Ubiquitination in intervertebral disc degeneration: from mechanisms to potential therapeutic strategies

Abstract

Intervertebral disc degeneration (IDD) is the primary cause of low back pain, imposing a heavy economic burden on individuals and society. The pathogenesis of IDD involves complex pathological processes. The interaction of extracellular matrix metabolism, regulated cell death (such as apoptosis, ferroptosis, pyroptosis, and autophagy), cell senescence, oxidative stress, and inflammatory response collectively contribute to the development of IDD. As a widespread post-translational modification, ubiquitination modulates various biological processes by regulating the activity and stability of proteins, including cell signal transduction, cell metabolism, and cell cycle. Recent studies have shown that ubiquitination plays an important role in the pathological process of IDD. This review systematically elucidates the molecular mechanisms by which ubiquitination regulates the progression of IDD. In addition, we summarize ubiquitination-based therapeutic strategies, including natural molecules, small-molecule compounds, extracellular vesicles, and bioactive materials. This review provides new insights and potential targets for understanding the molecular mechanisms of IDD as well as the treatment.

The translational potential of this article: This study systematically clarifies the regulatory network of ubiquitination in IDD and identifies specific E3 ligases and DUBs as potential targets. Furthermore, we critically evaluate the advantages and challenges of ubiquitination-based therapeutic strategies, providing novel insights with both mechanistic depth and broad application prospects for breaking through the bottlenecks in the clinical treatment of IDD.

Graphical abstract

The roles of ubiquitination in IDD. Studies have demonstrated that multiple E3 ubiquitin ligases and deubiquitinating enzymes coordinately regulate the stability and function of substrate proteins, thereby influencing the progression of IDD by modulating oxidative stress, inflammatory responses, cell death, cell senescence, and ECM metabolism. Natural molecules, small-molecule compounds, extracellular vesicles, and advanced biomaterials (hydrogels and nanoparticles) have beem shown to alleviate IDD by targeting the ubiquitination network. This provides novel strategies for the treatment of IDD.

1. Introduction

Intervertebral disc degeneration (IDD) is a common degenerative disease of the musculoskeletal system and a major cause of lower back pain [1]. It can lead to the development of various conditions, including disc herniation, spinal stenosis, and degenerative scoliosis [2]. These conditions severely affect patients' quality of life and impose a substantial economic burden on society [3]. With the progression of population aging, it is estimated that by 2050, over 800 million people worldwide will suffer from low back pain [4]. Current treatment strategies for IDD primarily focus on symptom relief and fail to effectively promote the repair of the intervertebral disc (IVD) microenvironment or reverse the pathological process of IDD [5]. Therefore, elucidating the pathological mechanisms of IDD and developing effective treatment strategies are crucial for improving the quality of life of patients and alleviating the public health burden.

The pathogenesis of IDD is multifactorial, involving mechanical stress, biological factors, and epigenetic influences [6,7]. These factors contribute to oxidative stress, cell senescence, cell death, inflammatory responses, and degradation of the extracellular matrix (ECM) [8]. Cell death is one of the key mechanisms in IDD. The death of nucleus pulposus (NP) cells leads to a gradual reduction in proteoglycans and water content within the NP, weakening its “cushion” function [9]. During degeneration, inflammatory factors such as tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) are released to induce an inflammatory response [10]. These inflammatory responses not only exacerbate IVD injury but may also induce nerve root edema, resulting in severe pain [11]. Chronic inflammation can further promote oxidative stress and induce cell death, thereby accelerating degeneration [12,13]. The inflammatory cascade mediates catabolism by upregulating the expression of ECM enzymes, while inhibiting anabolism through suppressing aggrecan expression [14]. These pathological mechanisms interact and ultimately contribute to IDD.

The normal function of cells depends on whether proteins function properly. Post-translational polypeptides undergo folding, cleavage, or specific chemical reactions to acquire normal function, a process known as post-translational modification (PTM) [15]. Common PTMs include methylation, ubiquitination, acetylation, glycosylation, phosphorylation, and SUMOylation, among others [16]. Ubiquitination is a conserved, widespread, and dynamic PTM that regulates the activity and stability of thousands of proteins, playing a vital role in modulating cellular signaling pathways, metabolism, and the cell cycle [17,18]. Ubiquitination involves a series of enzymatic reactions—including E1-mediated activation, E2-mediated conjugation, and E3-mediated ligation—through which ubiquitin is conjugated to target proteins [19]. E3 ligases are responsible for recognizing and interacting with specific substrates, determining the specificity of the ubiquitination reaction [20]. Ubiquitin-tagged proteins are either degraded by the 26S proteasome or exhibit cellular functions [21].

In recent years, accumulating evidence has demonstrated that ubiquitination plays a critical role in the progression of IDD. However, a systematic summary and discussion of this topic remain lacking. In this review, we comprehensively elucidate the molecular mechanisms by which ubiquitination regulates the progression of IDD and summarize potential therapeutic targets for IDD based on ubiquitination. This review provides insights for the development of novel therapeutic strategies for IDD.

2. Characteristics and pathological mechanisms of IDD

The IVD consists of three layers: the outer annulus fibrosus (AF), composed of fibroblast-like cells and collagen; the inner NP rich in aggrecan and water; and the cartilaginous endplates (CEP) that connect the upper and lower vertebral bodies [22]. This structure enables the IVD to buffer spinal pressure and increase spinal mobility [23]. Degenerated IVDs are characterized by disorganized AF, decreased water content in the NP, and sclerosis of the CEP [24]. These factors lead to decreased disc height and the development of IDD. As a complex degenerative disease, IDD involves multiple pathological processes (Fig. 1). The major pathological Characteristics include oxidative stress, inflammatory responses, regulated cell death (RCD), cell senescence, and ECM metabolic imbalance [8]. As one of the initiating factors of IDD, oxidative stress is induced by abnormal mechanical stress, aging, and metabolic disorders, leading to excessive accumulation of reactive oxygen species (ROS). Elevated ROS further activates multiple signaling pathways, such as NF-κB and NLRP3 inflammasome, thereby triggering and continuously amplifying inflammatory responses [6]. Inflammation and oxidative stress act synergistically to induce various patterns of regulated cell death (including apoptosis, pyroptosis, and ferroptosis) and facilitate cell senescence [25]. Moreover, senescent cells secrete senescence-associated secretory phenotype (SASP) factors, which in turn exacerbate inflammation and cell death. Ultimately, inflammatory cytokines and SASP factors upregulate the expression of Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), promoting ECM degradation [26]. These processes are interconnected and contribute to the structural and functional degeneration of the IVD. Understanding the pathological processes of IDD is crucial for developing novel treatment strategies.

Fig. 1.

Pathological Mechanisms of IDD. IDD is a complex degenerative disease involving multiple pathological processes. Its primary pathological features include oxidative stress, inflammatory response, regulated cell death, cell senescence, and imbalance in ECM metabolism. As one of the initiating drivers of IDD, oxidative stress triggers inflammatory responses, thereby inducing cell death and cellular senescence and accelerating ECM degradation. These factors are interrelated and collectively lead to the structural and functional degradation of IVD.

2.1. Oxidative stress

Redox balance is crucial for maintaining cellular function. Oxidative stress is a state of imbalance between oxidation and antioxidation, characterized by the abnormal accumulation of ROS in the body, leading to cellular dysfunction [27]. Abnormal accumulation of ROS within the disc accelerates the progression of IDD by inducing cell death, ECM metabolic imbalance, and inflammatory responses [28]. Mitochondria and the endoplasmic reticulum are considered the primary sources of ROS. Mitochondrial dysfunction disrupts normal electron transport, resulting in increased ROS production [29]. Protein misfolding causes endoplasmic reticulum stress. Endoplasmic reticulum stress increases Ca²⁺ concentration in mitochondria, disrupting ROS balance within mitochondria [30]. Additionally, another factor that regulates oxidative stress is the reduction in antioxidant activity. Multiple studies have shown that the expression levels of superoxide dismutase (SOD), catalase, and glutathione (GSH) are reduced in degenerated NP cells, which further promotes the accumulation of ROS [31]. ROS participates in the pathological processes of IDD through pathways such as MAPK, NF-κB, mTOR, and epigenetic regulation [6]. Small-molecule compounds, hormones, antioxidants, and biomaterials have been shown to effectively alleviate IDD progression by inhibiting oxidative stress [28].

2.2. Inflammatory response

Inflammatory response is a significant factor promoting the onset and development of IDD. The pathological process of IDD involves the infiltration of inflammatory cells, including neutrophils, mast cells, and macrophages [32]. These inflammatory cells secrete various cytokines, such as IL-1β, IL-6, and TNF-α. Inflammatory responses can also induce angiogenesis and nerve ingrowth, further exacerbating the deterioration of the IVD microenvironment and triggering low back pain [33]. Furthermore, various stress stimuli can stimulate NP cells to secrete endogenous inflammatory mediators, further promoting IDD progression [34]. Macrophage polarization is closely related to the inflammatory response of IVD. During IDD progression, M1 macrophage polarization exerts pro-inflammatory effects, whereas M2 polarization exhibits anti-inflammatory effects. Inhibiting M1 macrophage polarization and promoting M2 polarization can effectively alleviate IDD [35]. Targeting inflammatory responses in the IVD has been proposed as a potential therapeutic strategy.

2.3. Regulated cell death

During IDD, cell dysfunction results in reduced anabolic synthesis of ECM. RCD, such as apoptosis [36], ferroptosis [37], pyroptosis [38], and autophagy [39], plays a key role in reducing the number of cells and inhibiting ECM synthesis.

Apoptosis is an intrinsic cellular self-destruct mechanism that maintains normal cell function by removing senescent and abnormal cells. Excessive activation of apoptosis leads to NP and AF cells loss and ECM degradation, further accelerating IDD progression [40]. The extrinsic death receptor pathway, the intrinsic mitochondrial pathway, and the endoplasmic reticulum stress pathway, as classical apoptotic pathways, are involved in the initiation and development of IDD [41].

Ferroptosis, an iron-dependent form of cell death, is characterized by lipid peroxidation, ROS accumulation, and mitochondrial damage [42]. Iron overload is an independent risk factor for IDD and accelerates its pathological progression. Iron overload disrupts intracellular iron homeostasis, affecting the balance between oxidation and antioxidant systems. Increased peroxides damage the structure and function of NP, AF, and CEP cells, leading to cell death [43].

Pyroptosis is an inflammatory form of programmed cell death, characterized by NLRP3 activation [44]. Factors such as mechanical stimulation and oxidative stress act as damage-associated molecular patterns, activating the assembly of the NLRP3 inflammasome complex [45]. The inflammasome leads to oligomerization of the N-terminal domain of gasdermin D, forming pores in the plasma membrane, causing cell swelling and rupture, and releasing IL-1β and IL-18, which exacerbate the death of NP and AF cells [38].

Autophagy protects cells by clearing damaged proteins and dysfunctional organelles [46]. Autophagy may act as a double-edged sword in the progression of IDD. Restoring autophagic flux can alleviate IDD by reversing oxidative damage and mitochondrial dysfunction [47]. However, excessive autophagy can induce mitochondrial clearance, aggravating NP cell senescence [48]. The beneficial or detrimental effects of autophagy on cell function may depend on the cellular microenvironment. Moreover, mammalian target of rapamycin (mTOR), as a major regulator of autophagy, has emerged as a potential target for alleviating IDD [49].

2.4. Cell senescence

Cell senescence refers to a state of irreversible cell cycle arrest, where cells lose their proliferative capacity and physiological function. Cell senescence has been recognized as a key factor in IDD [50]. Mechanical stress, oxidative stress, and inflammatory responses can induce senescence in NP cells, AF cells, and mesenchymal stem cells [26]. Senescent cells carry a SASP and secrete pro-inflammatory cytokines, growth regulators, angiogenic factors, and MMPs, which promote IDD progression by inducing inflammatory responses and ECM metabolic imbalance [51]. NP cell senescence is the most extensively studied cell type in IDD. Studies have demonstrated that classic molecular signaling pathways such as p53, p21, p16, MAPK (mitogen-activated protein kinases), NF-κB (Nuclear factor kappa-B), and mTOR are involved in NP cell senescence. Targeting these molecules may represent therapeutic strategies to alleviate NP cell senescence and IDD progression [26]. Autophagy may be a factor modulating cell senescence. Activation of autophagy can mitigate NP cell senescence [52]. Conversely, inhibition of autophagy aggravates NP cell senescence [53]. However, the interplay between autophagy and cell senescence during IDD requires further investigation.

2.5. Extracellular matrix metabolism

The ECM of the IVD is mainly composed of collagen and aggrecan. Collagen provides tensile strength to the AF, while water-binding aggrecan is essential for buffering mechanical stress [54,55]. MMPs [56] and ADAMTS [57] are the primary enzymes that promote ECM degradation. In normal IVDs, ECM synthesis and degradation are in dynamic balance. One of the hallmarks of IDD is the imbalance between anabolism and catabolism within the IVD [58]. Accumulating evidence indicates that multiple factors, such as reduced nutrition, pressure overload, diabetes, smoking, and genetics, may contribute to ECM metabolic imbalance [59]. During IVD degeneration, proteoglycan content decreases, and type II collagen is replaced by type I collagen [60]. The reduction in type II collagen impairs the AF's ability to distribute mechanical stress, increases angiogenesis, induces neuropathic pain, and further disrupts tissue homeostasis. Aggrecan degradation leads to decreased water content and osmotic pressure in the IVD, which reduces the NP's ability to resist compression. Consequently, the NP is prone to protrusion, triggering disc herniation.

2.6. The interaction between the pathological mechanisms in IDD

Notably, these pathological processes do not occur in isolation. Key pro-inflammatory cytokines such as TNF-α and IL-1β act as central mediators that trigger downstream pathological events. The TNF-α and IL-1β produced not only promote ECM degradation by activating MMPs/ADAMTS [61], but also facilitate apoptosis, autophagy, and senescence [11]. The crosstalk between inflammation and pyroptosis forms a vicious cycle that exacerbates the inflammatory response of NP cells and the progression of IDD [38,44]. Senescent cells exhibit a SASP, which promotes the progression of IDD by triggering inflammatory responses and disrupting ECM metabolic homeostasis [51]. Meanwhile, inflammation-induced ROS accumulation further aggravates oxidative stress, which in turn activates the NF-κB signaling pathway, forming a positive feedback loop that collectively drives the progression of IDD [62].

3. Molecular mechanisms of ubiquitination

The homeostasis of the IVD relies on normal cellular function. Ubiquitination is a widespread PTM that participates in the regulation of various cellular functions. A growing body of evidence indicates that ubiquitination is closely associated with the progression of IDD. Elucidating the molecular mechanisms of ubiquitination is conducive to identifying potential therapeutic targets (Fig. 2).

Fig. 2.

Ubiquitination reaction. Ubiquitination is a protein modification mechanism involving a series of enzymatic reactions, primarily mediated by E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases. Driven by ATP hydrolysis, E1 ubiquitin-activating enzymes activate ubiquitin molecules; the activated ubiquitin then binds to E2 conjugating enzymes, which transfer the ubiquitin to E3 ligases; subsequently, E3 ligases mediate the transfer of ubiquitin to substrate proteins; finally, the ubiquitin-tagged substrate proteins are recognized and degraded by the proteasome. Ubiquitination is a reversible process, which is mediated by deubiquitinating enzymes (DUBs). DUBs eliminate ubiquitination modifications of substrate proteins by hydrolyzing the covalent bonds between ubiquitin and substrate proteins, and the free ubiquitin molecules are recycled for reuse. In addition, the structure of the ubiquitin chain determines the type of ubiquitination. A. Monoubiquitination, a single ubiquitin molecule is conjugated to the substrate protein. B. Polyubiquitination, a chain-like structure formed by the linkage of multiple ubiquitin molecules. C. Linear ubiquitination (M1-type) is characterized by the direct linkage between the N-terminal methionine of one ubiquitin molecule and the C-terminal glycine of another, forming an unbranched, end-to-end linear chain.

3.1. Ubiquitination

Ubiquitination is a process of a cascade of enzymatic reactions mediated by three types of enzymes: ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 [63]. First, in an ATP-dependent manner, a cysteine residue on the E1 enzyme forms a high-energy thioester bond with the C-terminal carboxyl group of ubiquitin, generating an E1∼ubiquitin complex [64]. Next, the activated ubiquitin is transferred to E2. E2 undergoes a trans-thioesterification reaction with ubiquitin to form an E2∼ubiquitin complex [65]. Finally, E2 carrying the activated ubiquitin binds to E3 ubiquitin ligase. E3 can recognize specific substrate proteins and transfer ubiquitin to the target protein [20]. The pathway by which ubiquitin binds to the substrate protein depends on the type of E3. For HECT-type E3, ubiquitin is first transferred to the cysteine residue of E3 itself and then to the substrate [66]. RING-type E3 ligases act as molecular scaffolds that bring ubiquitin-loaded E2 close to the substrate, directly catalyzing the transfer of ubiquitin from E2 to the substrate [66]. There is a large variety of E3 enzymes, which are the key determinants of substrate specificity and the core nodes of cellular signal integration and regulation.

Ubiquitin molecules can be repetitively linked to specific lysine residues or the N-terminal methionine of a ubiquitin molecule already conjugated to the substrate, thereby forming a polyubiquitin chain. The type of ubiquitin chain determines the fate of the substrate protein. K48-linked polyubiquitin chains are the most common degradation signals, mainly directing target proteins for complete degradation through the 26S proteasome [67]. K63-linked polyubiquitin chains are mainly involved in signal transduction (NF-κB pathway), autophagy, and inflammatory responses [68]. Other types (K11, K29, K33) are mainly involved in endoplasmic reticulum-associated degradation, lysosomal pathways, and membrane protein transport [69]. Linear ubiquitin chains (M1-linked), catalyzed by the linear ubiquitin chain assembly complex, play a crucial regulatory role in cell death and inflammatory responses [70].

3.2. Deubiquitination

Ubiquitination is a dynamically reversible process, primarily mediated by deubiquitinating enzymes (DUBs). As proteases, DUBs act like “erasers” by specifically hydrolyzing the covalent bonds formed between ubiquitin molecules and substrate proteins or between ubiquitin molecules themselves to remove ubiquitination modifications [71]. DUBs alter the fate of substrate proteins by altering the type of ubiquitin chains [72]. The human genome encodes approximately 100 DUBs, which are classified into two major classes and seven families [73]. Different types of DUBs undertake distinct functions. Ubiquitin-specific proteases (USPs) are the largest and most extensively studied family, responsible for recognizing and regulating substrates. Otubain proteases specifically recognize and remove K48- and K63-linked ubiquitin chains. The ubiquitin C-terminal hydrolase (UCH) family mainly processes small-molecule compounds and recycles free ubiquitin [74].

4. The role of ubiquitination in IDD

The pathogenesis of IDD involves multiple biological processes, including ECM metabolism, RCD, cell senescence, oxidative stress, and inflammatory responses. These factors interact and collectively promote IDD progression. Accumulating evidence indicates that E3 ligases and DUBs regulate IDD development through various pathways (Table 1). This section will discuss in detail the molecular mechanisms of E3 ligases and DUBs in the progression of IDD (Fig. 3).

Table 1.

Ubiquitination regulation mechanisms in IDD.

E3/DUB	Enzyme	Target	Signaling pathway	Function	References
E3	ZNF598	Nrf2	NF-κB	ROS ↑ Inflammation ↑	[75]
E3	KEAP1	Nrf2	NF-κB	Inflammation ↑	[76]
E3	TRIM21	HIF-1α	/	ROS ↑ ECM degradation ↑	[77]
E3	MAPL	DRP1	/	ROS ↑	[78]
DUB	USP11	Sirt3	Sirt3-GPX4	ROS ↓ Ferroptosis ↓	[79]
DUB	USP7	p65	NF-κB	ROS ↑ Inflammation ↑	[80]
DUB	A20	RIP1	NF-κB	Inflammation ↓	[81]
E3	RNF182	p65	NF-κB	Inflammation ↓	[82]
E3	UBR3	DUSP1	DUSP1/p38	Inflammation ↑	[83]
E3	PELI1	ASC	NLRP3	Pyroptosis ↑	[84]
DUB	USP14	NLRP3	/	Pyroptosis ↑ Inflammation ↑	[85]
E3	SIAH1	XIAP	NF-κB	Apoptosis ↑ Inflammation ↑	[86]
E3	CRL4	CtBP1/2	p300-FOXO3a	Apoptosis ↑	[87]
DUB	USP5	E2F1	/	Apoptosis ↓	[88]
DUB	USP15	FKBP5	PI3K/AKT	Apoptosis ↑	[89]
E3	FBXO2	LCN2	PINK1-Parkin	ROS ↓ Ferroptosis ↓	[90]
E3	FBXW7	mTOR	PINK1-Parkin	ROS ↑ Ferroptosis ↑	[91]
E3	RBX1	NCOA4	/	Ferroptosis ↓	[92]
E3	TRIM21	SLC7A11	/	Ferroptosis ↑	[93]
DUB	UCHL1	HSPA8	CMA	Ferroptosis ↓ Senescence ↓	[94]
E3	UBE3A	MCM7	p53-p21	Senescence ↑	[95]
E3	TRIM63	ACE	/	Senescence ↑	[96]
E3	HERC3	NCOA1	NCOA1-p300-pRunx2	ECM degradation ↓	[97]
E3	MARCHF8	TGFBI	NF-κB	ECM degradation ↓ Inflammation ↓	[98]
DUB	USP31	HIF-1α	/	ECM degradation ↓	[99]
DUB	USP24	pRunx2	NCOA1-p300-pRunx2	ECM degradation ↑	[100]
DUB	USP12	pPPRC1	pPPRC1-p300	ECM degradation ↑	[101]

Fig. 3.

Ubiquitination in IDD. E3 ligases and deubiquitinating enzymes influence the progression of IDD by modulating extracellular matrix metabolism, regulated cell death, cell senescence, oxidative stress, and inflammatory responses.

4.1. Ubiquitination and oxidative stress

Oxidative stress, caused by an imbalance between ROS production and antioxidant capacity, is a significant initiating factor for IDD. ROS accumulation leads to mitochondrial dysfunction, DNA damage, and protein oxidation, promoting cell death and ECM degradation [102]. Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) is a key transcription factor regulating the antioxidant response, and its expression is reduced in degenerated cartilaginous endplates [103]. Huang et al. [75] found that Zinc Finger Protein 598 (ZNF598) promotes oxidative stress and CEP degeneration by mediating Nrf2 ubiquitination. Kelch-like ECH-Associated Protein 1 (KEAP1) mediates the ubiquitination and degradation of Nrf2, inhibiting autophagic flux and leading to the accumulation of metabolites and ROS in NP cells [76]. HIF-1α plays an important role in maintaining the hypoxic adaptation and antioxidant capacity of NP cells. The degradation of HIF-1α reduces the expression of antioxidant enzymes, enhances oxidative stress, and promotes the degeneration of NP cells [104]. TRIM21 is upregulated under oxidative stress and mediates the ubiquitination and degradation of HIF-1α [77]. Mitochondria are a major source of ROS. Mitochondrial dysfunction increases ROS production. Lin et al. [78] discovered that the Mitochondrial-Anchored Protein Ligase (MAPL), an E3 ligase, promotes mitochondrial translocation and fission by mediating mitochondrial SUMOylation and SUMO1 modification of DRP1, resulting in intracellular ROS accumulation. Sirt3 regulates mitochondrial redox homeostasis by deacetylating mitochondrial proteins [105]. USP11 enhances the antioxidant capacity of NP cells by increasing the stability of Sirt3 protein [79].

4.2. Ubiquitination and inflammatory response

Chronic inflammation is a key pathological feature of IDD. TNF-α and IL-1β are core pro-inflammatory cytokines that trigger a cascade of inflammatory responses and promote IDD progression [106]. E3 ligases or DUBs regulate the inflammatory response by targeting key molecules in the NF-κB signaling pathway. USP7 is upregulated in activated monocytes and deubiquitinates the NF-κB subunit p65, promoting its nuclear translocation. This enhances the transcription of TNF-α, HMGB1, and IL-1β, further stimulating NP cells to secrete inflammatory factors and accumulate ROS, forming a vicious cycle of inflammation and oxidative stress [80]. A20 (also known as Tumor Necrosis Factor Alpha Induced Protein 3) is a DUB with critical anti-inflammatory functions [107]. Studies have demonstrated that A20 alleviates TNF-α-induced inflammation in NP cells by inhibiting the NF-κB signaling pathway [81]. RING finger protein 182 (RNF182) suppresses the release of IL-1β and TNF-α and alleviates IDD by mediating the degradation of p65 [82]. Jiang et al. [83] found that UBR3 is significantly elevated in NP tissues from IDD patients and correlates with IDD severity. Mechanistic studies showed that UBR3 mediates the ubiquitination of DUSP1 and promotes inflammation and apoptosis in NP cells via the p38 signaling pathway. Furthermore, pyroptosis can induce inflammatory responses. Targeting the ubiquitination of NLRP3 can influence cellular inflammation. PELI1 activates the NLRP3 inflammasome, promoting the release of IL-1β and IL-18. These inflammatory cytokines further amplify the inflammatory response [84]. USP14 promotes pyroptosis in human AF cells by deubiquitinating NLRP3, aggravating the inflammatory response [85].

4.3. Ubiquitination and regulated cell death

RCD is an important biological process that plays a key role in the progression of many diseases. The complex microenvironment within the IVD, such as mechanical loading, nutrient deprivation, low pH, and other biochemical factors, can induce RCD in NP and AF cells [8]. Various types of RCD have been reported to be involved in the IDD process, including apoptosis, ferroptosis, pyroptosis, and autophagy. Growing evidence suggests that E3 ligases or DUBs regulate these cell deaths by targeting key proteins, thereby influencing the progression of IDD.

4.3.1. Apoptosis

Apoptosis is the main form of RCD, characterized by cell shrinkage and nuclear condensation in the early stage, and nuclear fragmentation into apoptotic bodies in the late stage [108]. Studies have found that in both the extrinsic and intrinsic apoptotic pathways, E3 ligases or DUBs regulate apoptosis by controlling the stability of apoptosis-associated proteins [109]. Apoptosis is the most extensively studied form of cell death in IDD. Fang et al. [86] found that seven in absentia homolog 1 (SIAH1), an E3 ligase, is highly expressed in NP cells of IDD patients. Co-immunoprecipitation and ubiquitination assays confirmed that SIAH1 specifically targets and ubiquitinates X-linked inhibitor of apoptosis protein (XIAP). XIAP is a key anti-apoptotic protein that regulates cell death pathways and NF-κB-dependent immune signaling events [110]. XIAP prevents TNF-mediated apoptosis by controlling the ubiquitination of receptor-interacting protein kinase 3 (RIPK3) [111]. SIAH1-mediated ubiquitination and degradation of XIAP activate the caspase cascade and promote NP cell apoptosis [86]. Cullin-RING ligases (CRLs) are the largest family of E3 ubiquitin ligases, mediating the ubiquitination and degradation of approximately 20% of proteins [112]. CRL4 is composed of CUL4A/CUL4B, DDB1, and DCAF6, and is involved in inflammation-induced NP cell apoptosis. Chronic inflammation induces the assembly of CRL4 and degrades C-terminal-binding protein 1/2 (CtBP1/2). The degradation of CtBP1/2 dissociates it from the p300-FOXO3a complex, activating Bcl2-binding component 3 (BBC3)-dependent apoptosis [87]. E2F transcription factor 1 (E2F1) exerts an anti-apoptotic effect. Shi et al. [88] found that in degenerated NP cells, excessive cytoplasmic retention of USP5 leads to abnormal ubiquitination and degradation of E2F1. Transplantation of mesenchymal stem cell (MSC)-derived vesicles enriched in USP5 into degenerative IVD promotes the nuclear translocation of USP5 in NP cells, inhibits the ubiquitination and degradation of E2F1, and thereby reduces DNA damage and apoptosis of NP cells. Furthermore, the PI3K/AKT pathway is involved in apoptosis regulation. In degenerated NP cells, the expression level of USP15 is elevated, and it inhibits the deubiquitination and degradation of FKBP5 (FK506 Binding Protein 5) [89]. It has been reported that FKBP5 may function as a proto-oncogene or oncogene in different tumor tissues and is involved in kinase regulation [113]. Stabilized FKBP5 inhibits AKT phosphorylation, thereby promoting NP cell apoptosis through the PI3K/AKT pathway. Silencing USP15 can enhance AKT activation and reduce apoptosis, providing a potential therapeutic target for IDD [89].

4.3.2. Autophagy and ferroptosis

Autophagy-dependent cell death is a key form of RCD. Ferroptosis is a novel form of cell death characterized by iron overload and lipid peroxidation, which has been confirmed to be associated with IDD progression. Autophagy, especially selective autophagy (such as mitophagy, ferritinophagy, and chaperone-mediated autophagy), plays an important role in ferroptosis [114].

Importantly, E3 ligases and DUBs regulate ferroptosis by modulating autophagy-related genes. The PINK1/Parkin pathway is a canonical ubiquitin-dependent mitophagy pathway. As an E3 ubiquitin ligase, Parkin ubiquitinates outer mitochondrial membrane proteins and maintains mitochondrial homeostasis by clearing damaged mitochondria [115]. Wu et al. [90] identified through transcriptome sequencing that the F-Box Only Protein 2 (FBXO2) is downregulated in degenerated NP tissues. FBXO2 mediates the ubiquitination of Lipocalin-2 (LCN2), activating PINK1-Parkin-dependent mitophagy, reducing ROS accumulation, and inhibiting ferroptosis. FBXO2-knockout mice exhibited aggravated IDD. Additionally, as a central negative regulator of autophagy, mTOR degradation relieves its inhibitory effect on the ULK1 (autophagy-initiating kinase) complex, thereby promoting general autophagic flux, including PINK1/Parkin-mediated mitophagy [116]. F-Box and WD Repeat Domain Containing 7 (FBXW7) activates mitophagy and clears ROS through ubiquitinates and degrades mTOR [91]. Nuclear receptor coactivator 4 (NCOA4) is a selective cargo receptor that mediates the autophagic degradation of ferritin, a process known as “ferritinophagy” [117]. In the acidic microenvironment of IDD, NCOA4-mediated ferritinophagy was enhanced, leading to iron release and ferroptosis in NP cells [118]. A mechanistic study showed that ring-box 1 (RBX1) mediates the ubiquitination and degradation of NCOA4, ameliorating IDD progression by inhibiting ferritinophagy and ferroptosis [92].

Ubiquitin C-Terminal Hydrolase L1 (UCHL1) alleviates NP cell degeneration by blocking autophagy-dependent ferroptosis. UCHL1 deubiquitinates and stabilizes HSPA8, activating chaperone-mediated autophagy (CMA). Activated CMA promotes the degradation of HPCAL1, thereby inhibiting ferroptosis and alleviating NP cell degeneration [94]. Sirtuin 3 (Sirt3), an NAD⁺-dependent mitochondrial deacetylase, inhibits ferroptosis by promoting GSH expression [119]. Evidence indicates that Sirt3 is downregulated in IDD, and its knockout promotes ferroptosis and IDD progression [120]. Zhu et al. [79] further demonstrated that USP11 directly binds to and deubiquitinates Sirt3, enhancing the activity of mitochondrial antioxidant enzymes, increasing GPX4 synthesis, and inhibiting lipid peroxidation. Overexpression of USP11 can alleviate oxidative stress-induced ferroptosis and mitigate IDD. In addition, Solute carrier family 7 member 11 (SLC7A11), functioning as the primary transporter for cystine and glutamate, is involved in the synthesis of GSH, which in turn alleviates ferroptosis by inhibiting lipid peroxidation [121]. Yu et al. [93] found that TRIM21 binds to SLC7A11 and promotes its K48-linked ubiquitination and proteasomal degradation, which increases the level of lipid peroxidation in NP cells and facilitates ferroptosis.

4.3.3. Pyroptosis

Pyroptosis is an inflammatory form of cell death mediated by inflammasomes. Pyroptosis occurs mainly through the canonical pathway (Caspase-1-dependent) and the non-canonical pathway (Caspase-1-independent). Both pathways involve inflammasomes, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and the Gasdermin protein family [122]. NLRP3 is the most extensively studied inflammasome. Post-translational modifications of various pyroptosis-related proteins have been found, which can precisely control inflammasome activation [123]. Studies have shown that E3 ligases and DUBs affect pyroptosis by regulating the activation and assembly of inflammasomes [124]. Pellino E3 ubiquitin protein ligase 1 (PELI1) is a member of the Pellino family of E3 ubiquitin ligases, mediating necroptosis and inflammatory responses [84]. PELI1 is highly expressed in TNF-treated NP cells. PELI1 mediates the ubiquitination of ASC to activate the NLRP3 inflammasome, promotes ASC oligomerization and caspase-1 cleavage, and induces pyroptosis of NP cells [84]. Silencing PELI1 can reduce the expression of NLRP3, ASC, and cleaved caspase-1, inhibit the release of inflammatory cytokines, and alleviate IDD progression [125]. Hai et al. [85] found that USP14 protein levels were significantly elevated in AF cells from IDD patients. Further studies confirmed that USP14 promotes the activation of the NLRP3 inflammasome and the maturation of caspase-1 by deubiquitinating NLRP3. Knockdown of USP14 induces NLRP3 ubiquitination and degradation, inhibits AF cell pyroptosis, and alleviates IDD progression.

4.4. Ubiquitination and cell senescence

Cell senescence is characterized by irreversible cell cycle arrest and the secretion of SASP factors, and is a key driver of IDD. Cell senescence leads to the decline of cell function and further exacerbates IDD. E3 ligases or DUBs regulate NP cell senescence by targeting senescence-related proteins and signaling pathways. Ubiquitin-protein ligase E3A (UBE3A) promotes the degradation of substrate proteins by catalyzing K48-linked ubiquitin chains [126]. Studies have shown that UBE3A is involved in the regulation of the P53 gene [127]. As a tumor suppressor, P53 is involved in DNA repair and the expression of cell senescence and apoptosis genes, and is one of the markers of cell senescence [128]. A study demonstrated that UBE3A mediates the ubiquitination and degradation of minichromosome maintenance complex component 7 (MCM7), and then promotes NP cell senescence through the p53-p21 and p16 signaling pathways [95]. Tripartite motif-containing 63 (TRIM63) is considered to play a key role in skeletal function [129]. Guo et al. [96] found that TRIM63 inhibits NP cell senescence by mediating the ubiquitination and degradation of angiotensin-converting enzyme (ACE). In addition, USP5 alleviates NP cell senescence by stabilizing E2F1. USP5 promotes the nuclear translocation of E2F1 and stabilizes its expression, enhances DNA damage repair, and inhibits SASP secretion, thereby delaying NP cell senescence [88]. UCHL1 activates CMA by stabilizing HSPA8, promoting the degradation of senescence-related proteins [94]. This process can alleviate NP cell senescence and delay the progression of IDD.

4.5. Ubiquitination and extracellular matrix metabolism

An imbalance between the synthesis and degradation of the ECM is a hallmark of IDD. During IDD, type II collagen and aggrecan are reduced, while the expression of MMPs and ADAMTS is increased [130]. Together, they contribute to the ECM metabolic imbalance. E3 ligases and DUBs regulate ECM metabolism by targeting key transcription factors or signaling molecules that control the expression of matrix-degrading enzymes and ECM components, thereby influencing the progression of IDD.

HECT and RLD domain-containing E3 ubiquitin protein ligase 3 (HERC3) is a member of the HECT family of E3 ubiquitin ligases. It contains a regulator of chromosome condensation 1 (RCC1)-like domain (RLD) and a conserved catalytic HECT domain [131]. The expression level of HERC3 is significantly downregulated in IDD. HERC3 deficiency leads to the accumulation of nuclear receptor coactivator 1 (NCOA1), which forms a complex with histone acetyltransferase p300 and transcription factor Runx2, thereby activating the expression of 14 MMP genes. This promotes ECM degradation and accelerates IDD progression. Overexpression of HERC3 can ubiquitinate and degrade NCOA1, inhibit MMP activation, and reduce ECM degradation [97]. Membrane Associated Ring-CH-Type Finger 8 (MARCHF8) is an E3 ubiquitin ligase of the MARCHF family, which regulates cell apoptosis by ubiquitinating various receptors [132]. Transforming growth factor beta-induced protein (TGFBI) is an extracellular matrix protein that acts as a hub gene in the pathogenesis of IDD. It accumulates in lipopolysaccharide (LPS)-stimulated NP cells and promotes cell apoptosis and ECM degradation by activating the NF-κB pathway [98]. Zhang et al. [98] demonstrated that MARCHF8 mediates the ubiquitination and degradation of TGFBI, inhibits the expression of MMP13 and ADAMTS-5, and maintains the homeostasis of ECM metabolism in NP cells.

Hypoxia-inducible factor-1α (HIF-1α) is a key transcription factor regulating ECM synthesis. Downregulation of HIF-1α inhibits the expression of type II collagen and aggrecan, while promoting the expression of MMPs, ultimately leading to an imbalance in ECM metabolism [133]. Tripartite motif-containing protein 21 (TRIM21) is a member of the TRIM family, which shares multiple structural domains including a RING domain with E3 ubiquitin ligase activity [134]. Zheng et al. [77] found that the expression level of TRIM21 is upregulated in degenerated NP tissues and promotes ECM degradation by mediating the ubiquitin-dependent degradation of HIF-1α. DUBs are also involved in regulating HIF-1α ubiquitination. Su et al. [99] found that USP31 maintains ECM homeostasis by inhibiting the ubiquitin-dependent degradation of HIF-1α. Furthermore, USP24 deubiquitinates and stabilizes phosphorylated Runx2 in a chronic inflammatory microenvironment. The stabilized Runx2 recruits p300/NCOA3, activates the expression of 13 ADAMTS genes, and significantly promotes ECM degradation [100]. Phosphorylated Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1 (pPPRC1) can form a complex with p300 to enhance the expression of 12 MMP genes. USP12 interacts with pPPRC1 and promotes MMP gene expression by inhibiting its ubiquitin-dependent degradation, accelerating high glucose-induced ECM degradation and diabetes-associated IDD [101].

4.6. Ubiquitination regulatory network in IDD

In the research on ubiquitination regulation in IDD, TRIM21, USP5, USP11, PELI1, and USP14 are frequently reported and functionally versatile E3 ligases or DUBs. Among them, TRIM21 represents the most typical pleiotropic molecule. It not only disrupts ECM synthesis by mediating the ubiquitin-dependent degradation of HIF-1α but also induces ferroptosis by promoting the degradation of SLC7A11. These results demonstrate the cross-regulatory role of the same E3 ligase in two major pathological processes. Similarly, USP5 simultaneously inhibits apoptosis and senescence by stabilizing E2F1. USP11 synergistically resists oxidative stress and ferroptosis by stabilizing Sirt3. PELI1 and USP14 link pyroptosis and inflammatory responses by regulating the ubiquitination level of the NLRP3 inflammasome.

Further analysis reveals that these ubiquitination-modifying enzymes constitute a complex regulatory network with NF-κB, HIF-1α, and Nrf2 as core nodes. The NF-κB pathway occupies a central position and is jointly targeted by the NLRP3 inflammatory circuit regulated by USP7, A20, RNF182, and PELI1/USP14, dominating inflammatory responses and pyroptosis. As a hub between ECM metabolism and oxidative stress, HIF-1α is negatively regulated by TRIM21 but positively stabilized by USP31. Nrf2 serves as the core of antioxidant defense, and its stability is strictly controlled by ubiquitination modifications mediated by ZNF598 and KEAP1. Collectively, a ubiquitination regulatory network composed of diverse E3 ligases and DUBs, with the NF-κB/HIF-1α/Nrf2 signaling axis as the hub, exists in IDD. This network systematically drives the pathological progression of IDD by integrating ECM metabolism, multiple forms of cell death, cell senescence, oxidative stress, and inflammatory responses.

4.7. Stage-specific characteristics of the ubiquitination regulatory network in IDD

Notably, IDD is a progressive process, and the ubiquitination regulatory network may undergo dynamic changes. In the early stage of IDD, oxidative stress serves as the dominant pathological driver [6], with regulatory molecules including TRIM21 and KEAP1 exerting critical functions by modulating the stability of HIF-1α and Nrf2. With disease progression, inflammatory responses are aggravated [35], and E3 ligases/DUBs that regulate NLRP3 and NF-κB, such as PELI1 and USP7, may be activated preferentially. In the late stage of IDD, cell senescence and death become predominant [26], accompanied by increased expression of E3 ligases regulating the p53/p21 pathway, such as SIAH1 and UBE3A.

However, current studies are only cross-sectional observations, and there is a lack of time-series dynamic studies to verify this hypothesis. In the future, lineage tracing and spatiotemporal omics technologies should be employed to map the ubiquitination modification landscape at different stages of IDD, so as to reveal the key switches during disease progression.

5. The treatment strategies based on ubiquitination in IDD

Accumulating evidence indicates that strategies targeting ubiquitination can significantly alleviate IDD progression. Natural molecules, small-molecule compounds, extracellular vesicles, and biomaterials have been proven effective in vitro or in vivo, providing promising strategies for IDD treatment (Table 2). Although these therapeutic agents differ in nature, they all converge on regulating the ubiquitination system. Their effects are primarily achieved by either directly targeting the activity of E3 ligases/DUBs or indirectly influencing the expression or stability of these enzymes and their substrates, thereby restoring cellular homeostasis. In this section, we delve into the applications of these strategies in IDD to reveal novel potential targets for treating IDD (Fig. 4).

Table 2.

The treatment strategies based on ubiquitination in IDD.

Classification	Compound	Target	effects	Species	References
Natural molecules	Galangin	Nrf2	Inflammation ↓ ECM degradation ↓	Rat	[135]
	Naringin	Parkin	ROS ↓ Apoptosis ↓	mice	[136]
	Friedelin	RNF182	Inflammation ↓	Rat	[82]
	Astragalus	OGT	ECM degradation ↓	Rat	[137]
	Glutamine	Nrf2	Ferroptosis ↓ ECM degradation ↓	Rat	[138]
	Melatonin	Parkin	Apoptosis ↓	Rat	[139,140]
Small-molecule compounds	TSC01131	CRL4	Apoptosis ↓	mice	[87]
	ML264	KLF5	Inflammation↓ ECM degradation↓	Rat	[141]
	OICR-9429	KMT2D	ROS ↓	Rat	[142]
	SMTNP-191	NCOA1	ECM degradation ↓	Mice	[97]
	4-octyl itaconate	Nrf2	ECM degradation ↓	Rat	[75]
Extracellular vesicles (EVs)	BMSC-EV	miR-155-5p-Trim32	Apoptosis ↓	Mice	[143]
	BMSC-EV	miR-145a-5p/USP31	Inflammation↓ ECM degradation↓	Rat	[99]
	PRP-EV	NLRP3	Apoptosis ↓ Inflammation↓	Rat	[144]
	Apo-EV	USP5	Apoptosis ↓	Rat	[88,145]
	engineered BMSC-EV	UCHL1	Ferroptosis ↓ Senescence↓	Rat	[94]
	SF/GO-gel	CBL	Inflammation↓ Senescence↓	Rat	[146]
Bioactive materials	ROS-gel	TRIM16	Inflammation↓	Rat	[147]
	virus electrosorb-gel	TRIM63	ROS ↓	Rat	[96]
	Prussian Blue Nanoparticles	SOD1	ROS ↓	Rat	[148]
	Polydopamine Nanoparticles	GPX4	ROS ↓ Ferroptosis ↓	Rat	[149]

Fig. 4.

Ubiquitination-based therapy in IDD. Natural molecules, small-molecule compounds, extracellular vesicles, and biomaterials alleviate the progression of IDD by modulating ubiquitination- or deubiquitination-mediated processes.

5.1. Natural molecules or hormones

5.1.1. Galangin

Galangin is a natural plant-derived flavonoid with various biological activities. It exerts antioxidant and anti-inflammatory effects by regulating NF-κB and Nrf2 [150]. Additionally, Galangin can inhibit the degradation of type II collagen and aggrecan, maintaining ECM metabolic homeostasis [151]. Chen et al. [135] found that Galangin enhances the protein stability of Nrf2 by inhibiting its ubiquitin-dependent degradation, reduces the release of IL-1β-induced inflammatory factors (IL-1β, IL-6), and inhibits ECM degradation in NP cells. In vivo studies also confirmed that Galangin significantly alleviated IDD progression in rats.

5.1.2. Naringin

Naringin is a natural flavonoid glycoside primarily derived from citrus plants. It possesses a wide range of known pharmacological and biological activities, including antioxidant, anti-inflammatory, and bone/cartilage protective properties [152]. Studies show that Naringin at 20μg/mL significantly enhances NP cell proliferation and inhibits ECM degradation [153]. Gao et al. [154] further discovered that Naringin likely reduces the expression of MMP-3, ADAMTS5, IL-6, and TNF-α in IL-1β-treated human NP cells by inhibiting the NF-κB and p53 signaling pathways. Furthermore, Naringin exerts its protective effects by modulating ubiquitination-dependent processes. For instance, it activates SIRT3, a deacetylase that promotes K63-linked polyubiquitination and activation of Parkin, thereby enhancing mitophagy and protecting against oxidative stress-induced apoptosis of chondrocytes [136].

5.1.3. Melatonin

Melatonin is a hormone secreted by the pineal gland with anti-inflammatory and antioxidant effects [155]. Research indicates that Melatonin enhances ECM anabolism in NP cells by promoting autophagy, inhibiting oxidative stress, and suppressing pro-inflammatory factor release [156]. Parkin, an E3 ligase, plays a crucial role in mitophagy by mediating mitochondrial ubiquitination [157]. Studies have demonstrated that Melatonin can promote the clearance of damaged mitochondria by activating Parkin via increasing K63-linked polyubiquitination, thereby alleviating IDD in rats [139,140].

5.1.4. Friedelin

Friedelin is a natural pentacyclic triterpenoid widely found in various plants. It is reported to have anti-oxidative stress, anti-inflammatory, and anti-tumor effects [158]. Studies have proven that Friedelin promotes the binding of the E3 ligase RNF182 to p65, accelerating the K48 ubiquitination and degradation of p65, and inhibiting the NF-κB signaling pathway. This effect significantly reduces NP cell degeneration and inflammatory cytokine production, alleviating structural damage to intervertebral discs in rats [82].

5.1.5. Glutamine

Glutamine is an endogenous metabolite that serves as a carbon and nitrogen donor for nucleotides, amino acids, and the tricarboxylic acid cycle. It participates in various biological processes and is closely associated with human diseases [159]. Glutamine inhibits glycolysis, promotes autophagy, and reduces NP cell senescence [160]. It inhibits ferroptosis, pyroptosis, and ECM degradation in NP cells by deubiquitinating and stabilizing Nrf2 [138].

5.1.6. Astragalus polysaccharide

Astragalus Polysaccharide (APS) is a water-soluble heteropolysaccharide isolated from Astragalus membranaceus, possessing anti-inflammatory, antioxidant, and anti-apoptotic properties [161]. In osteoarthritis, APS exhibits anabolic effects and inhibits chondrocyte catabolism. In vitro experiments confirm that APS mitigates chondrocyte ferroptosis by modulating the Nrf2/HO-1 pathway [162]. Studies have proven that APS inhibits NP cell senescence and apoptosis by activating telomerase, thereby suppressing IDD progression [163,164]. Emerging evidence indicates that APS promotes OGT-mediated O-GlcNAcylation to stabilize Nrf2 expression through the ubiquitin-proteasome pathway, further promoting the synthesis of type II collagen and aggrecan, and alleviating IDD progression in rats [137].

5.2. Small-molecule compounds

Small-molecule compounds play an increasingly important role in the treatment of IDD. Their core strategy is to correct cellular homeostasis imbalance by specifically blocking abnormally activated signaling pathways or enzyme activities, thereby delaying degenerative progression [165]. Specific inhibitors targeting the ubiquitin-proteasome system are particularly noteworthy for their therapeutic potential in IDD. CUL4 recruits DDB1 to assemble CRL4, which ubiquitinates and degrades CtBP1/2, triggering BBC3-dependent apoptosis. TSC01131 is a small molecule specifically targeting the CUL4-DDB1 interaction, inhibiting CRL4 enzyme activity. TSC01131 inhibits CRL4-mediated ubiquitination and degradation of CtBP1/2 in vitro and in vivo, thereby reducing BBC3 expression levels and suppressing NP cell apoptosis. Administration of TSC01131 to mice significantly improved IDD progression [87]. Krüppel-like factor 5 (KLF5) plays an important role in skeletal development. Studies have shown that KLF5 mediates MMP9 expression to promote cartilage matrix degradation [166], and has been proven to mediate pro-inflammatory cytokine expression via the NF-κB pathway [167]. Xie et al. [141] found that SMAD Specific E3 Ubiquitin Protein Ligase 2 (SMURF2) mediates the ubiquitination and degradation of KLF5, alleviating inflammation and ECM degradation in rat NP cells. They further discovered that ML264 (a KLF5 inhibitor) synergizes with TGF-β to promote KLF5 ubiquitination and degradation, reversing the IL-1β-activated NF-κB pathway and restoring disc metabolic balance. Lysine Methyltransferase 2D (KMT2D) promotes ECM degradation by inducing the expression of MMP. ROS generated by H₂O₂ inhibit the ubiquitination and degradation of KMT2D in NP cells. OICR-9429 (a methylation inhibitor) inhibits NP cell degeneration under oxidative stress by targeting KMT2D [142]. As previously described, HERC3 deficiency inhibits the ubiquitination and degradation of NCOA1, promoting ECM degradation. SMTNP-191 is a compound targeting NCOA1 that reduces ECM degradation by inhibiting NCOA1 [97]. ZNF598 promotes oxidative stress and CEP degeneration by mediating the ubiquitination of Nrf2. 4-octyl itaconate (4OI) enhances the stability of Nrf2 protein by inhibiting ZNF598-dependent ubiquitination of Nrf2, thereby suppressing CEP catabolism and alleviating IDD [75].

5.3. Extracellular vesicle

Extracellular vesicle (EVs) is mainly produced by endocytosis and ultimately released through fusion with the cell membrane, playing key roles in various cellular processes. They are roughly classified into three categories based on their contents: exosomes, microvesicles, and apoptotic bodies (ApoEVs) [168]. EVs have emerged as a novel and promising strategy for the treatment of IDD [169]. Moreover, EVs targeting ubiquitination modifications are of particular interest in IDD therapy. MSC are the main source of EVs used for treating IDD. Studies have proven that BMSC-derived exosomes carrying miR-155-5p block TNF-α-induced NP cell apoptosis by inhibiting the expression of Trim32 [143]. Su et al. [99] found that BMSC-derived exosomes carrying miR-145a-5p target USP31, inhibit its deubiquitination of HIF-1α, and downregulate the expression of inflammatory factors and MMP13. PRP-derived exosomes promote the ubiquitination and degradation of NLRP3, inhibit M1 macrophage polarization and IL-1β secretion, reduce NP cell apoptosis, and alleviate the inflammatory microenvironment of IDD [144]. The ApoEV enriched with USP5 promotes USP5 nuclear translocation in NPCs, reducing NP cell apoptosis by stabilizing E2F1 [88,145]. Furthermore, engineered exosomes are natural exosomes modified to confer specific functions, including surface modification and loading of contents (drugs, proteins, and nucleic acids). Studies have confirmed that exosomes loaded with UCHL1 overexpression plasmids increase the stability of HSPA8 through deubiquitination, thereby activating CMA and inhibiting NP cell senescence [94].

5.4. Biomaterials

5.4.1. Hydrogels

Hydrogels, with their three-dimensional network and highly water content, not only provide mechanical support for the IVD but also support cell migration, adhesion, and proliferation [170].

Additionally, hydrogels can be loaded with drugs, small molecules, and stem cells to promote precise drug release and enhance therapeutic efficacy [171]. Therefore, hydrogels are considered ideal materials for IDD repair. In ubiquitination-based strategies for treating IDD, hydrogels are often used as carriers. Guo et al. [146] constructed an ultrasound-responsive graphene oxide hydrogel loaded with Troxerutin, enabling stable and sustained release of Troxerutin within the IVD. Troxerutin alleviates IDD progression by activating the Tie2 receptor, promoting CBL-mediated K48 ubiquitination and degradation of EGFR, thereby inhibiting inflammation and senescence in NP cells. Zhang et al. [147] constructed a ROS-responsive thermosensitive hydrogel loaded with MR409 (a growth hormone-releasing hormone analog) for controlled release. MR409 alleviates NPC inflammation and degeneration by inhibiting TRIM16-mediated autophagy and suppressing IL-1β secretion. Lentiviruses can efficiently and specifically target genes, and lentivirus-based therapy is considered a promising approach for IDD. Guo et al. [96] constructed an electroadsorptive hydrogel loaded with lentivirus specifically targeting the PTEN-TRIM63-ACE axis. It improves endoplasmic reticulum stress and alleviates IDD through TRIM63-mediated K48-linked ubiquitination and degradation of ACE.

5.4.2. Nanoparticles

Nanotechnology has broken the limitations of traditional delivery, offering significant advantages in targeted delivery, controlled and sustained release, and overcoming physiological barriers. Nanoparticles can effectively enhance the stability and solubility of encapsulated substances, promote intermembrane transport, and thereby improve therapeutic effects [172]. Additionally, some nanoparticles themselves possess mimetic enzyme activity, which can directly scavenge ROS and maintain redox balance [173]. Prussian blue nanoparticles improve mitochondrial quality by inhibiting the ubiquitination and degradation of SOD1, thereby enhancing the antioxidative capacity of NP cells and ultimately ameliorating ROS-induced IDD in rat models [148]. Polydopamine nanoparticles possess ROS-scavenging and Fe²⁺ chelation capabilities, and can inhibit oxidative stress-induced ferroptosis in NP cells in vitro. They co-localize with GPX4 and inhibit its ubiquitination and degradation, suppressing NP cell ferroptosis and improving AF puncture-induced IDD progression in rats [149].

6. Conclusion and future perspectives

This review systematically delineates the molecular networks through which E3 ubiquitin ligases and DUBs regulate IDD. These findings deepen our understanding of the complex pathogenesis of IDD. Furthermore, therapeutic strategies targeting the ubiquitination system hold broad promise. However, translating these discoveries into clinical applications still faces several challenges.

E3 ligases and DUBs play a central role in the pathological processes of IDD, mainly by forming a regulatory network of “enzyme-substrate-pathway-degenerative phenotype”. They precisely influence the progression of IDD by regulating the stability of key proteins in pathways such as HIF-1α, p65, Nrf2, NLRP3, PINK1/Parkin, and NF-κB. However, a single pathway often involves multiple pathological processes, and these processes are interconnected, which increases the complexity of ubiquitination-mediated regulation in IDD. Future research should employ systems biology approaches (e.g., proteomics, mass spectrometry) to comprehensively explore the ubiquitin enzyme-substrate networks and validate the specific functions of particular enzymes at different stages of IDD through animal experiments.

Various ubiquitination-based targeted strategies have been developed and demonstrated potential in treating IDD. However, these strategies face common challenges in clinical translation, such as delivery efficiency, safety, and long-term efficacy. The IVD is the largest avascular tissue in the human body. The nutrient supply of NP and AF mainly relies on diffusion through the endplate [174]. Drug diffusion from the endplate to the AF and NP is affected not only by distance but also by molecular weight [175]. Generally, only small molecules can effectively diffuse through the pores of the endplate or AF. Furthermore, since the NP matrix is rich in negatively charged chondroitin sulfate, positively charged drugs tend to be retained more easily within the NP [176]. Most natural compounds cannot efficiently penetrate the NP interior enclosed by the dense matrix, making it difficult for them to regulate cell phenotypes. Systemic administration is associated with first-pass effects and reduced bioavailability. Many natural compounds used for the treatment of IDD exhibit reduced in vivo drug concentrations due to metabolic instability [177]. Furthermore, natural compounds have attracted considerable attention in IDD therapy due to their multi-target and anti-inflammatory properties [178]. However, systemic inhibition of E3 ligases or DUBs may cause off-target effects and impair the function of other normal tissues. From the perspective of clinical translation potential, small-molecule inhibitors targeting specific E3 enzymes and engineered exosomes loaded with specific miRNAs or proteins represent the most attractive directions at present due to their well-defined components and ease of quality control. Biomaterials such as hydrogels and nanoparticles, as synergistic platforms, enable targeted, controllable, and long-term delivery, which will greatly enhance the in vivo efficacy of these therapies [179,180]. Developing inhibitors targeting specific E3/DUBs and combining them with novel biomaterials is expected to achieve more precise and sustained drug delivery within the IVD.

Furthermore, most current studies are limited to cell and animal models. Commonly used rodents (rats and mice) exhibit significant differences from humans in the IVD structure, biological characteristics, mechanical loading, and nutritional requirements [181]. In the future, it is necessary to conduct preclinical large animal model studies (such as goats, dogs, and non-human primates) that more closely mimic human pathological features, so as to evaluate the safety and efficacy of therapeutic strategies.

It is crucial to explore the crosstalk between ubiquitination and other PTMs. Complex crosstalk exists between ubiquitination and acetylation, which collectively determine cell fate. For example, the activity and stability of Sirt3 are dually regulated by deacetylation and deubiquitination. USP11 stabilizes Sirt3 through deubiquitination, and Sirt3 itself, as a deacetylase, modulates the acetylation levels of multiple proteins, jointly affecting mitochondrial function and ferroptosis. The specific roles and regulatory mechanisms of this complex PTM crosstalk network in IDD remain unclear. Future studies should employ techniques such as high-resolution mass spectrometry to delineate the multidimensional PTM landscape, including ubiquitination, acetylation, and SUMOylation. Deciphering these interactive networks will provide novel insights for the development of combination therapeutic strategies.

Authors’ contributions

Xuening Liu and Xuewen Kang conceived and designed the idea for this paper. Xuening Liu contributed to the visualization and the original draft preparation. Xuewen Kang contributed to revising the manuscript. All authors have read and approved the article.

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Declaration of generative AI in scientific writing

The authors declare that they have not used AI to write or edit this manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 82272536 and Grant No. 82460436), Natural Science Foundation of Gansu province (Grant No. 23JRRA1626), the Cuiying Scientific and Technological Innovation Program of The Second Hospital & Clinical Medical School, Lanzhou University (Grant No. CY2022-ZD-02).