Naringenin modulates the NO-cGMP-PKG signaling pathway through AKT binding to enhance osteogenic differentiation in hPDLSCs

Naringenin (NAR) is a prominent flavanone, and it has been recognized for its capacity to promote osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in prior research. The present study aimed to explore how NAR promotes the osteogenic differentiation of hPDLSCs and to assess its efficacy in repairing alveolar bone defects. In the present study, the protein-protein interaction (PPI) network of NAR action was established by mRNA sequencing and network pharmacological analysis. Gene and protein expression were evaluated by PCR and western blotting. Alizarin red and alkaline phosphatase staining were employed to observe the osteogenic capacity of hPDLSCs, and immunofluorescence was used to examine the co-localization of NAR molecular probes and AKT in cells. Repair of mandibular defects was assessed by micro computed tomography (micro-CT), Masson staining and immunofluorescence. Additionally, computer simulation docking software was utilized to determine the binding affinity of NAR to the target protein AKT. The results demonstrated that activation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway promoted the osteogenic differentiation of hPDLSCs. Inhibition of AKT, endothelial nitric oxide synthase and soluble guanylate cyclase individually attenuated NAR’s ability to promote the osteogenic differentiation of hPDLSCs. Micro-CT and Masson staining revealed more new bone formation at the defect site in the NAR gavage group. Immunofluorescence assays confirmed upregulated expression of Runt-related transcription factor 2 and osteopontin in the NAR gavage group. In conclusion, the present study suggests that NAR promotes the osteogenic differentiation of hPDLSCs by activating the NO-cGMP-PKG signaling pathway through its binding to AKT.