The Pivotal Role of Nrf2 Signal Axis in Intervertebral Disc Degeneration

Introduction

Intervertebral disc degeneration (IDD) is the most common and prevalent musculoskeletal disorder and one of the major causes of lower back pain (LBP), accounting for nearly 40% of the causes of LBP, resulting in severe socioeconomic burden worldwide.¹ The exact pathogenesis relevant to IDD progression is largely unknown, and the existent clinical treatments for IDD are limited to surgery or conservative approaches with the purpose of relieving clinical symptoms.² Therefore, there is an urgent need to address the pathological mechanism and successful treatment of IDD.

The intervertebral disc (IVD) is primarily composed of three tissue types: peripheral annulus fibrosus (AF), the internal nucleus pulposus (NP) and the upper and lower cartilaginous endplates.³ Compared with other two components, NP tissue is crucial for maintaining IVD physiological function by regulating the dynamic balance between extracellular matrix (ECM) synthesis and degradation.⁴ Dysregulation of NP cells results in their reduced ability to synthesize ECM components and increased capacity to secrete ECM degradative molecules.⁵ Additionally, inflammation, apoptosis, cell senescence and ECM degradation are termed as the hallmarks of IDD and are known to be interconnected and interdependent with each other contributing to the complicated pathological process of IVD degeneration.⁶

It is generally accepted that oxidative stress is another typical mediator in the initiation and progression of IDD.⁷ Redox homeostasis predominantly referred to keep the balance of reactive oxygen species (ROS) production and antioxidant system scavenging activity, which is crucial for maintaining physiological processes.⁸ Accumulating studies have reported that aged and degenerated discs exhibit decreased antioxidant activity and elevated concentrations of ROS during IDD development.⁹ Excessive accumulation of ROS can induce oxidative stress and cause damage to the regular function of NP cells, which triggers inflammatory responses and accelerates the senescence and apoptosis of NP cells, ultimately leading to ECM degradation.¹⁰

Moreover, ROS was regarded as messengers to activate various signaling pathways, including mitogen activated protein kinase (MAPK), nuclear factor kappa-B (NF-κB), and nuclear factor erythroid 2-related factor 2 (Nrf2).¹¹ Nrf2, which is involved in the regulation of the cellular redox homeostasis against oxidative stress and generating beneficial effects on anti-inflammatory and antioxidant response, plays an indispensable role in relieving joint diseases and preventing the progress of IDD.^12,13 The expression of Nrf2 progressively decreased in human NP tissue samples of patients with increased IVD degeneration.¹⁴ Interestingly, therapeutic approach targeting Nrf2 could alleviate oxidative stress-induced apoptosis and ECM decomposition of human NP cells, protecting against IDD progression.¹⁵

In this review, we attempt to introduce the biological characteristics and essential role of Nrf2 in IDD and provide new evidence, insights, and potential strategies for the prevention and treatment of IDD progression.

The Nrf2 Signaling Pathway

Nrf2 is a pivotal redox-sensitive transcription factor, is expressed in all cell types, which regulates antioxidant defense system by activating a great deal of cytoprotective genes including the regulation of glutathione (GSH) and thioredoxin (TXN) biosynthesis and utilization, as well as enzymes involved in nicotinamide adenine dinucleotide phosphate (NADPH) regeneration, heme and iron metabolism, and xenobiotic detoxification, such as glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1).¹⁶ In addition to oxidative and xenobiotic responses, Nrf2 has been viewed as an emerging regulator of other cellular processes, such as mitochondrial bioenergetics, autophagy, unfolded protein response, intermediary metabolism as well as stem cell quiescence.¹⁷

Nrf2 belongs to the Cap “n” Collar (CNC) subfamily and contains seven functional homology domains, arranging as follows from N- to C-terminus: Neh2, Neh4, Neh5, Neh7, Neh6, Neh1, Neh3.¹⁷ Neh2 is crucial for the interaction between Nrf2 and its negative regulator Kelch-like ECH associated protein (Keap1) associated with the highlighted amino acid motifs DLG and ETGE. Neh4 and Neh5 react as transactivation domains, which play a part in binding to transcriptional co-activator cAMP-response-element-binding protein-binding protein (CBP).¹⁸ The retinoid X receptor α (RXRα) suppresses Nrf2 transcriptional activity via interacting with Neh7 domain. Neh6 is related to the negative regulation of Nrf2 stability. NEH1 allows Nrf2 to be heterodimerized with small myofascial fibrosarcoma (MAF) protein, which is necessary for DNA binding. Neh3 also acts as transactivation domain to recruit the chromo-ATPase/helicase DNA-binding protein 6 (CHD6).¹⁹

Under normal conditions, Nrf2 exists in the cytoplasm, segregates by Keap1, a redox-sensitive Cul3 E3 ubiquitin ligase adaptor protein, which is responsible for the ubiquitylation and degradation of Nrf2 to inhibit the transcriptional activity.²⁰ Under oxidative stress conditions, Nrf2 dissociates from Keap1 and then translocates into nucleus, heterodimerizes with small Maf protein family (MafF, MafG, and MafK).²¹ The Nrf2-sMaf complex binds to the antioxidant response element (ARE) and activates the transcription of its target genes.²² In this context, the regulation of Nrf2 transcription depends on Keap1. Recently, emerging evidence has showed further Nrf2 regulation mechanism that is in a Keap1-independent manner.²³

The serine-rich Neh6 domain of Nrf2 contains two conservative peptide motifs (DSGIS and DSAPGS), which are recognized by β-transducing repeat-containing protein (β-TrCP), involving in Keap1-independent Nrf2 negative regulation.²⁴ β-TrCP, a substrate receptor, binds more effectively to the Neh6 domain after glycogen synthase kinase-3b (Gsk-3b)-mediated phosphorylation of the DSGIS motif and facilitates the collection of Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, which targets at the ubiquitination and subsequent proteasomal degradation of Nrf2²⁵ (Figure 1).

Figure 1 Nrf2 signaling pathway. Notes: (a) Structure of the Nrf2 transcription factor. Nrf2 is composed of seven Neh domains. Neh1 is a CNC-bZIP domain that interacts with sMAFs. Neh2 mediates the interaction and regulation with Keap1 through DLG and ETGE motifs. This binding leads to the ubiquitination of Nrf2 and its proteasomal degradation. Polyubiquitinated (Ub) lysine (K) residues contribute to NRF2 degradation by the proteasome. Neh3, 4, and 5 are crucial for transactivation. Neh6 is rich in serine residues and regulates Nrf2 stability. β-TrCP interacts with Nrf2 through this domain after being phosphorylated by GSK-3β. (b) Model of Nrf2 signaling pathway action. Under steady-state conditions, Nrf2 binds to its repressor Keap1 via two motifs, ETGE and DLG, leading to ubiquitination followed by proteasome-degradation. At this time, only a small amount of free Nrf2 translocate into the nucleus and activates basal expression of Nrf2 target genes. When cells are stimulated, Nrf2 dissociates from Keap1 and Nrf2 proteasome degradation is reduced. Dissociative Nrf2 forms heterodimers with sMaf proteins and binds to ARE to initiate transcription of various downstream genes. Thus, Nrf2-mediated defense against oxidative stress-induced imbalance of cellular redox status is strengthened.

In addition, accumulating evidence has revealed other non-classical Nrf2 regulatory pathway including the process of p62-dependent Nrf2 activation in which p62 isolates Keap1 to autophagic degradation that eventually leads to the liberation of Nrf2 and the transactivation of ARE-driven genes as well as the crucial role of micro-RNAs (miRNAs) in the regulation of Nrf2 activity.²⁶ Liao et al found that the interaction between long non-coding RNA MT1DP and miR-365 induces increased apoptosis of NP cells and damaged mitochondrial function by inhibiting Nrf2 pathway.²⁷

The Roles of Nrf2 in IVD Degradation

Nrf2 and Inflammation

It is generally accepted that increased production of inflammatory cytokines such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), which motivate the generation of a series of other pro-inflammatory cytokines and catabolic enzymes, such as nitric oxide (NO), cyclooxygenase-2 (COX-2), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), prostaglandin E2 (PGE2) and matrix metalloproteinase-13 (MMP-13), leading to IDD progresses.²⁸ A large number of experiments on Nrf2-knockout mice have demonstrated its pivotal role in the regulation of inflammation and activation of pro-inflammatory genes such as COX-2 and inducible nitric oxide synthase (iNOS).²⁹ NF-ĸB, a protein complex, which is responsible for DNA transcription. Abnormal regulation of NF-ĸB has been related to transcriptional upregulation of pro-inflammatory mediators such as IL-6, TNF-α, iNOS and IL-1β. As reported, activation of Nrf2 pathway could inhibit the overproduction of pro-inflammatory mediators as well as suppressing the activation of NF-ĸB signaling pathway.³⁰ HO-1 is rate-limiting enzyme catalyzing the degradation of heme into carbon monoxide (CO) and free iron, which has prominent anti-inflammatory properties. Elevation of HO-1 expression mediated by activated Nrf2 acts protective effects in regulating inflammatory conditions.³¹ Xie et al showed that activating the Nrf2/HO-1 signaling axis leading to the inhibition of NF-κB pathway could protect NP cells against IL-1β-induced inflammatory response and catabolism, which was beneficial to prevention and therapy of IVD degeneration.³ In other words, the reduction or deletion of Nrf2 and HO-1 genes may be key factors leading to IDD.³² Another study revealed that activation of Nrf2-ARE signaling pathway could suppress MAPK (P38 and JNK) pathway, contributing to ameliorate IL-1β-treated inflammation in NP cells.³³ Mechanically, Nrf2 binds to ARE and promotes the transcription of antioxidant genes such as HO-1 and NQO1 via translocating into the nucleus.

Taken together, these findings suggest that development of novel therapies targeted on Nrf2 for anti-inflammation activity could be advantageous of clinical management of IDD.

Nrf2 and ECM Metabolism

To the best of our knowledge, ECM degradation is one typical characteristic of IVD degeneration due to disturbing the balance of ECM anabolic and catabolic metabolism. During the process, the degradation of major components of ECM in NP including collagen-II (Col2), proteoglycans (PGs) and glycosaminoglycan (GAG) was accelerated, resulting from up-regulation catabolic proteinases, such as matrix metalloproteinase 3 (MMP3), MMP13 and ADMATS5, while their biosynthesis is decreased.³⁴ Therefore, maintenance of ECM homeostasis plays a crucial part in preventing and treating IVD degeneration. Chen et al observed that a moderate dose of fluid shear stress (FSS) could regulate ECM homeostasis through increased expressions of Col2, aggrecan and sulfated glycosaminoglycan (sGAG) and decreased expressions of MMP13 and ADMATS5 in rat NP cells.³⁵ It is interesting to note that Nrf2/HO-1 pathway acts as a protective regulator involved not only in the progression of joint destruction but also in IVD degeneration.³⁶ For instance, it has been shown that induction of HO-1 attenuates the increased ECM catabolism and ameliorates the reduced ECM anabolic activities in human NP cells under IL-1β stimulation conditions.³⁷ In summary, these findings put forward novel interest in targeting the Nrf2/HO-1 pathway as a trigger point to regulate relevant anti-ECM catabolism mechanisms involved in the degeneration of IVD as well as the possibilities of this signal path for the development of therapeutic strategies.

Nrf2 and Oxidative Stress

Numerous studies reinforce the notion that the presence of oxidative stress is regarded as a key intermediator resulting in pathological degeneration of IDD. Oxidative stress occurs when the balance between ROS production and scavenging is disrupted.³⁸ A large amount of ROS is produced due to mitochondrion dysfunction during oxidative stress response to various exogenous stimulation in disc cells including hydrogen peroxide (H₂O₂) and nitric oxide (NO).³⁹ Previous studies have shown that H₂O₂ may lead to IVD degeneration via oxidative stress damage, inflammatory response and apoptosis of NP cells.⁴⁰ Furthermore, the accumulation of ROS not only accelerates the degradation of peripheral matrix proteins but also disturbs the microenvironment of healthy intervertebral disc.⁴¹ Under regular conditions, ROS modulates the function and stability of cellular proteins such as nucleic acids, carbohydrates and lipids. However, the increased accumulation and decreased elimination of these species and disruption of the antioxidant/oxidant balance ultimately leading to oxidative stress and pathological changes of diseases.⁴² In recent decades, the activation of Keap1/Nrf2/ARE signaling pathway is the most noticeable discovery involving in the response to oxidative stress, which make sure that ROS is appropriately scavenged.²² Upon exposure to oxidative stress, Nrf2 rapidly dissociates from Keap1 and translocates into nucleus, where it interacts with ARE and accelerates the transcription of antioxidant genes to regulate DNA repair, xenobiotic metabolism and redox homeostasis.⁴³ In addition, the phosphorylation and nuclear translocation of Nrf2 may promote the expression of HO-1 by binding to ARE to protect NP cells from oxidative stress injury induced by high glucose.⁴⁴ Taken together, these studies reflect that oxidative stress has an adverse effect on the pathogenesis of IDD, and the therapeutic potential of targeting the Nrf2 pathways is deserved to highlight for the management of IDD.

Nrf2 and Cell Senescence

Cellular senescence is characterized by cell cycle arrest and secreting various inflammatory cytokines, chemokines and matrix proteases, which collectively are known as the senescence-associated secretory phenotype (SASP).⁴⁵ As reported, NP cell senescence is a crucial contributor to the initiation and progression of IVD.⁴⁶ Previous study demonstrated that inflammatory cytokine TNF-α promoted premature senescence of NP cells via PI3K/Akt signaling pathway, as indicated by decreased cell proliferation, increased senescence-associated beta-galactosidase (SA-β-gal) staining and the up-regulated expression of the senescence marker p16 and p53.⁴⁷

Obviously, protecting NP cells against senescence is conducive to the amelioration of IDD. Che et al exhibited that the deficiency of p16 could attenuate IVD by promoting cell cycle and inhibiting SASP, cell senescence, and oxidative stress.⁴⁸ Aged and degenerated IVDs have increased oxidative stress and NP cell senescence is associated with increased ROS level in IDD.⁴⁹ Therefore, molecular signaling modulation related to ROS could be targeted to control IVD senescence and degeneration. To date, research has shown that the activation of Nrf2 signaling pathway enhanced cellular antioxidant capacity and inhibited cellular senescence, thereby exerting protective effects against aging diseases.⁵⁰ A recent study by Dimozi et al demonstrated that prolonged exposure to H₂O₂ induces premature senescence in human NP cells and nuclear translocation of Nrf2 as a response to oxidative stress.³⁸

These researches revealed that the exploration of antagonistic cell senescence by regulating Nrf2 signaling pathway may be a potential strategy for the treatment of IDD.

Nrf2 and Autophagy and Mitophagy

Autophagy is mainly regarded as a process for the clearance of impaired organelles, misfolded proteins and intracellular pathogens that were wrapped in autophagosomes and degraded by lysosomes. Autophagy-mediated degradation plays an important role in maintaining cellular homeostasis and development and protecting organisms against oxidative or proteotoxic stress.⁵¹ Given its vital impacts, there is no doubt that damaged autophagic pathway is associated with the pathogenesis of diverse diseases such as cancer, cardiomyopathy, diabetes, osteoarthritis and IDD.⁵² A latest study has shown that Nrf2/Keap1 is a key pathway to regulate the expression of antioxidant proteins, and autophagy can promote Nrf2 expression and nuclear translocation and activate Nrf2/Keap1 signaling pathway. As a result, the Nrf2/Keap1 complex is disrupted and Nrf2 translocated to the nucleus, where it activates the transcription of genes encoding antioxidant enzymes, thereby affecting ROS clearance and cartilage endplate senescence.⁵³ Consistent with the above, Nrf2 is involved in the autophagic process in response to detrimental influence of H₂O₂-induced oxidative stress via Keap1/Nrf2/p62 feedback loop to protect intervertebral disc from degeneration.⁵⁴ Upon exposure to oxidative stresses, p62 works as an autophagy adaptor protein, sequestrated Keap1 into aggregates or autophagosomes, which harms the ubiquitylation of Nrf2, resulting in activating the Nrf2 signaling pathway.⁵⁵ Furthermore, damaged mitochondria can be degraded by a specific type of autophagy called mitophagy, and Nrf2 plays a part in maintaining mitochondrial homeostasis through modulating mitochondrial function and metabolism such as mitochondrial dynamics and mitophagy.⁵⁶ Recent evidences have shown that Parkin-mediated mitophagy for selectively removing dysfunctional mitochondria and Nrf2-mediated antioxidant system are critical to improve NP cells and endplate chondrocyte survival under pathological conditions.⁵⁷ Recently, a study has shown that Nrf2 is a regulator of Sirtuin 3 (SIRT3) expression, which maintains mitochondrial homeostasis by promoting mitochondrial dynamics, antioxidation and mitophagy, and deacetylation of manganese superoxide dismutase (MnSOD).⁵⁸ Hu et al proposed that Nrf2/Sirt3 signaling could activate mitophagy and restore the disruption of autophagic flux induced by oxidative stress, thereby protecting NP cells from apoptosis and ECM degradation.⁵⁹ Taken together, these studies suggest that activating Nrf2 signaling to accelerate autophagy and mitophagy in NP cells is a promising strategy to treat IDD.

Nrf2 and Cell Death (Apoptosis, Pyroptosis, Ferroptosis)

It is well known that excessive apoptosis of NP cells is regarded as the hallmarks of IVD degeneration.⁶⁰ In addition to the widely accepted apoptosis, other forms of cell death are newly discovered in the progress of IDD, such as pyroptosis and ferroptosis, which are obviously distinct from apoptosis according to the cellular morphological and biochemical characteristics.⁷ Apoptosis is a type of programmed cell death via eliminating damaged cells by phagocytes, which is critical for the development and homeostasis of organism.⁶¹ It is mainly caused by excessive ROS via increasing mitochondrial outer membrane permeability and pro-apoptotic cytochrome c release.⁶² It has been reported that excessive and sustained endoplasmic reticulum (ER) stress leads to disc ECM degradation and NP cell apoptosis.⁶³ Nrf2 activation inhibits oxidative stress-mediated ER stress and mitochondrial dysfunction.⁶⁴ Wang et al proposed that Nrf2/HO-1 signaling pathway is involved in regulating lipopolysaccharide (LPS)-induced NP cell dysfunction, including apoptosis caused by oxidative stress, inflammation, and ER stress.⁶⁵ Pyroptosis is a form of inflammatory cell death which is mediated by activated caspase 1, characterized by cell osmotic pressure changes and cell membrane rupture.⁶⁶ According to the report, the relationship was between the negative regulatory effect of Nrf2 and H₂O₂-induced NP cell pyroptosis through the expression of NLR family pyrin domain containing 3 (NLRP3) and PYD and CARD domain containing (PYCARD) inflammasome.⁶⁷ Studies have found that NLRP3 mediates pyroptosis and inflammation in H₂O₂-induced IVD degeneration. However, ferroptosis is a non-apoptotic cell death distinguished by mitochondrial shrinkage, enhanced mitochondrial membrane density, and accumulation of lipid peroxides. Nrf2 has been shown to participate in the development of IDD by reducing NP cell viability.^68,69 In recent years, it has been found that enhancing glutathione peroxidase 4 (GPX4) expression can alleviate human NP cell degeneration, while Nrf2 regulates endplate cell calcification and ECM homeostasis and inhibits endplate cell ferroptosis by upregulating GPX4 expression⁷⁰. As mentioned above, Nrf2 is known as a protective regulator, and it is not surprising that the activation of Nrf2 plays a critical role in mediating cellular death, including apoptosis, pyroptosis and ferroptosis.⁷¹

Therapeutic Strategies by Targeting Nrf2

Therapeutic Strategies by Targeting Nrf2, Inflammation, and ECM Metabolism

In recent years, numerous studies have demonstrated that Dimethyl fumarate (DMF) can activate Nrf2 effectively and promote the activation of its downstream antioxidant stress pathway.⁷² Zhu et al also revealed that DMF shown promising effects in the prevention of IDD by promoting the expression of Nrf2 and HO-1, significantly reducing the expression of inflammatory factors, increasing SOD activity, and reducing IL-1β-induced nuclear endoplasmic reticulum stress.⁷³ Moreover, evidence has suggested that moracin can antagonize LPS-mediated inflammatory mediators such as IL-1β, TNF-α and IL-6. Interestingly, treating with Nrf2-specific siRNA, the anti-inflammatory effects were significantly reversed. Moreover, they found that moracin treatment could remarkably reverse the increased expression of MMP-3 and MMP-13 levels and decreased expression of collagen-I, Col2 and aggrecan.⁷⁴

Furthermore, Wang et al demonstrated that Polydatin (PD) suppresses TNF‐α‐-induced ECM destruction via activating Nrf2/HO‐1 signaling pathway in rat NP cells to attenuate IVD degeneration.⁷⁵ Consistent with the above, Cyanidin-3-glucoside (C3G) prevents HG-induced ROS production in human NP cells and reversing apoptosis and degradation of extracellular matrix through regulating the Nrf2/HO-1 pathway. Besides, they also found that C3G could increase the proteoglycan and Col2 content and inhibit MMP3 and MMP13 expression in human disc cells.⁷⁶

Therapeutic Strategies by Targeting Nrf2 and Cell Senescence

Currently, it has been found that activation of Nrf2 signaling pathway may be an effective target for delaying cell senescence. Quercetin (QUE), a natural senolytic compound, could reduce the expression of senescence markers.^77,78 Shao et al discovered that QUE promoted ECM homeostasis and inhibited SASP factors expression and senescence phenotype in NP cells. Mechanistically, QUE ameliorated the progression of IDD via activating Nrf2/NF-κB axis.⁷⁹ PD, a resveratrol glucoside, possessed multiple pharmacological properties including anti-oxidation, anti-inflammation and anti-tumour. Wang et al found that PD prevented TNF-α-induced NP cell senescence by alleviating ROS-mediated mitochondrial dysfunction via promoting Nrf2 nucleus translocation and activating Nrf2/HO-1 signaling pathway.⁷⁵ Consistent with the above, a previous work reported that kinsenoside (Kin) could suppress tert-butyl hydroperoxide (TBHP)-induced NP cell senescence under oxidative stress by activating of AKT-ERK1/2-Nrf2 signaling pathway to delay the progression of IDD.⁸⁰ In summary, triggering Nrf2 activation to inhibit senescence of NP cells is a potential therapeutic method for IVD.

Therapeutic Strategies by Targeting Nrf2 and Apoptosis

Icariin, which is an isopentenyl flavonol glycoside isolated from epimedium, has been proved to have anti-inflammatory and antioxidant effects on NP cells.⁸¹ A recent study reported that icariin could activate Nrf2 signaling pathway, upregulate the protein expression of nuclear respiratory factor-1 and the mitochondrial transcription factor, promoting mitochondrial biogenesis to protect against H₂O₂-induced mitochondria-mediated human NP cell apoptosis.¹² A recent study by Tian et al revealed that bardoxolone methyl (BARD) protected NP cells from compression-induced mitochondrial apoptosis and ECM degradation. The authors found that BARD could promote Nrf2 protein nuclear translocation and Nrf2 target protein overexpression.⁸² In addition, Nrf2 can maintain redox balance in NP cells by reducing ROS production from the source by maintaining mitochondrial homeostasis.¹⁴ Interestingly, another study reported that Luteoloside, a flavonoid glycoside extracted from the plant luteolin, was able to maintain cell morphology and inhibit inflammation and apoptosis characterized by the reduced expression of cleaved caspase 3 in IL-1β-treated NP cells. In addition, the study also demonstrated the protective effects of luteoloside in NP cells through activating Nrf2/HO-1 signaling axis.⁸³ Selenium (Se), a vital component of antioxidant enzymes, is indispensable for maintaining redox homeostasis and promoting cell survival. It could suppress TBHP-induced oxidative stress and mitochondrial fission, rescued the imbalance of mitochondrial dynamics and inhibited apoptosis of NP cells to ameliorate IDD. Moreover, the effect of Se on protecting mitochondrial function was attributed to the activation of Nrf2 pathway.⁸⁴ Se reversed the excessive apoptosis of NP cells induced by TBHP via reducing the excessive production of intracellular ROS and the increase of malondialdehyde (MDA) level, as well as upregulating the expression of superoxide dismutase 2 (SOD2).⁸⁵ In addition, with the development of research, an increasing number of molecules have been shown to protect IVD cells from mitochondrial dysfunction and apoptosis by activating Nrf2 signaling, including VO-OHpic,⁸⁶ Andrographolide,⁸⁷ hyperoside,⁸⁸ Amobarbital⁹ and Procyanidin B2.⁸⁹

Therapeutic Strategies by Targeting Nrf2 and Pyroptosis

As a programmed necrosis different from classical apoptosis, pyroptosis can mediate the activation and release of inflammatory factors IL-1β and IL-18 by forming pores in the plasma membrane, which leads to inflammatory cascades and ECM degradation.⁶⁶ Increasing evidence indicates that a series of pyroptosis-occurring elements such as caspase-1, Gasdermin-D (GSDMD), and NLRP3 inflammasome were found to increase or activate in the pathological progression of IDD.⁹⁰ Existing evidence has revealed that application with melatonin significantly upregulated the expression of Nrf2 and downregulated the level of pyroptosis-related proteins such as NLRP3, cleaved caspase-1, gasdermin D, IL-18, and IL-1β to protect NP cells against ROS-induced pyroptosis.⁹¹ Milk fat globules-epidermal growth factor (EGF) factor 8 (MFG-E8) is an endogenous multifunctional glycoprotein, which is beneficial for anti-inflammatory, antioxidant, and modulation of NLRP3 inflammasome. Ma et al found that Nrf2/TXNIP/NLRP3 axis plays a crucial role in MFG-E8-mediated suppression of H₂O₂-induced oxidative stress, mitochondrial dysfunction, and NLRP3 inflammasome activation and protection of NP cells from pyroptosis and ECM degradation.⁹² Interestingly, consistent with the above, Xu et al showed that platelet-rich plasma (PRP)-derived exosomes miR-141-3p could reverse H₂O₂-induced NP cell pyroptosis by promoting the dissociation of Nrf2 from Keap1/Nrf2 complex and its further translocation contributing to achieve anti-oxidant biological functions and exert protective effects to slow down IVD translocation.⁹³

Overall, these findings suggested that the regulation of Nrf2 in NP cell pyroptosis plays a crucial part in IDD treatment.

Therapeutic Strategies by Targeting Nrf2 and Ferroptosis

After explaining the effect of Nrf2 on apoptosis and pyroptosis of NP cells, ferroptosis has to be mentioned. As a form of iron-dependent necrotic cell death, ferroptosis is characterized by iron ion-catalyzed disorder of cellular lipid metabolism and aggravates the degeneration of nucleus pulposus.⁹⁴ Yu et al found that bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs) carrying circ_0072464 could promote matrix synthesis and proliferation of NP cells via upregulating the expression of miR-431-mediated Nrf2 to inhibit IL-1β-induced NP cells ferroptosis. Collectively, BMSC-EVs alleviated intervertebral disc lesions through the circ_0072464/miR-431/Nrf2 axis.⁹⁵ Hesperetin-7-o-rutinoside (HRD) is a flavanone glycoside extracted from lime peel powder. It belongs to the subgroup of citrus flavonoids because of its anti-inflammation and anti-oxidation properties.⁹⁶ Previous studies have confirmed that HRD can alleviate oxidative stress and mitochondrial dysfunction through the Nrf2-ARE pathway.⁹⁷ Recently, Zhu et al confirmed that HRD may protect NP cells from degeneration by mitigating oxidative stress-dependent ferroptosis via enhancing the expression of Nrf2 and suppressing NF-κB pathway.⁹⁸ Astaxanthin (Ast), a natural lipid-soluble carotenoid, possesses antioxidant, anti-inflammatory, and anti-aging capacities. Additional supplement with Ast could protect vertebral cartilage endplate degeneration against TBHP-induced cartilage endplate chondrocytes ferroptosis including restoring the downregulated expression of GPX4, ferritin heavy chain (FTH1) and solute carrier family 7 member 11 (SLC7A11) via activating Nrf-2/HO-1 pathway.⁹⁹

Taken together, it is demonstrated that targeting Nrf2 signaling axis inhibits NP cell ferroptosis, which may be an effective therapeutic approach for IDD intervention and treatment (Figure 2). In addition to some of the molecular mechanisms described above, there are many mechanisms and related signaling pathways elaborating the role of Nrf2 activation in IDD therapies, see Table 1.

Table 1 Regulation of Nrf2 Signaling as Potential Therapeutic Options for IDD

Figure 2 Mechanism of Nrf2 targeted therapy for IDD. Notes: Extracellular inflammatory factors and oxidative stress induced NP cell produce excessive ROS, resulting in the increased expression of MMPs and ADAMTS, promoting inflammation or apoptosis of NP cells. At the same time, it activates Nrf2 transferring to nucleus and reacting, thereby affecting multiple downstream signaling pathways or molecules, promoting autophagy of NP cells to protect cells from oxidative damage, or inhibiting inflammation, senescence, apoptosis, pyroptosis and ferroptosis of NP cells.

Therapeutic Strategies without Targeting Nrf2

Given the limited known researches on the momentous role of Nrf2-targeted therapeutic strategies for alleviating IDD, complementary findings pave way for the treatment and prevention of IDD. Wang et al found that phosphatase and tensin homolog (PTEN)-induced putative kinase protein 1 (PINK1), a mitochondrial-targeted serine/threonine kinase, protects against mitochondrial dysfunction by activating PINK1/Parkin mediated mitophagy to clear damaged mitochondrial and alleviate H₂O₂-induced NP cell senescence.¹⁰⁰ In addition, according to published studies, the expression of proliferator-activated receptor gamma (PPARγ) is significantly decreased in degenerative disc tissue. However, its activator pioglitazone could significantly inhibit the activation of the NF-κB pathway induced by IL-17 or IL-17 combined with TNF-α, reduce the levels of metalloproteinases, and preserve the expression of key matrix components such as aggrecan and Col2.¹⁰¹ Interestingly, more studies have provided new insight into the beneficial effect of bone marrow mesenchymal stem cells (BMSCs) transplantation in the prevention and treatment of IDD.¹⁰² A recent study also demonstrated that the TNF-α-stimulated gene 6 protein (TSG-6) secreted by BMSCs displays notable effect to inhibit the activation of the TLR2/NF-κB inflammatory pathway. Mechanistically, BMSC reduced the expression of MMPS (such as MMP-3 and MMP-13) and simultaneously increased the expression of Col2 and aggrecan in IL-1β-treated NP cells by enhancing TSG-6 expression.¹⁰³ Consistent with the above, Zhang et al demonstrated that MSCs-exosomes could inhibit LPS-induced pyroptosis in NP cells. They found that administration of exosomal miR-3 downregulated NLRP410, resulting in decreased expression of caspase-1 and GSDMD, thereby weakening pyroptosis in NP cells to alleviate IDD.¹⁰⁴ Furthermore, it has been reported that some other important biologically active components also ameliorate IDD without targeting Nrf2 signal, these compounds included oxymatrine liposomes,¹⁰⁵ Caspase 3 siRNA,¹⁰⁶ Adenovirus-mediated growth and differentiation factor-5 (Ad-GDF5),¹⁰⁷ O-linked β-N-acetylglucosaminylation (O-GlcNAcylation),¹⁰⁸ Gamma-oryzanol,¹⁰⁹ ascorbic acid,¹¹⁰ prolactin,¹¹¹ see Table 2.

Table 2 Potential Therapeutic Options for IDD without Targeting Nrf2

In summary, there are still many studies that provide new insights into IDD treatment through different molecular mechanisms and pathways. It is well known that Nrf2 is considered a central hub modulating a variety of physiological and pathological processes, and the interaction between Nrf2 and further signal path and molecules remains an urged need to be clearly investigated in favour of providing more feasible method for the clinical treatment of IVD.

Conclusion

In this review, we synthesize and conduct deep understanding of the key mechanisms of Nrf2 in regulating the pathophysiological phenotypes of IDD. Inflammatory response, imbalance of ECM metabolism, oxidative stress-induced mitochondrial dysfunction, cell senescence and apoptosis, pyroptosis and ferroptosis of NP cells are the key characteristics of IDD. In general, the activation of Nrf2 or Nrf2-related signal axis can decrease the expression of inflammatory cytokines, inhibit ECM degradation, overexpression of ROS, cell senescence and NP cell death and promote the expression of antioxidant genes, matrix synthesis and mitophagy, which play a protective role in delaying the pathogenesis of IDD. This leads to the possibility that activation of this signaling pathway may open up new therapeutic approaches to support targeting Nrf2 for prevention and treatment of IDD. Although this review has proven the beneficial effects of Nrf2 for preventing IDD development in vitro and in vivo models, there is a lack of clinical application. Therefore, further systematic studies are demanded to clarify complicated mechanisms involved in IDD therapy depending on Nrf2 or the interaction with additional key molecule.