Potential Mechanism of Luteolin in Promoting MC3T3-E1 Proliferation and Osteo-genic Differentiation by Activating the PI3K/AKT Signaling Pathway
Objective Based on network pharmacology and experimental validation,this study aims to explore the potential mechanism by which luteolin promotes the proliferation and osteogenic differentiation of MC3T3-E1 Subclone 14 cells via the PI3K/AKT signaling pathway,thereby providing a rational basis for luteolin's potential to promote osteogenesis and improve postmenopausal osteoporosis(PMOP).Methods Using network pharmacology,the target genes regulating os-teogenic differentiation by luteolin were identified.A protein-protein interaction(PPI)network was constructed,followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,as well as molecular docking.Cell cytotoxicity was assessed by CCK-8 and cell colony formation assays.The macro-regulatory mechanism of luteolin on osteogenic differentiation was studied by alkaline phosphatase(ALP)staining and activity measurement,alizarin red staining,and calcium salt quantification.Furthermore,Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the differential expression of mRNA and proteins related to the PI3K/AKT signaling pathway.Finally,immunofluorescence was used to examine the translocation of β-catenin to the cell nucleus,clarifying the impact of luteolin on the expression of key genes and proteins in the PI3K/AKT signaling pathway.Results A total of 109 target genes related to osteogenic differentiation regulated by luteolin were identified.These genes were primarily in-volved in biological processes such as cell proliferation and regulation of cell proliferation.Four target genes were found to act on the PI3K/AKT signaling pathway,and all were able to bind well with luteolin.Additionally,luteolin(5 μmol/L,10 µmol/L)significantly enhanced the viability of MC3T3-E1 Subclone 14 cells and promoted their proliferation(P＜0.05).Moreover,luteolin increased ALP activity and calcium deposition(P＜0.05).Luteolin(5 µmol/L,10 µmol/L)up-regulated the mRNA expression of Pi3k,β-catenin,C-myc,and Cyclin D1 in MC3T3-E1 Subclone 14 cells(P＜0.05),and promoted the phosphorylation of PI3K,AKT1,and GSK3β,subsequently upregulating the protein expression ofβ-catenin,C-MYC,and CYCLIN D1,as well as increasing the ratios of p-PI3K/PI3K,p-AKT1/AKT1,and p-GSK3 β/GSK3β(P＜0.05).Additionally,luteolin facilitated the translocation of β-catenin to the cell nucleus,thereby promoting osteogenic differentiation of MC3T3-E1 Subclone 14 cells.Conclusion Luteolin regulates the expression of osteogenesis-related genes through the PI3 K/AKT signaling pathway,significantly promoting the osteogenic differentiation of MC3T3-E1 Subclone 14 cells.
万方数据
维普
