Introduction

Low back pain (LBP) is considered as pain or discomfort in the area below the rib margin and above the buttock crease, and it is a common musculoskeletal disorder.1 With the intensification of global population aging, the incidence rate of LBP is on the rise.2 According to relevant studies, the global number of people suffering from LBP was 377.5 million in 1990, which increased to 577 million in 2017.3,4 In low- and middle-income countries, the prevalence of LBP is even higher. Based on limited data, the annual prevalence of LBP in adults is 57% and 67% in Africa and Latin America respectively,5,6 with a lifetime prevalence reaching up to 93%.7,8 LBP is the leading cause of disability, with a 54% increase in disability caused by LBP globally between 1990 and 2015.3,9 The LBP has also brought about a significant economic burden. According to estimates, the direct medical costs for LBP in the UK and the US are approximately £19.77 billion and $100 billion respectively, while in Japan it is 1.2 trillion yen.4,10 Although there are many factors contributing to LBP, IDD is the main cause, accounting for approximately 40% of symptomatic LBP.11,12

Figure 1 The main pathogenic factors of IDD.

Figure 2 Different developmental stages of IDD and their manifestations.

Figure 3 Diagnosis and current IDD treatments.

Figure 4 Redox disorders and their pathogenic mechanisms in degenerative IVD.

Figure 5 Complex signalling networks in degenerating IVD cells and their interaction with the oxidative stress microenvironment.

The intervertebral disc (IVD) is a fibrocartilaginous tissue located between the vertebrae, consisting primarily of the nucleus pulposus (NP), annulus fibrosus (AF), and cartilaginous endplate (CEP), which plays a crucial role in maintaining spinal stability, cushioning spinal pressure, and enhancing spinal mobility.13 In the development process of IDD, multiple risk factors are involved in the progression of IDD, including genetic susceptibility, aging, trauma, immune disorders, occupational exposure and abnormal non-physiological mechanical load14–17 (Figure 1). However, the exact pathogenic mechanism of IDD is unknown. Currently, IDD is considered to be a degenerative process involving molecules, cells, tissues and organs mediated by IVD cells in a specific genetic context, which impairs the normal tissue structure and biomechanical properties of the IVD, ultimately destroying its ability to withstand loading.8,18 The latest research indicates that the oxidative stress microenvironment plays a crucial role in the occurrence, development, and inhibition of regeneration in IDD.19 Oxidative stress is caused by an imbalance in intracellular and extracellular redox reactions, which induces cellular senescence, death, and imbalanced metabolism of the extracellular matrix (ECM) through various signaling cascades and intracellular signal transduction pathways, thereby participating in the progression of IDD.19 Current treatments for IDD are pharmacological and surgical to alleviate symptoms and reduce the incidence of disability, but both have the disadvantages of high complications, high costs and unknown efficacy.20 These methods can only act on the terminal stage of IDD and cannot delay or reverse the progression of IDD by improving the pathological changes of IVD. In addition, some recently emerging preclinical treatment methods, such as small molecule drugs, proteins, and nucleic acids, have shown excellent therapeutic effects in improving the survival and functional impairments of IVD cells.21 However, the short half-life and rapid elimination of these drugs limit their treatment efficacy.11 In this regard, approaches based on nano-drug delivery systems (NDDSs) may be promising for the treatment of IDD.22 Currently, NDDSs are mainly involved in the delivery of antioxidant agents through physical encapsulation (liposomes and exosomes, etc.) or chemical binding [inorganic and polymeric nanoparticles (NPs), etc.].23 Among them, some NDDSs possess excellent antioxidant capacity by themselves (polydopamine NPs, MnO2 NPs, fullerenes, and fullerols), and their combination with antioxidant agents can exert stronger synergistic antioxidant effects.24 Secondly, NDDSs also have the ability to control release, which mainly depends on physical conformation changes or chemical bond breaks of nanocarriers.25 In particular, some carriers can respond to internal (ROS, pH, enzymes, etc.) or external stimuli (temperature, light, magnetic field, etc.) to achieve precise controlled release of drugs in time and space.25,26 In addition, NDDSs can increase drug accumulation at the lesion through passive and active targeting capabilities, thereby improving efficacy and reducing side effects.27 These properties make NDDSs an effective method for improving the oxidative stress microenvironment within degenerating IVDs.

The aim of this review is to briefly review the changing pathophysiology of IDD and the limitations of current treatments. In addition, we also emphasized the oxidative stress microenvironment in degenerative IVDs and its role in IDD. Importantly, we demonstrated that biomedical engineering approaches can be used to ameliorate the oxidative stress microenvironment of IDD, thereby promoting regeneration and recovery of IDD, which may help advance the development of NDDSs in the treatment of IDD.

The Pathophysiology of IDD Normal Structure and Function of IVD

The IVD is an integral part of the spinal tissue and biomechanical composition. The normal spine comprises a minimum of 23 highly hydrated IVDs that connect adjacent vertebrae, playing a crucial role in enhancing spinal mobility and optimizing load distribution.28 The complete IVD comprises NP, AF, and CEP on both sides.

NP is located between the center and posterior of IVD, serving as the core of IVD.29 In general, NP is a soft and elastic gel-like substance. The healthy IVD typically exhibits a relatively high-water content, ranging from approximately 80% to 90%, whereas degenerated IVDs demonstrate a reduction in water content to around 70–75%, or even lower.30,31 In terms of cellular composition, NP cells are a group of mixed cells at different stages of maturation, used to maintain the metabolic balance of ECM.4 Before the age of 10, the mixed cell population consisted of larger vacuolated notochord cells (NC) (mainly expressing CK-8, LGALS3, STMN2, RTN1, PRPH, CXCL12, IGF1, MAP1B, ISL1, CLDN1 and THBS2) and smaller chondrocyte-like cells (mainly expressing HIF1α, GLUT1, proteoglycans, type II collagen, KRT18/19, CA12 and CD24).31–34 In adulthood, these cells all differentiate into chondrocyte-like cells with reduced metabolic activity.35 Research has shown that NC contributes to the regulation of proteoglycan production and proliferation activity in IVD chondrocytes, suggesting a potential association between NC degradation and IDD.36 In terms of the organization composition of NP, 35–65% is composed of proteoglycans, 5–20% is composed of collagen II (Col II) and elastin protein.37 The high specific gravity and negative charge properties of proteoglycans contribute to the highly hydrated characteristics of NP tissue, while also facilitating the dispersion of loads.11,38 The mesh composed of Col II and elastic protein fibers is embedded in the gel formed by proteoglycans to maintain the elasticity of ECM.39

The AF is located on the periphery of NP, consisting of well-arranged concentric rings or lamellae composed of collagen fibers and proteoglycans.4 The lamellae are inclined approximately 30° from one vertebra to another and cross diagonally at an angle of >60°between adjacent lamellae, which helps to limit rotation and bending between adjacent vertebrae while providing sufficient resistance to axial loads.37,40 The AF can be further divided into an inner fibrocartilage region and an outer fibrous region, which have different cellular and tissue compositions.21 The inner region mainly consists of circular chondrocyte-like cells that produce proteoglycans and Col II, while the outer region contains elongated fibroblast-like cells that primarily produce Col I.21,41 Correspondingly, the organizational structure of both the internal and external regions has also undergone excessive changes. AF tends to gradually lose proteoglycans, Col II, and water content from the inside out, resulting in a large amount of Col deposition, which effectively restricts NP during axial compression, extension, and bending.42–44

The endplate is the boundary between the IVD and the adjacent vertebral bodies, including the bony endplate and CEP. The main cell type within the CEP is the chondrocyte, which primarily secretes Col II and proteoglycans.45 Compared to NP, the proportion of proteoglycans and Col II in CEP is relatively low, which can provide a solid mechanical barrier between IVD and vertebral body, thereby preventing the protrusion of NP tissue into the trabecular bone of the vertebral body.46,47 In addition, CEP is also the main channel for IVD to exchange substances with the external environment.48

The IVD is the largest avascular tissue in the body.49 Compared to the lifelong avascularity in NP, CEP and AF have abundant blood vessels during fetal and infant stages.50 However, with age, the blood vessels in CEP will completely regress, while only the outer layer of AF maintains vascularization.51 The limited distribution of blood vessels in the IVD primarily facilitates substance exchange through diffusion (for small molecular solutes such as glucose, oxygen, and lactate) or convection (for large molecular solutes such as growth factors, hormones, and proteoglycans).52,53 Therefore, IVD is in a hypoxic microenvironment. According to the study of canine IVD samples, the oxygen tension in the IVD (0.53–1.06 kPa) is lower compared to that in muscle (3.8±0.8 kPa) and brain (3.4±0.2 kPa), and it decreases as it gets closer to the center of the IVD.54,55 The hypoxic microenvironment makes NP cells partially dependent on anaerobic glycolysis to produce ATP, which contributes to the formation of an acidic microenvironment.56,57 Regarding the innervation within the IVD, the NP has no innervation, whereas the outer layer of the AF is innervated by branches of the sinus vertebral nerve.58

Degenerative IVD: Molecular, Tissue and Mechanical Functions

IDD is an age-related degenerative process that is influenced and accelerated by other unfavourable factors, such as genetic and environmental factors.59 The process of degeneration can be divided into three closely connected stages (Figure 2). Firstly, under specific genetic backgrounds, abnormal mechanical load induces functional impairment of IVD cells, leading to an imbalance in ECM metabolism. Subsequently, the microenvironment of IVD continued to deteriorate, leading to further decrease in the number and activity of IVD cells, resulting in a cascade reaction of damage. In the later stage, IVD loses its mechanical function and is accompanied by infiltration of nerves and blood vessels, eventually leading to IVD-related LBP.

Although the traditional view holds that IDD is an age-related disease, it cannot be denied that genetic factors may play a significant role in determining this condition.60 The existence of genetic susceptibility to IDD is supported by preliminary evidence from twins and family studies. In 1999, Sambrook et al evaluated the MRI characteristics of cervical and lumbar IVD in 172 identical twins and 154 dizygotic twins.61 The results indicated a heritability rate of 74% for lumbar IVD degeneration and 73% for cervical IVD degeneration. Another prospective study involving 116 identical twins found that the estimated heritability of IDD within 5 years was between 47–66%.62 In addition, in a 10-year longitudinal study involving 234 pairs of twins, Williams et al found that disc herniation has a genetic component across all age groups.63 Recently, genome-wide association studies (GWAS) have made significant contributions to the etiology of complex diseases and have indicated that genetic susceptibility to IDD may be widespread.64–66 In 2022, Bjornsdottir et al conducted a (58,854 cases, 922,958 controls) GWAS study on IDD and identified 41 genetic variants in 33 loci that were associated with cartilage, bone biology, and inflammatory processes.67

In fact, IVD had already undergone degradation in the first decade of life.68 The reduction of vacuolar NCs in the NP is considered to be the initiation process of IVD degeneration, because NCs can increase the anabolic and proliferative activity of NP cells.36,69,70 In all cases, early IDD is mainly characterized by an imbalance between anabolic and catabolic processes in the ECM, which is mainly caused by a phenotypic transition of IVD cells.71,72 In terms of anabolism, there was a significant reduction in the production of proteoglycans, along with a shift in the composition of their side chains from chondroitin sulphate to keratan sulphate, which led to diminished hydration of NP tissues.73,74 The balance of collagen production shifts from Col II to Col I, accelerating the process of IVD fibrosis.74 In terms of catabolism, the expression of matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-13, MMP-14, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 significantly increased.74–76 In contrast, the levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were decreased.76 These factors have led to a further decline in the quality of ECM.

Early IVD degeneration lasts for a considerable period of time (10 or 20 years or more), which may be accelerated by other unfavorable factors (genetics, trauma and abnormal loads) and enters the radical phase. During the radical phase, the microenvironment within the IVD is severely deteriorated, accompanied by inflammation and oxidative stress storms.77 NP cells secrete various cytokines, including tumor necrosis factor α (TNF-α), interleukin-1 β (IL-1β), IL-6, IL-8, IL-17 and prostaglandin E2 (PGE2).47,78 Simultaneously, NP cells stimulate the synthesis of chemokines, specifically CCL2, 3, 4, 5, 7, 10 and 13, which activate and recruit immune cells to infiltrate the IVD.22,32 This further exacerbates the cellular inflammatory response. In addition, degenerating IVDs undergo intense local oxidative stress, with large amounts of reactive oxygen species (ROS) being generated.19 Excessive ROS can attack the mitochondrial respiratory chain, endoplasmic reticulum, and inhibit the production of reductases, further promoting the accumulation of ROS.19,79,80 Inflammatory response and oxidative stress promote each other, thus forming a positive feedback loop and acting directly or indirectly on a dynamic signaling network with complex feedback loops composed of different signaling pathways.72,81,82 Aberrant activation or inhibition of these signaling networks may exacerbate NP dysfunction, including apoptosis, senescence, hypertrophy and calcification, which in turn exacerbate the imbalance of ECM synthesis and degradation.18,83 In addition, hypertrophy and calcification of CEP cells and sclerosis and occlusion of the bone marrow lumen will lead to loss of CEP permeability, which will result in impaired energy supply and accumulation of metabolic wastes within the IVD, ultimately accelerating IDD.84–86

When entering the late stage of IDD, IVD will gradually lose its mechanical function and result in IVD-related LBP. At this time, the normally active NP cells are almost completely damaged, and proteoglycan and Col II are replaced by Col I.87 As a result, the highly hydrated and elastic jelly-like NP is replaced by stiff fibrous tissue, which results in an inability to effectively distribute the load.87 Meanwhile, the imbalance in ECM metabolism leads to structural disorder in AF, resulting in a significant reduction in its ability to resist loads and the occurrence of cracks.87 Once the integrity of AF is compromised, internal NP tissue is exposed to the circulatory system.88 The immune system will recognize these “foreign” tissues and generate an autoimmune response.89 IgG antibodies and antigen-antibody complexes against proteoglycans and collagen were found in human degenerative IVD samples, providing direct evidence for autoimmune reactions.90,91 In addition, NP cells and immune cells secrete a large amount of IL-1, TNF-α, IL-8, and PGE2, which can stimulate the production of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF).92–94 These growth factors induce infiltration of blood vessels and nerves, leading to IVD-induced LBP.94,95 Moreover, the herniated NP can exert direct compression on the adjacent nerve roots, resulting in significant low back and leg pain.96 Meanwhile, the dysfunctional IVD causes spinal instability, leading to degeneration of surrounding small joints, formation of bone spurs, and hypertrophy of surrounding ligaments.97,98 This in turn results in spondylolisthesis, spinal stenosis, and even degenerative scoliosis.98

Diagnosis and Treatment of IDD

The initial diagnosis of IDD is mainly based on the patient’s symptoms and physical signs. Patients with IDD typically present with LBP. This type of pain is often characterized by an unclear location, primarily concentrated in the central region of the lower back or adjacent to the IVD, and worsens after prolonged standing or sitting. When the protruding NP directly compresses or the inflammatory response irritates the nerve root, LBP may worsen and radiate to one or both limbs. In addition to clinical symptoms and signs, the diagnosis of IDD is mainly based on X-ray, CT scan or MRI, and its severity is further assessed by IVD hydration status (Pfirrmann) or CEP Modic changes.99,100

Currently, the treatment of IDD includes conservative treatment and surgical treatment according to the patient’s symptoms (Figure 3). Conservative treatment includes drug therapy and non-drug therapy, which are mainly suitable for early-stage IDD patients and can alleviate symptoms and improve quality of life. When the degeneration is mild, bed rest, appropriate and regular exercise, weight loss, and physical therapy (such as cold compresses, electrical stimulation, ultrasound therapy, and traction) are simple and effective measures to improve symptoms.101–103 These methods are designed to reduce stress on the IVD, strengthen the low back muscles and improve spinal stability. When patients experience significant pain in the lower back, medication can be used for treatment, including nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, muscle relaxants, benzodiazepines, antidepressants, corticosteroids and antiepileptic drugs.102 For some advanced patients, some invasive methods can be used, such as chemical nucleolysis, radiofrequency ablation, nucleotomy and nerve root blockade.21 Once conservative treatment fails, symptoms of nerve root compression are severe, cauda equina syndrome and severe spinal deformity or instability occur, surgical treatment is required. Surgical treatment mainly consists of interbody fusion, partial or total IVD removal and total disc replacement (TDA).103 Among them, interbody fusion has always been regarded as the surgical gold standard for symptomatic IVD protrusion and is suitable for the majority of patients with disc herniation.103,104 TDA has similar clinical results to interbody fusion, but is usually reserved for single-level situations without facet joint disease.104–106

Oxidative Stress and IDD Oxidative Stress in IDD

ROS are a class of unstable and highly reactive molecules, including superoxide anion (O2−), hydroxyl radical (OH-), hydrogen peroxide (H2O2), and hypochlorite ion (OC1−).107 In addition, some nitrogen-containing compounds (nitric oxide and nitrogen dioxide) are also considered as members of the ROS superfamily due to their similar effects.108 As a byproduct of aerobic metabolism, the generation of ROS in cells is inevitable Although IVD cells are in a hypoxic environment due to lack of direct blood supply, aerobic metabolism is still an important way for IVD cells to obtain energy and thus generate ROS.80 In order to maintain redox balance, the human body has developed a set of ROS scavenging systems, which remove excess ROS to keep them at a level that is harmless to the organism.109 In normal levels, ROS is an important intracellular signaling molecule that participates in various physiological processes through intracellular signal transduction.110 As IVD degeneration progresses, aging, trauma, abnormal mechanical load, and smoking cause an imbalance between oxidants and antioxidants, resulting in oxidative stress, which in turn leads to damage to molecules, cells, and tissues111 (Figure 4). At the molecular level, biomacromolecules such as proteins, nucleic acids, lipids, and carbohydrates in cells are inevitably exposed to ROS and suffer from oxidative damage.111 Currently, there are reports indicating a significant elevation of advanced oxidative protein products (AOPPs), 8-oxoguanine (an oxidative product derived from guanine), malondialdehyde (MDA), and advanced glycation end products (AGEs) in degenerative human IVD tissue.112–115 At the cellular level, large amounts of ROS lead to restricted survival and altered metabolic phenotype of IVD cells by damaging cellular macromolecules, which in turn accelerates IDD progression.79 At the tissue level, oxidative stress causes damage to normal IVD tissue structure and biomechanical dysfunction, which in turn leads to corresponding clinical symptoms.116

Redox Imbalance Within the IVD Increased ROS Production in Degenerative IVD

Mitochondria are the center of oxidative metabolism and the main site of ROS production, because 1–2% O2 is converted into ROS during oxidative phosphorylation, which depends on the mitochondrial electron transport chain (ETC).117,118 Indeed, under physiological conditions, 0.2–2% of the electrons do not follow the normal ETC transfer sequence (complex I/III/IV and complex II/III/IV with succinate as substrate), but rather leak directly from the ETC and interact with O2 to produce O2 -.119 The complexes I and III are the main sites where electrons transfer to O2 to generate O2-.120 For complex I, ROS are thought to originate from reduced flavin mononucleotide or N-1a and N-1b iron-sulfur clusters, whereas ROS for complex III are thought to originate from ubiquinone oxidation sites.121,122 When the activities of complex I and complex III are inhibited or the concentration of succinate is low, complex II becomes the main source of ROS.42 Interestingly, under hypoxic conditions, complex II switches its catalytic activity from succinate dehydrogenase to fumarate reductase, and this change is associated with increased ROS generation.123,124 Therefore, it is possible that complex II has a unique association with increased ROS production in degenerative IVD. In degenerative IVD, the adverse microenvironment can lead to abnormal opening of mitochondrial permeability transition pores (mPTP) in IVD cells, decreased membrane potential, and damage to the respiratory chain, ultimately leading to mitochondrial dysfunction.125,126 Mitochondrial dysfunction leads to massive electron leakage, which increases ROS production.127 A large amount of ROS attacks mitochondrial DNA, proteins, and membrane lipids, which further exacerbates mitochondrial damage, thus forming a vicious cycle.127,128

The NADPH oxidase (Nox) family comprises seven isoforms (Nox1, Nox2, Nox3, Nox4, Nox5, Duox1, and Duox2), which exhibit distinct expression patterns across various cell types and subcellular compartments.129 Nox generates O2− or H2O2 by catalyzing NADPH electron transfer.130,131 The significant increase in the NADPH/NADP+ ratio in degenerated IVD cells suggests that NOX is involved in the formation of the oxidative microenvironment in IDD.132,133 In IDD, the most extensively studied are Nox 2 and Nox 4.134–136 Chang et al found that 15% high cyclic stretch upregulated Nox 2 expression in AF cells, which led to a significant increase in ROS levels.137 In addition, 20% O2 increases the production of ROS by upregulating Nox4 in NP cells, thereby activating the p53-p21-Rb and p16-Rb pathways to induce cellular senescence.138 The endoplasmic reticulum (ER) is also an important source of ROS.139,140 Under the influence of nutritional deficiency, oxidative stress, and genetic mutations, the accumulation of unfolded/misfolded proteins leads to ER stress, which in turn activates the unfolded protein response (UPR).141,142 This phenomenon is widely observed in IDD.142,143 During the UPR process, the involvement of protein disulfide isomerase, endoplasmic reticulum oxidoreductin-1 and Nox complex can trigger ROS cascade reactions.42 In addition, ER stress leads to Ca2+ overload and increases intra-mitochondrial Ca2+ levels through the mitochondria-associated membrane (MAM), which in turn leads to mitochondrial dysfunction and increased ROS generation.139,144 In addition to the above pathways, xanthine oxidase (XO), uncoupled endothelial nitric oxide synthase, cytochrome P-450 monooxygenases, and cyclooxygenases are also sites of ROS generation,42,120 collectively contributing to endogenous ROS accumulation. In addition to endogenous pathways, external stimuli such as smoking, UV radiation, ionizing radiation, air pollution and drugs can also lead to the accumulation of ROS.145–147

Reduced Antioxidant Defense in Degenerative IVD

The complete intracellular antioxidant defence system mainly consists of antioxidant enzymes, non-enzymatic small molecules and redox signal transduction.116 Antioxidant enzymes are the first line of defense system, mainly including superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), and methionine sulfoxide reductase (Msr).148,149 SOD is an important antioxidant enzyme, which can be classified into Cu/Zn-SOD based on the types of metal ions it binds to.150 These two enzymes are prevalent in the mitochondria of NP cells and alleviate oxidative stress by efficiently catalysing the conversion of O2− to H2O2 and O2.151,152 Similarly, GPX and CAT are members of the antioxidant defence within the IVD, both of which can catalyze the decomposition of H2O2 into H2O.151 However, multiple studies have indicated a significant decrease in the expression of SOD, GSH, and CAT in degenerated IVD tissues and cells.153–155 In addition, Msr is an repair enzyme that can remove ROS by reducing the methionine residues in oxidized proteins.156 The expression of Msr is significantly reduced in aging AF cells, which makes AF cells vulnerable to oxidative stress damage.157 Following antioxidant enzymes, non-enzymatic small molecules are a class of compounds that do not rely on the enzyme system in the organism to scavenge free radicals and combat oxidative stress, and mainly include vitamins C and E, β-carotene, lipoic acid, ubiquinone, melatonin (MT), carotenoids, ascorbic acid and uric acid.97,158 They are widely present in the cytoplasm of IVD cells, and the reduction of their expression levels can also lead to oxidative stress in IVD. In addition to the classical antioxidant system, intracellular redox signaling is also an important way to maintain intracellular redox homeostasis, which mainly includes the Kelch-like ECH-associated protein 1 (Keap1) - Nuclear factor E2- related factor 2 (Nrf2) - Antioxidant response element (ARE) signaling pathway.159 When the intracellular ROS levels increase, Keap1 dissociates from CUL-E3 ligase, causing a conformational change in Keap1 and releasing Nrf2, which leads to the accumulation of Nrf2 in the cytoplasm and subsequent nuclear translocation.160 High levels of Nrf2 bind to the ARE, which activates the transcription of antioxidant genes, including heme oxygenase-1 (HO-1), GSH, SOD, and CAT, and thus exerts antioxidant effects.116,160 I In human degenerated IVD and aged NP cells, Nrf2 expression levels were significantly decreased and correlated with the severity of IDD.161,162 In addition, high levels of Nrf2 promote mitochondrial biosynthesis by up-regulating the expression of nuclear respiratory factor 1/2 (NRF-1/2), PGC-1α, and mitochondrial transcription factor A, as well as mitochondrial fusion by degrading the mitochondrial fission protein dynamin-related protein 1 (Drp1), which contribute to protection against oxidative stress.163,164

Role of Oxidative Stress in IDD

ROS is an important signaling molecule within cells, which means it can directly or indirectly interact with dynamic signal networks composed of different signaling pathways and complex feedback loops, thereby regulating the physiological and pathological processes of IDD151 (Figure 5). During this process, there are various phenotypic changes in IVD cells, which accelerate the progression of IDD.

Oxidative Stress and Cell Death

The balance of anabolic and catabolic metabolism within the IVD is dependent on the metabolic capacity conferred by a sufficient number of cells and normal cellular function. When excessive cell death occurs within the IVD, this balance is disrupted and promotes the progression of IDD. ROS are potent inducers of cell death, and here we focus on three cell death types, including apoptosis, pyroptosis, and ferroptosis.165,166

Apoptosis is a controlled cell death that includes two major pathways: intrinsic and extrinsic,167 the former of which is often associated with mitochondrial dysfunction. Intrinsic apoptosis depends on the abnormal activation of BCL-2 family proteins on the mitochondrial membrane,168 which leads to increased mitochondrial outer membrane permeability and leakage of cyt-c, ultimately inducing cell apoptosis by activating a series of caspases.169 ROS is involved in IVD cell apoptosis through the regulation of multiple signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt), NF-κB, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), Sirtuin (SIRT) protein family, Keap1/Nrf2, and AMP-activated protein kinase (AMPK).170–173 The activation or inhibition of these signaling pathways leads to the differential expression of pro-apoptotic proteins (such as Bak, Bax, and Bok) and anti-apoptotic proteins (such as Bcl-XL, Bcl-2, Bcl-w, and Mcl-1), thereby inducing cell apoptosis.168 For example, when IVD cells were co-cultured with oxidants (H2O2 and TBHP) in vitro, a decrease in the expression of PI3K/Akt,174 AMPK,175 SIRT,114 and Nrf2162 and an increase in the expression of MAPK/ ERK176 and NF-κB177 were observed. This resulted in up-regulation of BAX, Caspase-9 and Caspase-3 expression and down-regulation of Bcl-2 expression, which in turn induced apoptosis. In fact, these signaling pathways are not isolated but intertwined with each other to form positive or negative feedback loops. For example, when the PI3K/Akt signaling pathway is activated, Akt can phosphorylate and inhibit the degradation of IκBα (a member of the IκB protein family and a specific inhibitor of NF-κB), thereby suppressing the activation of NF-κB.178 In specific circumstances, NF-κB can in turn promote the activation of Akt, forming a positive feedback loop that enhances cell survival capability.179 In addition, the roles of these signaling pathways in cell apoptosis are diverse and may depend on the cell type, stimulus type, and intracellular environment.180

Pyroptosis is a type of programmed cell death between apoptosis and necrosis that depends on the activation of inflammasomes.181 Inflammasomes are a class of protein complexes, the most widely studied of which is the NLRP3 inflammasome,182 which can sense pathogen-associated molecular patterns or damage-associated molecular patterns and then recruit and activate Caspase-1.182 The activated Caspase-1 can cleave and activate other proteins, such as Gasdermin D (GSDMD), IL-1β, and IL-18.183 After being cleaved, the N-terminal fragment of GSDMD forms pores on the cell membrane, leading to leakage of cellular contents and triggering a strong inflammatory response.184 Compared with non-degenerated cells, high levels of ROS and caspase-1 were observed in degenerated human IVD cells.185 In vitro, after NP cells were co-cultured with H2O2, the expression of ROS, NLRP3 inflammasome and caspase-1 in the cells was significantly upregulated, indicating that oxidative stress can effectively induce cell pyroptosis.186 In fact, oxidative stress is involved in the induction of cell death through various mechanisms, including activation of inflammasomes, mitochondrial dysfunction, enhanced inflammatory response, and instability of cell membrane.187 ROS can directly promote the activation of NLRP3 inflammasome, with mitochondrial ROS (mtROS) being the most important signal for NLRP3 inflammasome activation,188 which has also been demonstrated in cardiovascular disease.189 In addition, ROS can damage IVD cell mitochondria, leading to abnormal opening of mPTP and leakage of mitochondrial DNA (mtDNA).190 Released mtDNA promotes cell pyroptosis by activating the Toll-like receptor 9 (TLR9)-NF-κB-NLRP3 axis.191 Recent studies have also shown that thioredoxin-interacting protein (TXNIP) is a crucial bridging molecule for the activation of NLRP3 inflammasome.192 When ROS is overloaded in IVD cells, TXNIP dissociates from Thioredoxin (Trx) and binds to NLRP3, thereby initiating cell pyroptosis, but this process is inhibited by Nrf2 signaling.186,193 A possible explanation is that Nrf2 inhibits TXNIP function by up-regulating antioxidant proteins and regulating the balance of Trx/TXNIP complexes.194–196 In addition, the SIRT protein family is involved in the regulation of cellular pyroptosis.197 Overexpression of SIRT1 can improve IL-1β-induced mitochondrial dysfunction and NLRP3 inflammasome activation in NP cells by promoting PINK1/Parkin-mediated mitophagy, thereby alleviating NP cell pyroptosis.188

Ferroptosis is a form of iron-driven cell death characterized by intracellular iron accumulation and oxidative stress, resulting in lipid peroxidation and membrane damage.198 In fact, there is a vicious cycle of mutual promotion between oxidative stress and iron overload.199 Iron is a catalyst for many oxidation reactions, especially the Fenton Reaction, in which Fe2+ can combine with H2O2 to form OH-, accelerating the accumulation of intracellular ROS.200 Furthermore, iron overload can induce the depletion of antioxidant defense systems, rendering cells more vulnerable to oxidative stress-induced damage.201,202 On the other hand, oxidative stress can exacerbate iron overload. Oxidative stress can lead to the oxidation modification of ferritin, weakening its normal iron storage capacity and exacerbating the accumulation of iron within cells.203 Compared with the normal control group, the expression of GPX4 and ferritin in degenerated IVD tissues was significantly decreased, accompanied by increased oxidative stress and ferroptosis.204 In vitro, co-culture of oxidants with IVD cells also leads to oxidative stress, lipid peroxidation and iron overload in a dose-dependent manner.204–206 Multiple molecules and signaling pathways are involved in regulating iron death within the IVD. Ferroportin (FPN) is an iron efflux protein responsible for the translocation of intracellular iron to the extracellular space.207 In TBHP-induced human NP cells, FPN is severely dysfunctional, which leads to intercellular iron overload and ferroptosis.208 Nrf2 is also an important signaling molecule that regulates ferroptosis in IVD cells.194 In vitro, Nrf2 activation can improve Parkin-mediated mitophagy, which helps to alleviate mitochondrial dysfunction and oxidative stress, thereby inhibiting ferroptosis.209 Similarly, Nrf2 can also upregulate the expression of HO-1 and inhibit the nuclear translocation of NF-κB, thereby alleviating oxidative stress, inflammatory response, and ferroptosis in IVD cells.84,210,211 In addition, SIRT3 can also alleviate oxidative stress-induced ferroptosis in IVD cells by upregulating the expression of antioxidant genes HO-1, NQO1, SOD2, and GPX4.212

Oxidative Stress and Cellular Senescence

Cellular senescence is the process by which cells gradually lose their function and proliferative capacity during their life cycle, including replicative senescence and stress-induced senescence.213 The replicative senescence is caused by the shortening of telomeres and exacerbated by factors that promote the accumulation or inhibit the breakdown of cellular aging, with ROS being the most important factor inducing cell senescence.113 In both human and rat degenerated IVD tissues, the proportion of senescence-associated β-galactosidase (SA-β-gal) positive cells significantly increases, which is positively correlated with oxidative stress levels within the IVD.214 Cellular senescence not only leads to a decrease in the number of functionally normal IVD cells, but also deteriorates the IVD internal microenvironment through the paracrine effect of senescence-associated secretory phenotype (SASP).16 These SASP-associated proteins include ECM-degrading enzymes (MMPs and ADAMTS), pro-inflammatory cytokine factors (IL-1β, TNF-α, IL-7, IL-8), chemokines, and other biologically active substances, which can further exacerbate the mitochondrial dysfunction and the production of ROS, thus generating a vicious circle.39,215 More importantly, high levels of SASP-associated MMPs have been shown to induce autocrine ligand shedding, which renders senescent cells resistant to immune surveillance and clearance.216 Multiple signaling molecules are involved in oxidative stress-induced senescence of IVD cells. For example, ROS can promote the ubiquitin-dependent degradation of IκB through ROS-Hsp27-IκB kinase (IKK), leading to increased nuclear translocation of NF-κB (such as p65) and subsequent activation of downstream target genes to regulate cellular functions.19,217 Compared with normal NP cells, the p65 level in the nucleus of oxidant-induced degenerative NP cells increased, and induced cell senescence by regulating p53-p21-Rb and p16-Rb.218,219 The PI3K/Akt signaling pathway is an important intracellular signaling pathway that participates in regulating cell apoptosis, aging, growth, and metabolism.220 When the PI3K/Akt signaling pathway is activated, it activates downstream signaling molecules to alleviate mitochondrial dysfunction and intracellular ROS levels, while downregulating the p53-p21-Rb signaling axis to mitigate cellular senescence in IVD cells.174,220 Recently, Zhang et al found that high concentrations of lactic acid can induce senescence and oxidative stress in NP cells.221 Mechanistically, lactate can bind to lysine 39 and leucine 52 residues in the Akt PH domain to inhibit Akt kinase activity, thereby inducing NP cell senescence and oxidative stress by regulating p21/p27/cyclinD1 and Nrf2/HO-1 signaling. In addition, the SIRT protein family is also involved in regulating cellular aging in IVD. Activation of SIRT1 has been reported to ameliorate oxidative stress-induced cellular senescence and ECM degradation.222,223 Similarly, several studies have found that SIRT3 activation can improve mitochondrial function and SOD activity, and alleviate IVD cell senescence by inhibiting p16-Rb signaling.224,225 In addition, SIRT2 overexpression can also inhibit oxidative stress by upregulating the expression of SOD1/2 and inhibit NP cell senescence by downregulating the level of p53-p21-Rb.226

Oxidative Stress and Cellular Autophagy

Autophagy is an evolutionarily conserved self-degradation system that degrades and recycles intracellular proteins and damaged organelles to maintain cell homeostasis and function.227 Compared with healthy IVD tissues, the expression levels of autophagy-related genes (Atg) in degenerated IVD tissues were significantly changed, indicating that autophagy plays an important role in IDD.228,229 As a stress response system, almost all stress factors that affect cellular homeostasis can induce autophagy. In recent years, an increasing amount of evidence has shown that ROS is a crucial intracellular signaling molecule that affects autophagy.230,231 However, the relationship between autophagy and oxidative stress is not a simple upstream-downstream signaling regulation relationship, but rather a complex network composed of numerous signaling pathways and crossroads. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that responds to nutrient levels and growth signals.82 It is a negative regulator of autophagy initiation and its activity is regulated by PI3K-Akt, AMPK and MAPK.232–234 Excessive ROS down-regulated the ratios of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR, resulting in the inhibition of the PI3K/Akt/mTOR signaling pathway, which in turn induced autophagy in NP cells.235 In addition, ROS can also downregulate the level of p-AMPK, leading to the downregulation of ULK1 phosphorylation and upregulation of mTOR phosphorylation, thereby inhibiting NP cell autophagy activation and autophagic flux.235 Interestingly, under oxidative stress conditions, the upregulation of p38MAPK phosphorylation promotes mTOR phosphorylation, thereby inhibiting autophagy in NP cells.236 In addition to being regulated by upstream signaling molecules, mTOR can also regulate autophagy through its target genes. Downregulation of mTOR signaling can lead to increased nuclear translocation of EB transcription factor (TFEB) and downregulation of p70S6K levels, thereby enhancing cellular autophagy to combat oxidative stress.237–239 Mitophagy is a type of cellular autophagy that plays a key role in maintaining mitochondrial quality control.240 PINK1/Parkin signaling pathway is an important signaling axis regulating mitophagy.116 In NP cells, upregulation of the PINK1/Parkin signaling pathway induces mitophagy, thereby delaying the progression of IDD by eliminating damaged mitochondria.162,188 In addition, the SIRT protein family and Nrf2 can also regulate mitochondrial autophagy in IVD cells.241,242

Oxidative Stress and Inflammation

The inflammatory response is a defence mechanism against pathogens, damaged cells or specific stimuli, which is induced by a stressor and leads to the expression of inflammatory cytokines through a series of intracellular signal transduction.243 As a complex immune response, inflammatory response can maintain tissue homeostasis by eliminating pathogens and repairing damaged tissues. However, excessive activation and persistence of inflammatory responses may lead to tissue damage and a variety of diseases. ROS can act as signaling molecules to activate the expression of inflammation-related genes, thereby initiating or exacerbating inflammatory responses.244 In vitro, co-culture of rat NP cells with H2O2 can lead to the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and nitric oxide synthase (iNOS).245 In addition, ROS can cause damage to intracellular biomacromolecules and organelles (such as mitochondria), which will trigger the activation and assembly of NLRP3, further aggravating the inflammatory response.246,247 ROS regulates inflammatory responses through a variety of signaling molecules, including PI3K/Akt, MAPK, Nrf2, and NF-κB, among which MAPK and NF-κB pathways play the most important roles.248,249 ROS can activate MAPK kinase, which in turn phosphorylates and activates downstream MAPK, leading to the expression of pro-inflammatory cytokines in IVD cells.249,250 In addition, oxidative stress can directly or indirectly activate NF-κB, promoting the expression of inflammatory genes such as IL-1, IL-6, TNF-α in IVD cells and exacerbating the inflammatory response.251,252 Cell damage caused by oxidative stress can trigger an inflammatory response, while inflammation can also exacerbate oxidative stress, forming a vicious cycle.19 For example, TNF-α, IL-1β and lipopolysaccharide (LPS) co-cultured with NP cells can lead to oxidative stress by promoting intracellular ROS production and inhibiting the expression of SOD1, SOD2, CAT and GPX3.248,253,254

Oxidative Stress and ECM Degradation

The complete biomechanical function of IVD depends on the integrity of its tissue structure. With the progression of IVD degeneration, there have been significant changes in the quality and composition of ECM. Due to the death and aging of a large number of IVD cells, the synthesis of ECM is significantly reduced.255 At the same time, the catabolic phenotype of IVD cells is enhanced, which further exacerbates the metabolic imbalance of ECM.139 As an important stressor, ROS can regulate cellular behavior through multiple signaling pathways, thereby influencing the expression and secretion of ECM proteins.77 According to the current study, excessive ROS can downregulate PI3K/Akt, SIRT, AMPK and Nrf2 and upregulate the expression of NF-κB and MAPK, which in turn promotes the degradation of ECM.18,151,225,256 In addition, ROS can disrupt the components of ECM through oxidation, such as causing cross-linking and degradation of collagen protein, affecting its structural stability and functional integrity.257 In conclusion, oxidative stress can lead to abnormal ECM metabolism and oxidative damage of related molecules, promoting the progression of IDD.

Oxidative Stress and Epigenetics

Epigenetics refers to the mechanisms that can influence gene expression without altering the DNA sequence, and these changes can be passed on to future generations.258 Common epigenetic regulations include DNA methylation, histone modification, and non-coding RNA (ncRNA) regulation.259 These changes not only play an important role in the normal physiological processes of cells, but are also involved in driving the pathological processes of various diseases.260 Oxidative stress and epigenetic regulation have a bidirectional control relationship and are involved in the progression of IDD.

DNA methylation is a crucial epigenetic modification involving the addition of a methyl group at specific positions on the DNA molecule, predominantly occurring at the 5′ carbon position of cytosine (C) bases, resulting in the formation of 5-methylcytosine (5-mC).261 This process is catalyzed by DNA methyltransferases (DNMTs), especially DNMT1, DNMT3A, and DNMT3B.262 DNA methylation induces gene silencing by interfering with the binding of transcription factors to highly methylated promoter regions, modifying chromatin structure, or reactivating the domains of methyl-binding proteins.261,263 During oxidative stress, ROS can affect DNA methylation through oxidative DNA damage formed by pyrimidine and 5-methylcytosine (5mC) hydroxylation.264 In addition, ROS can also affect DNA demethylation through DNA oxidation and hydroxymethylation mediated by ten-eleven translocation (TET).264 Meanwhile, DNA me