxiv2026

Matrix Gla Protein deficiency reveals a previously unrecognized mucosal calcification in the mouse airways

Tracheal calcification is a common finding that is considered to have limited clinical significance when detected most often in elderly individuals. Despite its frequent occurrence, the cellular and molecular mechanisms at the origin of tracheal calcification remain largely unexplored. In this context, we aimed to characterize the development of tracheal calcification, and to determine the role of matrix Gla protein (MGP), a major inhibitor of ectopic calcification whose genetic or pharmacologic deficiency is associated with premature tracheal calcification in humans. We found that wild-type mice undergo an unexpected early physiologic calcification of the tracheal cartilage rings beginning around one month after birth. This calcification progresses in a dynamic rostro-caudal sequence through a process involving terminal differentiation of tracheal chondrocytes. These findings challenge the prevailing view that tracheal cartilage is a permanent hyaline cartilage and instead reveals an early and highly organized physiologic calcification process. Furthermore, our findings further identify MGP as a key regulator of calcification within the respiratory tract. Indeed, MGP deficiency not only markedly accelerates tracheal cartilage calcification through a similar pattern and mechanism but also induces a previously unrecognized calcification of the tracheal mucosa, a lesion absent in wild-type mice. These alterations could partly explain the respiratory manifestations observed in MGP-deficient patients having Keutel syndrome, which include dyspnea, cough, stridor, and recurrent infections, symptoms previously being mainly attributed to calcification of the tracheal cartilage rings.

cell biology↗