Abstract：Objective: To investigate the transcriptional activation effect of the transcription factor Forkhead box O1 (FoxO1) on the autophagy-related protein Beclin-1 in human dental periodontal stem cells, as well as the regulatory mechanisms of FoxO1 on autophagy, inflammation, and osteogenic differentiation in human periodontal ligament stem cells (PDLSCs). Methods: Human PDLSCs were divided into four groups: control group (human PDLSCs were cultured in Dulbecco's Modified Eagle Medium (DMEM) for 24hours, then replaced with fresh DMEM and cultured for one week), osteogenic induction group (human PDLSCs were cultured in DMEM for 24hours, then replaced with DMEM containing 100nmoL/L dexamethasone, 10mmoL/L beta-glycerophosphate, and 80 mg/L vitamin C and cultured for one week), empty vector group (EV; human PDLSCs were transfected with an empty vector plasmid for 24hours, then replaced with fresh DMEM and cultured for one week), and FoxO1 overexpression group (FoxO1-OE; human PDLSCs were transfected with a recombinant plasmid overexpressing FoxO1 for 24hours, then replaced with fresh DMEM and cultured for one week). The expression of FoxO1 and Beclin-1 in the cell lysate was analyzed using quantitative PCR (qPCR) and Western blot. Dual-luciferase assays were utilized to verify the binding of FoxO1 to the Beclin-1 promoter region. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure the levels of inflammatory cytokines (Interleukin-1β (IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α)) in the culture supernatants of each group. Western blotting was employed to detect the expression of autophagy markers p62, Microtubule-associated protein 1 light chain 3-I (LC3-I), and Microtubule-associated protein 1 light chain 3-II (LC3-II). Alkaline Phosphatase (ALP) staining and Alizarin Red S staining were used to assess osteogenic differentiation and calcium nodule formation in each group. Immunofluorescence was utilized to detect the expression of the transcription factor Runx2. Results: Compared to the control group, the osteogenic induction group exhibited increased mRNA and protein levels of FoxO1 and Beclin-1 (P<0.05), and the same trend was observed when comparing the EV group to the FoxO1-OE group (P<0.05). The dual-luciferase assay demonstrated that FoxO1 significantly increased the luciferase activity of the Beclin-1 wild-type (WT) group (P<0.05), but had no effect on the Beclin-1 mutant (MUT) group (P > 0.05). Compared to the control group, the osteogenic induction group showed a decrease in p62 protein levels and an increase in the LC3-II/LC3-I ratio (P<0.05); similar differences were observed between the EV group and the FoxO1-OE group (P<0.05). Osteogenic induction did not significantly affect the levels of inflammatory cytokines (IL-1β, IL-6, TNF-α) (P>0.05), but FoxO1 overexpression significantly reduced these cytokines' levels (P<0.05). Both osteogenic induction and FoxO1 overexpression resulted in increased ALP and calcium nodule staining areas, accompanied by upregulated Runx2 expression (P<0.05). Conclusion: FoxO1 promotes autophagy through the activation of Beclin-1 expression in human PDLSCs, reduces inflammation, and thereby enhances osteogenic differentiation.
[1] Bee SL,Hamid ZAA.Hamid,Asymmetric resorbable-based dental barrier membrane for periodontal guided tissue regeneration and guided bone regeneration:a review,Journal of biomedical materials research[J].Biomed Mater Res B Appl Biomater,2022,110(9):2157-2182. [2] Zhang Y,Wang P,Wang Y,et al.Gold nanoparticles promote the bone regeneration of periodontal ligament stem cell sheets through activation of autophagy[J].Int Nanomedicine,2021,6(16):61-73. [3] Ying S,Tan M,Feng G,et al.Low-intensity pulsed ultrasound regulates alveolar bone homeostasis in experimental periodontitis by diminishing oxidative stress[J].Theranostics,2020,10(21):9789-9807. [4] Wang Q,Shi W,Lin S,et al.FOXO1 regulates osteogenic differentiation of periodontal ligament stem cells through the METTL3 signaling pathway[J].Orthop Surg Res,2023,18(1):637. [5] Parry TL,Tichy L,Brantley JT.Cardioprotective effects of preconditioning exercise in the female tumor bearing mouse[J].Front Cell Dev Biol,2022,1(10):950479. [6] Xie L,Li W,Zheng XQ,et al.Treponema pallidum membrane protein Tp47 induced autophagy and inhibited cell migration in HMC3 cells via the PI3K/AKT/FOXO1 pathway[J].Cell Mol Med,2023,27(20):3065-3074. [7] Huang W,Cheng C,Shan WS,et al.Knockdown of SGK1 alleviates the IL-1β-induced chondrocyte anabolic and catabolic imbalance by activating FoxO1-mediated autophagy in human chondrocytes[J].FEBS,2020,287(1):94-107. [8] Xu K,Zhu W,Xu A,et al.Inhibition of FOXO1-mediated autophagy promotes paclitaxel-induced apoptosis of MDA-MB-231 cells[J].Mol Med Rep,2022,25(2):72. [9] Huang X,Su X,Ma Q,et al.FoxO1 agonists promote bone regeneration in periodontitis by protecting the osteogenesis of periodontal ligament stem cells[J].Stem Cells Dev,2023,32(15-16):491-503.
2025, Vol. 31 
冀公网安备13080202000786号