Aging induces chondrocytes to secrete a senescence-associated secretory phenotype (SASP), leading to mitochondrial dysfunction and imbalanced regulation of cell death through multiple mechanisms, including oxidative stress, inflammatory response, apoptosis, and autophagy, thereby promoting the development of OA (34).

Redox homeostasis is a dynamic balance of oxidation and reduction reactions within cells, maintained through various antioxidant enzymes, thioredoxin systems, and small molecule antioxidants (35–37). Oxidative stress refers to the imbalance between the production of reactive oxygen species (ROS), nitric oxide (NO), iNOS, and superoxides, and the antioxidant defense system. Excessive ROS can damage DNA, proteins, and lipids, induce chondrocyte apoptosis and senescence, provoke inflammatory responses, and promote cartilage matrix degradation (38). Antioxidant enzymes include SOD, CAT, and glutathione peroxidase (GPx). Notably, oxidative stress is not purely detrimental; moderate levels of ROS can function as signaling molecules that activate protective responses, exemplifying the ‘hormesis’ effect in homeostatic regulation (37, 39).

When chondrocytes are subjected to oxidative stress, FoxO3a is typically activated and translocated into the nucleus, leading to the upregulation of a series of antioxidant enzymes, including SOD, CAT, and GPx, which are crucial for eliminating intracellular ROS and mitigating oxidative damage to cells (40, 41). AMPK (5’ adenosine monophosphate-activated protein kinase) is a protein kinase found in eukaryotic organisms. Under oxidative stress conditions, AMPK establishes a multi-layered network for DNA repair and protection by directly regulating repair enzymes, activating SIRT3 to safeguard mitochondrial DNA, coordinating energy metabolism and autophagy, and interacting with DNA damage response factors (42, 43). AMPK activates FoxO3a in chondrocytes, resulting in the downregulation of various oxidative stress and catabolic markers, which alleviates mitochondrial oxidative damage and promotes DNA repair (30). FoxO3a-NETT@SMs have been shown to restore mitochondrial function and inhibit apoptosis in H₂O₂-induced OA chondrocytes through the overexpression of FoxO3a (31). Furthermore, DL-3-n-Butylphthalide (NBP) enhances FoxO3a expression by inhibiting the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which increases the expression of MnSOD and CAT, thus improving the chondrocytes’ ability to withstand oxidative stress and alleviating oxidative damage-induced apoptosis and matrix degradation (44). In contrast, knocking down FoxO1 and FoxO3a using tBHP resulted in a significant reduction in GPx-1 and CAT levels, exacerbating oxidative damage in chondrocytes (29). However, a study by Wang et al. indicated that silencing FoxO3a could reduce the upregulation of iNOS induced by IL-1β in rabbit chondrocytes, thereby providing protection against oxidative stress ( Table 1 ) (33). Nevertheless, maintaining or enhancing the antioxidant function of FoxO3a remains a promising strategy for protecting chondrocytes from oxidative stress and delaying the progression of OA.

The extracellular matrix (ECM) is primarily composed of macromolecules, including collagen, glycoproteins, and ACAN, and its synthesis and degradation are regulated by various cytokines and growth factors (45). The function of articular cartilage is dependent on the integrity of the cartilage ECM. Chondrocytes play a crucial role in maintaining cartilage homeostasis by balancing the expression of matrix synthesis-related genes (e.g., ACAN, COL2A1, and Sox9) and matrix degradation-related genes including MMPs and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) (46, 47).

The homeostasis of the cartilage ECM is closely associated with the activity of FoxO3a. In ATDC5 cells and human OA chondrocytes, overexpression of FoxO3a significantly increased the expression of Sox9, ACAN, and COL2A1, while its downregulation inhibited the expression of these markers (31, 48). Furthermore, NBP reduced the expression of ADAMTSs and MMPs in human OA chondrocytes by activating FoxO3a (44). In mouse models, cartilage-specific knockout of FoxO1/3a/4 resulted in spontaneous cartilage degradation and exacerbated OA lesions ( Table 1 ) (18). In summary, the regulation of cartilage ECM homeostasis by FoxO3a contributes to the maintenance the integrity of articular cartilage integrity, thereby mitigating OA progression.

Apoptosis, also known as programmed cell death, is a highly ordered physiological mechanism, characterized by distinctive morphological changes, including cell membrane shrinkage, chromatin condensation, and DNA fragmentation (49, 50). Chondrocyte apoptosis is a significant feature of cartilage degeneration in OA, leading to a reduction in both the number and activity of chondrocytes, as well as a decline in ECM synthesis capacity (51).

In various cell types, the accumulation of activated FoxO3a in the nucleus can upregulate the expression of pro-apoptotic genes, thereby inducing cell apoptosis (52). By inhibiting the active expression of FoxO3a, circFoxO3a enhances the expression of apoptosis markers, including Cleaved PARP, Cleaved caspase-3, and BAX, which are induced by IL-1β in ATDC5 cells, thus exacerbating cell apoptosis (53). Additionally, siFoxO3a can also induce chondrocyte apoptosis, accompanied by caspase activation (29). Punicalin ameliorates IL-1β and TNF-α-induced chondrocyte growth inhibition and apoptosis by preserving the transcriptional activity of FoxO3a (32). The protein phosphatase SCP4 promotes the nuclear translocation of FoxO3a through its dephosphorylation, thereby reducing chondrocyte apoptosis and promoting cartilage development (54). However, a study has indicated that FoxO3a, under IL-1β stimulation, exacerbates inflammatory responses by positively regulating tenascin-c (Tnc), promoting iNOS expression, and inducing apoptosis in rabbit chondrocytes (33). In summary, FoxO3a exerts anti-apoptotic effects on chondrocytes through antioxidation or inhibition of hypertrophic differentiation, especially when homeostasis or specific protective regulations are present ( Table 1 ) (44).

Autophagy is an evolutionarily conserved intracellular degradation mechanism that recycles damaged proteins and organelles through the lysosomal pathway. This process involves autophagosome formation, substrate encapsulation, and fusion with lysosomes, thereby maintaining cellular homeostasis (55–57). In articular cartilage, moderate autophagy is crucial for maintaining chondrocyte function and matrix balance (58, 59).

Activated FoxO3a can enter the nucleus and upregulate the expression of various autophagy-related genes (Atgs), such as LC3, Beclin-1, Gabarapl1, and ULK1, thereby initiating or enhancing autophagic flux (60). The synergistic effect of FoxO3a and SIRT3 alleviates oxidative stress in mouse endplate chondrocytes by enhancing mitophagy and inhibiting NLRP3 inflammasome activation (61). Glucosamine activates autophagy in human chondrocytes by inhibiting the AKT/FoxO3a/mTOR pathway (62). Furthermore, adenosine A2A receptors enhance the autophagic flux of Gabarapl1 and Beclin-1 by activating FoxO1/3a, which improves cartilage metabolism and reduces apoptosis—a mechanism validated in obese OA mouse models ( Table 1 ) (63). Therefore, maintaining appropriate FoxO3a activity may promote chondrocyte autophagy and delay the progression of OA.

In addition to the aforementioned functions, FoxO3a also participates in regulating processes such as differentiation and senescence in chondrocytes. Overexpression of FoxO3a can induce the expression of various chondrocyte differentiation markers, including Sox9, ACAN, and COL2A1, thereby enhancing early chondrogenesis and terminal hypertrophy of cartilage stem cells (48). This suggests that FoxO3a may be involved in the differentiation of mesenchymal stem cells (MSCs) into chondrocytes. However, some studies have indicated that upregulation of FoxO3a can inhibit the hypertrophic differentiation of MSCs by reducing the expression of type X collagen (COL X), while also limiting excessive proliferation by promoting early apoptosis (64). The natural flavonoid 5,7,3’,4’-tetramethoxyflavone (TMF) effectively inhibits OA cartilage hypertrophy by activating FoxO3a (65). Furthermore, fibroblast growth factor 18 (FGF18) can inhibit the expression of P16, P21, and P53 by activating FoxO3a, thus protecting chondrocytes from degeneration and aging effects (66). Ubiquitin-specific protease 3 (USP3) upregulates SIRT3 to deacetylate FoxO3a and attenuates IL-1β-induced senescence in SD rat chondrocytes ( Table 1 ) (67). These studies further expand our understanding of the complex roles of FoxO3a in OA.

The PI3K/AKT signaling pathway serves as a classic negative regulator of FoxO3a. Upon activation of the PI3K/AKT pathway by growth factors, AKT phosphorylates conserved amino acid sites on FoxO3a. Phosphorylated FoxO3a binds to 14-3–3 proteins and remains in the cytoplasm, inhibiting its entry into the nucleus and thereby preventing its transcriptional regulatory functions (21). In chondrocytes, AKT mediates the phosphorylation of FoxO3a at specific sites (e.g., Ser253), leading to its retention in the cytoplasm and subsequent inactivation, which affects the antioxidant and autophagy functions of chondrocytes (44, 68, 69).

The AMPK/SIRT pathway has the capacity to activate FoxO3a. Deacetylases, such as SIRT1, activate FoxO3a through deacetylation modifications, which enhance its DNA-binding ability and transcriptional activity, thereby promoting cellular resistance to oxidative stress and extending lifespan (70). TMF can facilitate the deacetylation of FoxO3a by activating SIRT1 in chondrocytes, which enhances its nuclear translocation and transcriptional activity (71). Similarly, USP3 promotes the deacetylation of FoxO3a by upregulating SIRT3, thus inhibiting IL-1β mediated senescence in rat chondrocytes ( Table 2 ) (67).

As a transcription factor, FoxO3a’s intracellular localization is heavily dependent on its amino acid modifications. The removal of these modifications allows FoxO3a to enter the nucleus, where it binds to the promoter regions of target genes and regulates transcription (72). Phosphorylation and acetylation are the most extensively studied post-translational modifications (PTMs) of FoxO3a. Besides AKT, other kinases can also phosphorylate FoxO3a. For instance, transglutaminase 2 (TG2) induces phosphorylation of FoxO3a at Ser253, inhibiting its nuclear translocation and consequently promoting the synthesis of MMP3 and MMP13 (73). Deacetylases SIRT1 and SIRT3 enhance the nuclear localization and activity of FoxO3a by removing its acetyl modifications ( Table 2 ) (67, 71). Dynamic modifications of protein structures, including phosphorylation and acetylation, are key mechanisms regulating FoxO3a activity and chondrocyte fate.

In tumor research, the epigenetic modifications of FoxO3a have become a significant area of study (74). Certain microRNAs (miRNAs) can target the 3’UTR of FoxO3a, thereby regulating its transcriptional activity (75). Additionally, DNA methylation, histone acetylation, and three-dimensional chromatin remodeling can influence the expression of FoxO3a from various perspectives (76). Similar phenomena have also been observed in the musculoskeletal system. For instance, miR-182 found in synovium-derived exosomes targets the 3’UTR of FoxO3a, inhibiting its expression in human OA synovial stromal cells ( Table 2 ) (77). Consequently, the precise regulation of FoxO3a’s expression and localization at the molecular level—through the utilization of signaling pathway cascades, dynamic regulation of PTMs, and specific epigenetic modifications—may represent a highly promising avenue for research.

A study integrating bioinformatics analysis and machine learning strategies identified downregulated FoxO3a as a biomarker for OA aging and validated its reliability in the peripheral blood of OA patients (90). Obtaining synovial fluid from the joints of experimental rats is extremely challenging and yields minimal quantities; however, this study found that FoxO3a is downregulated in the joint fluid of OA rats (77). Furthermore, the expression of FoxO3a varies in the joint fluid of OA patients depending on the severity of their condition. Single nucleotide polymorphisms (SNPs) of FoxO3a also contribute to OA diagnosis, with male carriers of the minor allele of FoxO3a quantitative trait loci (QTL) rs4946936 exhibiting a lower risk of developing hip OA ( Table 4 ) (91). This study suggests that the expression levels and genetic variations of FoxO3a in peripheral blood and synovial fluid may serve as novel diagnostic biomarkers for OA.

Currently, several natural or synthetic small molecules have been identified that can directly or indirectly activate FoxO3a in chondrocytes. For instance, NBP and glucosamine activate FoxO3a by inhibiting the PI3K/AKT pathway, which in turn reduces chondrocyte apoptosis and promotes autophagy, demonstrating beneficial effects in both OA rats and human chondrocytes (44, 62). TMF facilitates the nuclear translocation of FoxO3a through the SIRT1/FoxO3a axis, inhibits the expression of ADAMTSs, reduces matrix degradation, and suppresses cartilage hypertrophy (65, 71). USP3 induces the deacetylation of FoxO3a by activating SIRT3, thereby attenuating IL-1β-induced senescence in rat chondrocytes (67). AMPK agonists, such as AICAR and A-769662, upregulate FoxO3a expression by activating the AMPK/FoxO3a signaling pathway, which inhibits oxidative stress and catabolism in chondrocytes, further mitigating cartilage damage in OA (30, 83). Punicalin, a pomegranate extract, exhibits therapeutic effects on IL-1β and TNF-α-induced metabolic disorders in mouse chondrocytes and cartilage by enhancing the transcriptional activity of FoxO3a (32). Additionally, platelet-rich plasma (PRP) has been shown to inhibit apoptosis and promote autophagy by enhancing FoxO3a expression in human OA chondrocytes ( Table 5 ) (92).

It is noteworthy that the application of small molecule compounds often requires high bioavailability. For instance, Zhang et al. achieved a high local concentration through intra-articular injection in rats. However, the delivery specificity of FoxO3a agonists presents another challenge. Currently, most systemic administrations of FoxO3a agonists do not utilize specific tools targeting the knee joint, making it unclear what effective concentration reaches the knee joint cartilage ( Table 6 ). These studies have laid a foundation for the development of FoxO3a-targeted drugs, but further preclinical and clinical trials are necessary to evaluate their efficacy and safety in OA treatment.

As a revolutionary medical technology, gene therapy has demonstrated significant potential in treating both inherited and acquired diseases (93–95). In the context of OA, gene therapy primarily involves the localized delivery of genes to joint tissues to modulate inflammatory responses or promote cartilage repair (96–98).

Studies have demonstrated that lentivirus-mediated overexpression of FoxO3a can induce the expression of ACAN and COL2A1, and reverse IL-1β-induced chondrocyte apoptosis and inflammatory responses (29, 48). Additionally, the delivery of FoxO3a to the knee joint cavity using FoxO3a-NETT@SM effectively ameliorates progressive OA in DMM rats (31). Conversely, the siRNA knockdown of FoxO3a results in increased apoptosis and decreased anabolism in ADTC5 cells ( Table 5 ) (48).

Gene therapy faces numerous challenges, particularly the immunogenicity induced by viral vectors and gene editing tools, which can easily trigger host immune responses. This reaction may reduce therapeutic efficacy or even cause toxic side effects (99). Additionally, achieving long-term stable expression of FoxO3a presents a significant technical hurdle that must be addressed in gene therapy. Chen et al. utilized hydrogels to deliver nano-engineered FoxO3a plasmids, leveraging the sustained-release properties of the material to enhance long-term stable expression of FoxO3a. Furthermore, gene editing technology is still mired in ethical controversies ( Table 6 ). While gene therapy encounters challenges related to delivery efficiency, targeting specificity, and long-term safety, continuous technological advancements are anticipated to establish it as a crucial direction in future OA treatment.

It is noteworthy that current clinical research on FoxO3a in OA primarily focuses on assessing its expression levels and genetic polymorphisms, with active regulatory strategies for FoxO3a still in the animal experimentation phase. Consequently, more preclinical research data are necessary to support the application of FoxO3a in clinical studies.

As a critical transcription factor, FoxO3a plays a vital role in regulating ECM homeostasis, oxidative stress response, and cell survival in chondrocytes. However, its systemic activation may pose uncontrollable risks. To develop more precise, efficient, and safe regulatory approaches for FoxO3a, the following strategies are expected to be the focus of future research.

The first strategy involves the tissue-specific regulation of FoxO3a. On one hand, tissue-specific delivery systems, such as nanoparticle carriers and ligand-coupling technology, can be developed to precisely deliver FoxO3a activators to OA joints or cartilage, thereby avoiding systemic exposure. On the other hand, cartilage-specific promoters, such as COL2A1, can be utilized to construct gene therapy vectors that achieve specific expression of FoxO3a in chondrocytes (100).

The second strategy is the multi-target combination therapy involving FoxO3a. Combining FoxO3a with upstream and downstream pathway regulators, such as AMPK or PI3K/AKT, can facilitate personalized treatment for OA. For instance, the combination of PI3K/AKT inhibitors MK-2206 and LY294002 in human chondrocytes has been shown to inhibit dexamethasone-induced FoxO3a upregulation and apoptosis (101).

Lastly, the dynamic regulation of FoxO3a activity was discussed. The development of light-controlled and chemically inducible regulatory systems has enabled spatiotemporal control of FoxO3a activity (102). For instance, pH-responsive drug delivery systems can leverage the acidic characteristics of the hypoxic microenvironment in cartilage to modulate the release rhythm of FoxO3a activators.

A comprehensive identification and validation of the downstream target gene network of FoxO3a in chondrocytes is fundamental for understanding and harnessing its functions. In addition to known antioxidant enzymes, apoptosis/autophagy-related proteins, and matrix metabolic enzymes, high-throughput sequencing technologies (e.g., ChIP-seq, RNA-seq) should be employed to systematically explore new target genes that are crucial to OA pathological processes (103). Systematic identification and validation of the downstream target genes of FoxO3a not only elucidates its role in OA but also provides potential targets for the development of innovative therapeutic strategies.

With the rapid advancement of omics technologies, investigating the mechanisms of FoxO3a in OA through multi-omics research is poised to become a significant focus for future studies. By integrating transcriptomic, proteomic, and metabolomic data, researchers can achieve a more comprehensive understanding of the precise regulatory network of FoxO3a within the specific pathological microenvironment of OA. For instance, RNA-seq technology can be utilized to identify transcriptomic changes associated with FoxO3a expression, and when combined with proteomic analysis, it can elucidate alterations in its downstream signaling pathways (83). Single-cell omics further facilitates the exploration of the expression, functional differences, and intercellular communication of FoxO3a across various cell types. Additionally, network biology approaches, including network pharmacology, assist in constructing the regulatory network of FoxO3a and identifying its interactions with other key molecules, thereby revealing its systemic role in OA.