FoxO1 Agonists Promote Bone Regeneration in Periodontitis by Protecting the Osteogenesis of Periodontal Ligament Stem Cells

Abstract

Protecting the function of periodontal ligament stem cells (PDLSCs) is crucial for bone regeneration in periodontitis. Forkhead box protein O1 (FoxO1) has been previously reported as a crucial mediator in bone homeostasis, providing a favorable environment for osteoblast proliferation and differentiation. In this study, we investigated the effect and mechanism of FoxO1 agonists on the osteogenesis of PDLSCs under inflammatory conditions. In this study, we screened FoxO1 agonists by detecting their effects on the osteogenic differentiation of PDLSCs. Then, the function of these agonists in bone regeneration was analyzed in the periodontitis model. We found that hyperoside or 2-furoyl-LIGRLO-amide trifluoroacetate salt (2-Fly) promoted osteogenic differentiation under inflammation by simultaneously inhibiting nuclear factor κB (NF-κB) activation, β-catenin expression, and reactive oxygen species (ROS) production. Moreover, local injection of hyperoside or 2-Fly rescued the expression of FoxO1 and runt-related transcription factor 2 (Runx2) in vivo, alleviating alveolar bone loss and periodontal ligament damage. These findings suggested that FoxO1 agonists exerted a protective effect on osteogenesis in PDLSCs, as a result, facilitating bone formation under inflammatory conditions. Taken together, FoxO1 might serve as a therapeutic target for bone regeneration in periodontitis by mediating multiple signaling pathways.

Introduction

Periodontitis is an infectious disease caused by periodontal pathogenic microbiota, which eventually results in tooth loss [1]. Lipopolysaccharide of pathogenic microbes stimulates Toll-like receptors, leading to inflammatory response activation and proinflammatory cytokine production. Potent proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), and interferon γ (IFN-γ), modulate osteoclast differentiation and activation, resulting in alveolar bone resorption and attachment apparatus damage [2]. TNF-α stimulation inhibits the expression of osteogenic genes and upregulates nuclear factor κB ligand (RANKL) expression in osteocytes [3 –5]. IFN-γ could facilitate osteoclast differentiation and trigger local inflammation [6]. Moreover, TNF-α and IFN-γ inhibit osteogenic differentiation of periodontal ligament stem cells (PDLSCs) through p38 MAPK and JNK signaling, which is implicated in the initiation and progression of periodontitis [7,8].

PDLSCs are the critical cellular constituent of the periodontal ligament and are closely related to alveolar bone and periodontal tissue repair [9]. However, osteoblast gene expression, alkaline phosphatase (ALP) activity, and mineralization nodule formation are decreased in PDLSCs obtained from periodontitis patients [10]. Furthermore, an increased RANKL/OPG ratio in PDLSCs exacerbates bone resorption and periodontal inflammation [11,12]. Inflammatory factors hinder the osteogenesis of PDLSCs through complex molecular signaling pathways, including oxidative stress, nuclear factor κB (NF-κB), Wnt/β-catenin, and RANKL/RANK/OPG pathways [9,13].

Activation of the NF-κB signaling pathway exaggerates the release of inflammatory cytokines and production of reactive oxygen species (ROS), leading to a depression of osteogenesis in PDLSCs in inflammatory environment. Furthermore, NF-κB signaling upregulates the expression of RANKL in the osteoblast lineage, promoting osteoclast differentiation and causing alveolar bone destruction [14,15].

Under inflammatory conditions, phosphorylation of GSK3β increases the accumulation of nuclear β-catenin, which impairs the osteogenic differentiation of PDLSCs induced by runt-related transcription factor 2 (Runx2) [4]. Moreover, depressing the Wnt/β-catenin pathway using ICG001 reduces the expression of MMP-7 and MMP-9, which is beneficial for alleviating inflammation [16]. The production of ROS was increased to defend against invading pathogenic microorganisms. However, excessive ROS production can further exacerbate the inflammatory response, resulting in decreased osteogenesis of PDLSCs and ultimately, periodontal tissue destruction [13,17]. Inhibiting the ROS/JNK signaling pathway could decrease the RANKL/OPG ratio of PDLSCs and facilitate periodontal regeneration [11].

Therefore, the key issue in periodontitis therapy is to block the inflammatory pathways and protect the osteogenesis of PDLSCs.

Forkhead box protein O1 (FoxO1), a member of the FoxO family, regulates several cellular processes, including redox balance, apoptosis, immune response, and autophagy, through multiple signaling pathways [18,19]. FoxO1 is highly expressed in osteoblasts and positively regulates osteoblast proliferation and bone formation by interacting with ATF4. The activation of the FoxO1/β-catenin pathway by natural antioxidants improves osteogenic differentiation, indicating that increased expression of FoxO1 could facilitate bone formation [20]. Furthermore, FoxO1 is involved in mediating different inflammatory-related signaling pathways. AKT-mediated FoxO1 translocation to the cytoplasm induces NF-κB activation and inflammation progression [21]. Upregulation of FoxO1 could reverse the imbalance of Th17/Treg and alleviate inflammation [22]. In our previous study, we found that FoxO1 overexpression could enhance the antioxidative ability and osteogenic differentiation of PDLSCs under inflammatory conditions, suggesting that FoxO1 might serve as a therapeutic target for bone regeneration in periodontitis [23].

In the present study, we first investigated whether these FoxO1 agonists could block the downstream signaling cascades of proinflammatory factors in PDLSCs. We then detected the effect of small-molecule-induced FoxO1 activation on the osteogenic differentiation of PDLSCs under inflammation. Finally, we constructed a rat periodontitis model to clarify whether FoxO1 agonists protect against inflammatory bone destruction in vivo.

Materials and Methods

Cell isolation and culture

Primary cultures of PDLSCs were obtained from premolars for orthodontic reasons and impacted third molars of 12- to 20-year-old healthy donors (n = 12). All samples were collected at the Oral and Maxillofacial Surgery Clinic of West China Hospital of Stomatology. The study was approved by the Ethics Committee of West China Hospital of Stomatology in Sichuan University (No. WCHSIRB-D-2022-292). More detailed descriptions of PDLSCs isolation and culture are located further within the Supplementary Data.

Intracellular ROS measurement

After 24 h of stimulation, the intracellular ROS level of PDLSCs was detected by flow cytometric analysis. Further information regarding the detailed procedure for ROS measurement is provided in the Supplementary Data.

CCK-8 assay

Cell viability was determined by the CCK-8 assay after 48 h of treatment. The Supplementary Data contain a more comprehensive description of the CCK-8 assay.

Relative quantification for quantitative real-time PCR

After a 7-day osteogenic induction, the mRNA levels of the osteogenic genes were measured by quantitative real-time PCR (RT-qPCR). A complete process of RT-qPCR is provided in the Supplementary Data and the primer sequences are listed in Supplementary Table S1.

Western blot analysis

We extracted nuclear proteins to detect the activity of NF-κΒ/p65, β-catenin, and FoxO1 after 1 h of treatment. Whole cell lysates were prepared to detect protein levels after 24 h of treatment. More detailed descriptions of the western blot procedure can be found in the Supplementary Data.

ALP and Alizarin Red S staining

ALP staining was performed to assess the osteogenic potential after 7 days of osteogenic differentiation induction. Alizarin Red staining was conducted to assess mineralized nodule formation after 21 days of osteogenic induction. A detailed description of the ALP and Alizarin Red S staining procedure can be found in the Supplementary Data.

Animal study

Twelve adult female Sprague‒Dawley rats (225.875 ± 19.043 g) were purchased from DS Experimental Animals Co. Ltd. Animal treatments were conducted after getting the permission from the Ethics Committee of Sichuan University (No. WCHSIRB-D-2022-411). The periodontitis model was established as previous studies described [24,25]. Briefly, a 4-0 silk ligature was tied around the maxillary right second molar of each rat for 30 days, and the left side served as the baseline control. The ligatures were checked every 3 days. Ligature-induced periodontitis rats were randomly assigned to three groups: PBS (periodontitis + vehicle, periodontitis group), 0.45 mg/kg hyperoside (periodontitis + hyperoside, hyperoside group), 1.5 μg/kg 2-furoyl-LIGRLO-amide trifluoroacetate salt (periodontitis +2-Fly, 2-Fly group). Subperiosteal injections were received in three areas: the distal of the molar, the mesial of the molar, and the middle of the molar every 3 days according to previous studies [26 –29]. After 30 days of treatment, the animals were sacrificed, and maxillary tissues were harvested.

Microcomputed tomography imaging

Maxillary samples were scanned using microCT 50 (Scanco Medical, Switzerland) with medium resolution (500 projections/180°) and 8 μm voxel size. Three-dimensional images and multiplanar reconstructions (MPR) of maxillary specimens were reconstructed and obtained by the MicroCT system software. The distance from the cement–enamel junction (CEJ) to the alveolar bone crest (ABC) was measured to evaluate alveolar bone loss using the MPR images. The area between furcation and the root apexes of the maxillary second molar was selected as the region of interest (ROI), and bone/tissue volume (BV/TV, %) in the ROI was calculated.

Histological analysis and immunofluorescent staining

A more detailed description of histological analysis and immunofluorescent staining is located within the Supplementary Data.

Statistical analysis

Data were analyzed using SPSS26 statistical software (SPSS, Inc., Chicago, IL), and Student's t test or one-way ANOVA was used to evaluate significant differences. P values of <0.05 were considered statistically significant.

Results

Inflammatory factors inhibit the osteogenic differentiation of PDLSCs

The destiny of mesenchymal stem cells (MSCs) is modulated by cues in the microenvironment. Inflammatory factors in periodontal tissues play critical roles in the impairment of PDLSC-mediated alveolar bone regeneration. To investigate the molecular pathways that inhibit osteogenic differentiation of PDLSCs during periodontitis, we applied an in vitro model of PDLSCs treated with inflammatory factors. The PDLSCs used in this study positively expressed (>95%) CD73, CD90, and CD105 and did not express (<5%) CD11, CD19, and CD45 (Supplementary Fig. S1A). Colony-forming assays showed that PDLSCs displayed colony-formation ability (Supplementary Fig. S1B). Alizarin Red S staining and Oil Red O staining confirmed that PDLSCs possessed osteogenic and adipogenic differentiation potential (Supplementary Fig. S1C, D), indicating that the isolated PDLSCs presented the characteristics of MSCs.

Under normal osteogenic conditions, PDLSCs highly expressed ALP, a marker of osteogenesis, and generated abundant mineral nodules, as indicated by ALP staining and Alizarin Red S staining. To investigate the effect of the inflammatory microenvironment on the osteogenic differentiation of PDLSCs in vitro, we treated PDLSCs with TNF-α and INF-γ as previously reported [7,30,31]. After treatment, we observed a significant decrease in ALP activity and mineralized nodule formation (Fig. 1A, B), indicating that the osteogenic potential of PDLSCs was severely impaired.

FIG. 1.

Impaired osteogenesis was accompanied by inflammatory signaling pathway activation and decreased FoxO1 expression under inflammation. (A) Entire plate views of ALP activity and mineralized nodules in PDLSCs. ALP and Alizarin Red staining, respectively, was performed after the 7-day and 21-day osteogenic induction. (B) Qualitative assessment of mineralized nodules. (C) The level of FoxO1 in the nucleus and whole cell extract, respectively, was analyzed after 1 and 24 h inflammatory cytokines stimulation. (D) Quantitative and statistical analyses of FoxO1 level. Nucleus protein levels were normalized to TBP, and whole-cell protein was normalized to β-actin. (E) Western blot analysis of β-catenin and p65 in the nucleus (1 h stimulation) and whole cell extract (24 h stimulation). (F) Quantitative and statistical analyses of β-catenin and p65 level. (G) ROS levels were measured by flow cytometry in PDLSCs treated with TNF-α and INF-γ for 24 h. (H) Schematic diagram of the relationship among FoxO1, osteogenesis, and several signaling pathways in the inflammatory environment. FoxO1 might participate in regulating NF-κB/p65 activation, β-catenin expression, and ROS production. However, TNF-α and IFN-γ inhibited the expression of FoxO1 and activated these inflammatory and osteogenic signaling pathways, leading to impaired osteogenesis of PDLSCs. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. FoxO1, forkhead box protein O1; PDLSC, periodontal ligament stem cell; ALP, alkaline phosphatase; ROS, reactive oxygen species; TNF-α, tumor necrosis factor α; IFN-γ, interferon γ.

FoxO1 is a central target of inflammatory factors to inhibit osteogenic differentiation of PDLSCs

FoxO1 has been reported to be a critical regulator of bone formation [18]. We extracted nuclear protein and whole cell lysates to measure the activity and total protein expression of FoxO1, respectively. Our findings showed that the total protein expression of FoxO1 was decreased after TNF-α and INF-γ stimulation, as shown in Fig. 1C and D. Although FoxO1 nuclear translocation was reduced, there was no statistically significant difference observed (Fig. 1C, D). These results suggest that inflammation decreases the total expression of FoxO1 rather than its activity.

By checking published articles and the KEGG database, we found that FoxO1 participates in mediating inflammatory- and osteogenic-related signaling, including the oxidative stress pathway, Wnt/β-catenin pathway, and NF-κB pathways. According to previous reports, FoxO1 can activate the antioxidant system to alleviate oxidative stress and block the NF-κB p65-mediated inflammatory response. Moreover, FoxO1 inhibits the Wnt/β-catenin-signaling pathway by directly binding with β-catenin [32 –34]. The oxidative stress pathway, Wnt/β-catenin pathway, and NF-κB signaling pathway are involved in the inhibition of PDLSCs osteogenesis. Treatment with TNF-α and IFN-γ led to an upregulation of NF-κB/p65 and β-catenin translocation to the nucleus, as well as an increase in their expression levels in whole cell lysates, suggesting that their activity and expression were increased under inflammatory conditions (Fig. 1E, F). In addition, inflammation resulted in increased production of ROS (Fig. 1G).

Therefore, we speculated that FoxO1 participates in regulating multiple signaling pathways correlated with inflammation and osteogenesis under inflammatory conditions. The suppression of FoxO1 might lead to inflammatory signaling pathway activation and further impair the osteogenic differentiation of PDLSCs (Fig. 1H). We then evaluated whether increasing FoxO1 expression by drugs could recover PDLSCs function and reduce inflammatory bone loss in periodontitis.

FoxO1 agonists depressed β-catenin, NF-κB signaling, and oxidative stress in PDLSCs

To investigate whether FoxO1 is a therapeutic target of periodontitis, we selected four FoxO1 agonists, hyperoside, β-hydroxybutyric acid (BHB), 2-Fly, and paclitaxel, to treat PDLSCs in the inflammatory microenvironment.

First, we assessed the cytotoxicity of each FoxO1 agonist by CCK-8 assay. The results showed that high concentrations of hyperoside and paclitaxel significantly inhibited the viability of PDLSCs (Fig. 2A). To avoid the inhibitory effect of FoxO1 agonists on PDLSC proliferation, 150 μM hyperoside, 10 mM BHB, 10 μg/mL 2-Fly, and 50 nM paclitaxel were used for subsequent experiments.

FIG. 2.

FoxO1 agonists depressed β-catenin, NF-κB signaling, and oxidative stress in PDLSCs. (A) Cell viability was measured by CCK-8 assay in PDLSCs treated with various concentrations of FoxO1 agonists for 48 h. (B) FoxO1 levels in the nucleus (1 h stimulation) and whole cell extract (24 h stimulation). (C) Quantification and statistical analysis of FoxO1 level. (D) The levels of β-catenin and p65 proteins in the nucleus (1 h stimulation) and whole cell extract (24 h stimulation). (E) Quantification and statistical analysis of β-catenin and p65 level. (F) ROS levels were measured by flow cytometry in PDLSCs treated with FoxO1 agonists for 24 h. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Western blot analysis demonstrated that hyperoside and 2-Fly enhanced FoxO1 translocation to the nucleus, indicating an increase in the activity of FoxO1. Additionally, BHB and 2-Fly promoted the protein expression of FoxO1 (Fig. 2B, C), consistent with previous findings [35 –37]. Although there was no statistical difference, NF-κB/p65 and β-catenin nuclear translocation showed a downward trend after FoxO1 agonist treatment. Moreover, hyperoside, BHB, 2-Fly, and paclitaxel reduced the total protein levels of β-catenin and NF-κB/p65 (Fig. 2D, E). In particular, hyperoside and 2-Fly, notably the former, decreased ROS levels under inflammation (Fig. 2F). These results indicated that FoxO1 agonists could depress inflammatory signaling by reducing the total protein levels of β-catenin and NF-κB/p65 and decreasing ROS production.

FoxO1 agonists protected the osteoblast differentiation of PDLSCs in inflammatory environments

Under homeostatic conditions, FoxO1 agonists promoted the mRNA expression of osteoblast markers (Fig. 3A). To further evaluate the protective effect of FoxO1 agonists under inflammatory conditions, we measured the osteogenic potential of PDLSCs. RT-qPCR results showed that hyperoside increased the expression of OCN, BSP, and BMP4. BHB induced the expression of OCN and BSP. Two-Fly upregulated the level of BMP4, OCN, and Runx2. Paclitaxel increased OCN and Runx2 expression (Fig. 3B). Furthermore, hyperoside and 2-Fly promoted the ALP activity and bone-mineralized nodule formation under inflammation (Fig. 3C, D). The results suggested that hyperoside and 2-Fly could rescue the dysfunction of PDLSCs and were thus selected for subsequent experiments.

FIG. 3.

FoxO1 agonists protected the osteoblast differentiation of PDLSCs in inflammatory environments. (A) Relative mRNA levels of osteogenic markers were detected by RT-qPCR in PDLSCs incubated with osteogenic induction medium containing hyperoside, BHB, 2-Fly, or paclitaxel under homeostatic conditions for 7 days. (B) Osteogenic marker level of PDLSCs was detected under inflammatory conditions for 7 days. (C) Entire plate views of ALP activity (7 days osteogenic induction) and mineralized nodules (21 days osteogenic induction) under inflammatory conditions. (D) Qualitative assessment of mineralized nodules. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Hyperoside and 2-Fly protected the osteoblast differentiation ability of PDLSCs through FoxO1

To confirm the role of FoxO1 in protecting osteogenic differentiation under inflammatory conditions, PDLSCs were treated with 1 μM FoxO1 inhibitor AS1842856 (Fig. 4A). The effects of hyperoside and 2-Fly on ALP activity and mineralized nodule formation could be depressed by AS1842856 (Fig. 4A). RT-qPCR results showed that the depression of FoxO1 significantly decreased the expression of BMP4 (Fig. 4B). These results demonstrated that hyperoside and 2-Fly regulated osteoblast differentiation through FoxO1.

FIG. 4.

Hyperoside and 2-Fly protected the osteoblast differentiation ability of PDLSCs through FoxO1. (A) Entire plate views of ALP activity (7-day osteogenic induction) and mineralized nodules (21-day osteogenic induction) with or without FoxO1 inhibitor AS1842856 treatment under inflammatory conditions. (B) Relative mRNA levels of BMP4 were detected by RT-qPCR in PDLSCs under inflammatory conditions for 7 days. Data are shown as the mean ± SD. ****P < 0.0001.

Hyperoside and 2-Fly reduced bone loss in the periodontitis model

Encouraged by these results, we further verified the effect of hyperoside and 2-Fly on bone regeneration in the periodontitis model of rats (Fig. 5A). Micro-CT scanning results revealed that the vehicle group showed significant alveolar bone destruction at the ligature site, whereas hyperoside or 2-Fly injection partially rescued inflammation-mediated bone loss (Fig. 5B). To further evaluate the extent of alveolar bone loss, the CEJ-ABC distance was measured, which is an indicator of the severity of alveolar bone loss. The vehicle group exhibited an increased CEJ-ABC distance and lower bone quantity (BV/TV) compared with the sham group. However, hyperoside or 2-Fly administration decreased the CEJ-ABC distance and increased BV/TV (Fig. 5C, D). Furthermore, immunofluorescence staining showed increased expression of Runx2 after injection of these FoxO1 agonists (Fig. 5E). These results indicated that hyperoside and 2-Fly could alleviate alveolar bone loss in periodontitis rats.

FIG. 5.

Hyperoside and 2-Fly reduced bone loss and periodontal inflammation in the periodontitis model. (A) Establishment of the periodontitis model in rats. A 4-0 silk ligature was tied around the maxillary right second molar of each rat for 30 days. Injection sites are indicated with blue arrows. (B) 3D images and MPR of maxillary specimens were reconstructed. The red continuous dotted line shows alveolar bone absorption. The blue line with an arrow indicates the CEJ-ABC distance of the ligature site. (C) BV/TV of the alveolar bone. (D) CEJ-ABC distance of mesial side. (E) Representative photomicrographs of Runx2 expression in maxillary tissues were determined by immunofluorescence staining (Scale bar = 100 μm). (F) Representative photomicrographs of maxillary tissue sections were presented by HE staining (Scale bar = 100 μm). B, bone; D, dentin; DP, dental pulp; PDL, periodontal ligament. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

To investigate whether hyperoside and 2-Fly could relieve inflammation-mediated periodontal soft tissue damage, a histological assessment was performed. Attachment loss and gingival destruction were observed in the vehicle group, whereas drug injection alleviated ligature-induced tissue damage (Fig. 5F).

We observed that FoxO1 levels declined after periodontitis induction, but hyperoside and 2-Fly injection rescued the expression of FoxO1 (Fig. 6A). Immunofluorescence results showed that β-catenin, p65, and p-p65 expression were reduced after hyperoside and 2-Fly injection (Fig. 6B–D), suggesting that FoxO1 agonists could depress β-catenin expression and NF-κB signaling activation, which contributes to bone regeneration and local inflammation control.

FIG. 6.

Hyperoside and 2-Fly depressed β-catenin and NF-κB signaling in vivo. Representative photomicrographs of immunofluorescence staining for FoxO1 (A), β-catenin (B), p65 (C), and p-p65 (D) levels. Scale bar = 100 μm.

Taken together, these results demonstrated that recovering FoxO1 expression by hyperoside or 2-Fly could promote bone and soft tissue regeneration in vivo by mediating β-catenin and NF-κB signaling, improving regeneration efficiency in the periodontitis model.

Discussion

As reported previously, the inflammatory microenvironment suppresses the osteogenic differentiation potential of PDLSCs by activating multiple signaling pathways in periodontitis [9,13]. Thus, protecting PDLSC's functions is the key factor in reversing bone destruction. In the present study, we found that FoxO1 expression was decreased both in PDLSCs and local periodontal tissues of mimic periodontitis models. However, restoring FoxO1 expression using hyperoside or 2-Fly increased osteoblastic differentiation and bone regeneration in the inflammatory environment. Additionally, these FoxO1 agonists inhibited inflammation-related signaling pathways, including NF-κB signaling, oxidative stress, and Wnt/β-catenin pathway. Our results suggest that FoxO1 is a common mediator that represses these inflammatory signaling pathways to protect the osteogenesis of PDLSCs. Therefore, increasing FoxO1 expression using drugs may be beneficial for inflammation resolution and bone regeneration (Fig. 7).

FIG. 7.

The schematic diagram depicts the effect of FoxO1 agonists on osteogenesis of PDLSCs through several signaling pathways under inflammatory conditions. Inflammatory cytokines, such as TNF-α and IFN-γ, can inhibit osteoblast differentiation by activating β-catenin, NF-κB signaling, and oxidative stress pathways. However, FoxO1 agonists could block these inflammatory and osteogenic signaling pathways, leading to the protection of PDLSC osteogenesis. As a result, periodontal inflammation and bone destruction can be alleviated.

FoxO1, recently reported as a positive mediator in osteogenesis, controls bone remodeling and cartilage tissue maintenance [38,39]. The interaction of FoxO1 with ATF4 promotes protein synthesis and confers redox resistance in osteoblasts through the p19/p16/p53 signaling cascade, regulating osteoblast proliferation and bone homeostasis [18]. FoxO1 also mediates cell viability, autophagy, and apoptosis of osteoblast precursor cells through the target genes, cyclin D1, Bim, Gadd45a, and Rab7. Curculigoside, a natural compound from plants, was reported to promote the expression of osteogenic markers and mineralized nodule formation of MSCs and rescue bone loss in osteoporosis mice by increasing the expression of FoxO1 [40].

In the present study, we found that FoxO1 agonists promoted the expression of the osteogenic genes. Hyperoside and 2-Fly, among the FoxO1 agonists, increased ALP activity and mineralized nodule formation and this effect could be depressed by FoxO1 inhibitor. Furthermore, hyperoside and 2-Fly partly rescued bone loss in periodontitis rats, indicating that improving FoxO1 expression contributes to bone destruction repair.

Multiple signaling pathways are involved in periodontal inflammation progression. Proinflammatory cytokines induce the activation of NF-kB and Wnt/β-catenin signaling, which influences the osteogenic differentiation potential of MSCs. Our findings suggest that FoxO1 agonists could suppress NF-κB signaling by reducing p65 expression. Under inflammatory conditions, the NF-κB pathway has been shown to inhibit osteoblast activity and mineralization. Therefore, by inhibiting NF-κB/p65, FoxO1 agonists can promote osteogenic differentiation of PDLSCs, thus preventing bone destruction [41 –43]. Wnt/β-catenin, which is closely linked to bone formation, exerts dual effects on the osteogenesis of MSCs [44 –47]. Wnt signaling activation could promote osteogenic gene expression and protect against bone loss in osteoporosis [48]. However, the compromised osteogenic potential of PDLSCs might be related to Wnt/β-catenin signaling activation under inflammatory conditions.

Previous studies have shown that inflammation-induced β-catenin activation suppresses the osteogenic differentiation of PDLSCs through the noncanonical Wnt/Ca²⁺ pathway, while β-catenin knockdown accelerates nodule formation [49]. Consistent with these findings, our study showed that decreasing β-catenin expression is associated with increased osteogenic potential and bone formation, suggesting that β-catenin may act as a negative mediator of osteogenesis in the inflammatory environment.

Oxidative stress is a crucial pathophysiological mechanism associated with periodontitis progression and plays an important role in cellular therapy. Targeting oxidative stress has been proven to promote the therapeutic potential of PDLSCs in periodontitis [13,50,51]. Resveratrol and curcumin, which function largely as antioxidants, control periodontal inflammation and reverse periodontal tissue destruction by decreasing IL-1β, IL-6, and MMP-9 production. Resveratrol could reestablish the redox balance, suppress apoptosis, and promote osteogenesis through the SIRT1/FoxO1 pathway [52]. In the present study, hyperoside and 2-Fly reduced the level of ROS in PDLSCs. These drugs may confer oxidative resistance by increasing FoxO1 expression, facilitating osteogenic differentiation in PDLSCs, and periodontal tissue regeneration in vivo.

Involvement in regulating the proliferation, differentiation, and function of different immune cells, FoxO1 mediates the immune response in the progression of many diseases and has emerged as a therapeutic target for tumors, neoplastic parasites, and diabetes [19,53 –55]. On the one hand, the potential therapeutic effect of FoxO1 on cancer metastasis and cancer immunity by participating in NK cell activation and IL-27 upregulation has been previously proven [56,57]. On the other hand, FoxO1 is considered a control point for T cell differentiation-related gene expression, controlling chronic infection by mediating the differentiation and survival of T cells [58]. Furthermore, FoxO1 mediates dendritic cell recruitment and adaptive immunity activation, increasing susceptibility to periodontitis. Deletion of FoxO1 in dendritic cells activates the immune response by upregulating the levels of inflammatory cytokines and RANKL expression, leading to osteoclast formation and periodontitis development [59].

We found that FoxO1 agonists exert a protective effect on osteogenic differentiation in PDLSCs and bone formation in vivo by inhibiting inflammation-related pathways, indicating that FoxO1 might be a potential target for bone regeneration in periodontitis. Because FoxO1 participates in osteoblast proliferation, osteoclast differentiation, and the immune response, it is reasonable to speculate that the effect of FoxO1 on bone formation might be associated with immune cell regulation. However, the link between the immune response and osteogenesis regulated by FoxO1 under pathological conditions is unclear, and the role of FoxO1 in regulating the immune response in the pathogenesis of periodontitis needs further exploration.

Conclusions

Our work showed that increasing FoxO1 expression could protect the functions of PDLSCs in the inflammatory environment and partly rescue alveolar bone destruction. FoxO1 might simultaneously block β-catenin, NF-κB signaling, and oxidative stress, exerting protective effects on osteoblast differentiation and bone formation under inflammatory conditions. The present study provided novel insight into the potential of FoxO1 agonists in periodontitis treatment, which might also help to increase the therapeutic effect in other inflammation-related bone diseases, such as osteoporosis, osteoarthritis, and rheumatoid arthritis.

Footnotes

Author Disclosure Statement

The authors declare that there are no conflicts of interest regarding the publication of this article.

Funding Information

This study was supported by National Natural Science Foundation of China (82170947, 81600912, and 82071092), the Technology Innovation Research and Development Project of Chengdu (2022-YF05-01388-SN), the Key Project of Sichuan province, China (No. 2023YFS0056 and 2023YFS0151).

Supplementary Material

Supplementary Data

Supplementary Table S1

Supplementary Figure S1

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