Targeting oxidative stress, iron overload and ferroptosis in bone-degenerative conditions

Oxidative stress and bone homeostasis

Oxidative stress has become an important modulator of bone homeostasis, exerting profound effects on various bone cell types and their functions [18]. In osteoclasts, the bone-resorbing cells, elevated ROS levels were found to promote their differentiation and functional activity, contributing to accelerated bone loss [19], 20]. Conversely, in osteoblasts, the bone-forming cells, oxidative stress can impair their differentiation, mineralization capacity, and survival, thereby compromising bone formation [21]. Additionally, chondrocytes, the cellular components of cartilage, are vulnerable to oxidative injury, which can lead to cartilage degradation in osteoarthritis [22]. Notably, oxidative stress also impinges on cell fate determination of mesenchymal stem cells, hindering their osteogenic differentiation potential while favoring adipogenic lineage commitment, ultimately disrupting bone homeostasis [23]. These findings highlight the intricate interaction between oxidative burden and various bone lineages, underscoring the potential benefits of mitigating oxidative stress in the management of bone-degenerative conditions.

Iron homeostasis in bone and cartilage lineages

Iron metabolism is meticulously regulated in different cell types within the bone and cartilage to maintain proper cell differentiation and functional homeostasis. In osteoclasts, which are responsible for bone resorption, iron uptake is critically important for their differentiation and function [24], 25]. During osteoclastogenesis, the expression of key genes facilitating iron import, such as transferrin receptor 1 (TFR1) and the non-transferrin-bound iron (NTBI) transporter solute carrier family 39 member 14 (SLC39A14), is significantly upregulated [26]. This increased iron acquisition is essential for supporting the high energy demands of osteoclasts, as iron is a crucial cofactor for mitochondrial respiration and biogenesis [27]. Notably, TFR1 depletion in osteoclasts leads to a substantial decrease in labile iron levels and attenuated bone resorption activity [28]. Conversely, iron chelation by agents like deferoxamine (DFO) dose-dependently inhibits osteoclast development and activity [29], 30]. Osteoblasts, on the other hand, exhibit a more complex relationship with iron. While moderate levels of iron are necessary for osteoblast function, excessive iron can impair their differentiation, mineralization, and survival [31]. Iron overload conditions lead to the upregulation of iron-importing factors like divalent metal transporter 1 (DMT1) and TFR1 in osteoblasts, accompanied by increased oxidative stress and potential ferroptosis induction [32], 33]. Furthermore, iron overload suppresses osteogenic master regulators such as runt-related transcription factor 2 (Runx2) and its target genes, including osteocalcin and alkaline phosphatase (ALP), hampering osteoblast differentiation and matrix mineralization [34].

In mesenchymal stem cells (MSCs), the precursors of osteoblasts and chondrocytes, iron dysregulation can break the balanced differentiation between osteogenesis and adipogenesis. Iron overload impairs the osteogenic commitment of MSCs [35], while iron chelation can rescue this anti-osteogenic effect [36]. Chondrocytes, the key cellular component of cartilage, are also susceptible to iron-induced damage. Excessive iron induces catabolic markers like matrix metalloproteinases (MMPs) in chondrocytes, leading to extracellular matrix degradation and cartilage degeneration [37]. Iron chelators and antioxidants can alleviate these detrimental effects, underscoring the involvement of iron-dependent oxidative burden in cartilage deterioration [38], 39]. In summary, iron metabolism can functionally impinge on different cell types within the bone and cartilage microenvironment. Maintaining an optimal balance of iron levels is critical for preserving bone homeostasis, as both iron overload and deficiency can contribute to pathological conditions like osteoporosis and cartilage degeneration.

Iron regulation and ferroptosis induction

Iron is a mediator of oxidative stress and indispensable for ferroptosis initiation. Iron ions within cells mainly originate from heme group or non-heme iron found in dietary food. Non-heme iron can be present as Fe²⁺ or Fe³⁺. The enzymatic action of duodenal cytochrome B (DCYTB) in the intestine converts Fe³⁺ to Fe²⁺, facilitating its uptake through epithelial cells via DMT1 [40]. Ferritin iron and heme iron can also be internalized by intestinal epithelial cells via ferritin receptors and the cluster of differentiation 91 (CD91), respectively [41]. Excess Fe²⁺ can bind to ferritin within intestinal epithelial cells for storage [42]. In times of need, Fe²⁺ can be extruded from intestinal epithelial cells through ferroportin 1 (FPN1) and undergo oxidation to Fe³⁺ by hephaestin (HEPH) for onward transportation [43], 44]. Hepatic transferrin (TF) is loaded with Fe³⁺, with each TF molecule capable of binding two Fe³⁺ ions [45]. Fe³⁺-TF undergoes translocation to target cells through the interaction with transferrin receptor 1 (TFR1), ushering Fe³⁺ to endosomal compartments [46]. Within the acidic milieu of endosomes, Fe³⁺ is converted to Fe²⁺ by metal-containing reductases like six-transmembrane epithelial antigen of prostate 3 (STEAP3) before being delivered to the cellular cytoplasm by DMT1 [47].

Intracellular regulation of Fe²⁺ is tightly controlled to maintain homeostasis. Cellular insufficiency of Fe²⁺ can hinder various biological processes, while an abundance of Fe²⁺ triggers the Fenton reaction. This reaction involves the excess Fe²⁺ reacting with hydrogen peroxide [48], resulting in ROS production. The accumulation of iron and ROS can trigger ferroptosis [49] (Figure 1) Furthermore, an excess of Fe²⁺ can stimulate lipid peroxidation through participation in the enzymatic subunit of lipoxygenase (LOX) [50], 51]. Additionally, surplus intracellular Fe²⁺ can be converted to Fe³⁺ by ferritin and stored in the structure of ferritin heavy chain 1 (FTH1) or ferritin light chain (FTL) [52]. Ferritin protein may undergo degradation by nuclear receptor coactivator 4 (NCOA4), releasing labile iron [53]. Cells can also eliminate excessive Fe²⁺ through the action of FPN1. FPN1, along with its governing factor hepcidin, has a pivotal role in iron metabolism. FPN1 has currently been recognized as the sole iron export avenue [54]. Irregular expression or malfunction of these proteins can result in heightened levels of intracellular labile iron.

Figure 1:

Current understanding of ferroptosis induction and inhibition. Ferroptosis induction mechanisms (left panel) and the inhibition mechanisms (right panel) are summarized. Iron metabolism: Extracellular Fe³⁺ binds to TF, forming the Fe³⁺-TF1 complex, which interacts with TFR1 for Fe³⁺ entry into the cytosol. Fe³⁺ is converted to Fe²⁺, catalyzing hydroxyl radicals (^·OH) production through the Fenton reaction. Both ^·OH and Fe²⁺-containing LOX enzyme induce lipid peroxidation. Mitochondria: the central organelle in iron homeostasis, the mitochondrion, requires iron for heme groups and sulfur-iron clusters essential for mitochondrial ETC components. In cancer cells, increased iron levels enhance electron transport and ROS production in mitochondria. Lipid metabolism: PUFAs are converted to PUFA-CoA by ACSL4 and then to PUFA-PL by LPCAT. LOXs or ^·OH oxidize PUFA-PL to produce PLOOH. GPX4: a selenoprotein, GPX4, utilizes GSH to reduce PLOOH and prevent ferroptosis. GSH metabolism: the antiporter system Xc− exchanges cystine for glutamate, converting cystine to cysteine for GSH biosynthesis. Blocking system Xc− leads to GSH depletion, promoting ferroptosis. Mevalonate pathway: IPP, synthesized from acetyl-CoA, activates GPX4 with a selenocysteine residue. FSP1-dependent pathway: FSP1 converts CoQ10 to ubiquinol to counteract PLOOH. DHODH-dependent pathway: DHODH reduces CoQ10 to ubiquinol-10, suppressing mitochondrial lipid peroxidation and ferroptosis. GCH1-dependent pathway: GCH-1 generates BH4 from BH2 to counteract PLOOH and inhibit ferroptosis. ACSL4, acyl-CoA synthetase long-chain family member 4; BH2, dihydrobiopterin; BH4, tetrahydrobiopterin; CoQ10, coenzyme Q10 (also known as ubiquinone); DHODH, dihydroorotate dehydrogenase; ETC, electron transport chain; FSP1, ferroptosis suppression protein 1; GCH1, GTP cyclohydrolase 1; GPX4, glutathione peroxidase 4; IPP, isopentenyl pyrophosphate; LPCAT3, lysophosphatidylcholine acyltransferase 3; LOX, lipoxygenase; NOX, NADPH oxidase; PLOOH, phospholipid hydroperoxides; PUFA, polyunsaturated fatty acid; PUFA-PL, polyunsaturated phospholipid; RTA, radical-trapping antioxidant; TF, transferrin; TFRC, transferrin receptor.

Another important process in ferroptosis induction is iron-dependent lipid peroxidation. Phospholipids (PLs) are essential constituents of plasma membranes, with the ability to form diverse fatty acyl chains [55]. Polyunsaturated fatty acyl (PUFA) can form PUFA-PLs by attaching to the sn2 position of PL with the assistance of acyl-CoA synthetase long-chain family member 4 (ACSL4) and Lys phosphatidylcholine acyltransferase 3 (LPCAT3) [56]. Deletion of these enzymes could suppress the generation of PUFA-PLs [57], 58]. PUFA-PLs are significant for maintaining cell membrane fluidity and functional flexibility [59]. However, PUFAs with bis-allylic hydrogen atoms are highly susceptible to oxidation by potent oxidants. This reaction produces phospholipid radicals (PL^·) which are further transformed into phospholipid peroxyl radicals (PLOO^·) [60]. It is noteworthy that PLOO^· could remove hydrogen atoms from the bis-allylic positions of a different PUFA-PL, leading to the production of PUFA-PL-OOH [61]. The continual synthesis and buildup of PUFA-PL-OOH, influenced by intracellular iron and ROS levels, undermines the plasma membrane integrity to induce cell death [62]. Additionally, lipoxygenases (LOXs) can promote the oxidation of PUFA-PLs and support ferroptosis [63].

Negative regulators of ferroptosis

Cells, under normal conditions, can counteract lipid peroxidation efficiently to avoid excessive accumulation. One crucial enzyme responsible for detoxifying PL-OOH is Glutathione peroxidase 4 (GPX4) from the GPXs family (Figure 1). GPX4 can transform PL-OOH into harmless phospholipid alcohol (PL-OH) through the reducing power of glutathione (GSH) [64]. GSH acts as a vital reducing cofactor to facilitate lipid peroxidation detoxification mediated by GPX4 [65]. The activity of GPX4 is impaired when there is a decrease in GSH synthesis. GSH is composed of glutamate, glycine, and cysteine, with cysteine being a key intermediate amino acid that must be obtained extracellularly for GSH synthesis [66]. Cellular import of cysteine relies on the cystine/glutamate transporter (system Xc−), consisting of the light chain subunit solute carrier family 7 member 11 (SLC7A11) and the heavy chain subunit solute carrier family 3 member 2 (SLC3A2). The exchange of intracellular glutamate with extracellular cystine is facilitated by SLC7A11 [67]. Intracellular cystine is further converted to cysteine to contribute to GSH production [68]. Thus, the primary defense mechanism against ferroptosis involves GSH biosynthesis and the GPX4-mediated detoxification of GSH-dependent PL-OOH, forming the SLC7A11-GSH-GPX4 network. Disruption of this network, such as through inhibition of SLC7A11-GSH-GPX4, can trigger ferroptosis. Notably, Dixon et al. demonstrated that inhibitors like Ras-selective lethal small molecule 3 (RSL3) and Erastin induce ferroptosis by blocking GPX4 and System Xc-, resulting in cellular PL-OOH overload [69].

Additional pathways have been identified to combat ferroptosis. One such mechanism involves ubiquinone (UQ, also known as coenzyme Q10, or CoQ10), a lipophilic molecule that is prevalent across various cell types. Coenzyme Q10 (CoQ10), primarily produced in mitochondria, plays a crucial role in the electron transport chain due to its redox properties [70]. CoQ10 exists in various oxidation states, with its fully reduced form ubiquinol (CoQ10H2) acting as a potent antioxidant that combats lipophilic free radicals and suppresses ferroptosis. The enzyme ferroptosis suppressor protein 1 (FSP1) catalyzes the reduction of CoQ10 to CoQ10H2, utilizing NAD(P)H in the process [71]. FSP1’s N-terminal myristoylation motif is crucial for its plasma membrane localization and function, with modifications to this region potentially impairing its activity [72]. The FSP1-CoQ10H2 system functions as an alternative ferroptosis defense mechanism, complementing the SLC7A11-GSH-GPX4 pathway by continuously generating CoQ10H2 and eliminating lipid free radicals. Studies by Bersuker et al. demonstrated FSP1’s synergistic action with GPX4 in ferroptosis prevention. While FSP1 knockout did not affect GSH levels, it led to increased lipid peroxidation. Moreover, GPX4-deficient H460 cells remained viable with functional FSP1, but simultaneous knockout of both GPX4 and FSP1 resulted in pronounced ferroptosis [73]. Further research by Doll et al. showed that FSP1 overexpression mitigated lipid peroxidation in GPX4-deficient cells [74]. Another protective pathway against ferroptosis involves GTP cyclohydrolase-1 (GCH1) and tetrahydrobiopterin (BH4). BH4 serves as a crucial cofactor, possessing significant antioxidant qualities that aid in reducing lipid peroxidation directly and enhancing CoQ10 synthesis [75], 76]. GCH1 functions as a key factor catalyzing the reduction of guanosine triphosphate (GTP) to BH4 [76]. Research has demonstrated that the GCH1-BH4 pathway can inhibit ferroptosis by facilitating lipid remodeling [77], 78].

Diabetic bone loss

Iron dysregulation and ferroptosis have been documented in the development of diabetic bone loss. Under high glucose conditions, iron metabolism is disrupted, culminating in iron over-accumulation and excessive ROS production in bone cells. This iron-dependent oxidative stress induces cell death featured by lipid ROS generation and mitochondrial dysfunction [96]. Several ferroptosis-related proteins have also been implicated in this process. For example, GPX4, a key antioxidant enzyme that protects against lipid peroxidation, is downregulated in the bone tissue of diabetic osteoporosis models. The FtMt protein, which sequesters iron in mitochondria, can suppress high glucose induced ferroptosis in osteoblasts, and FtMt knockdown triggers mitophagy and ferroptosis [86]. The iron exporter ferroportin (FPN) is also involved, as its inhibition by hepcidin causes an increase in intracellular iron and increased ROS production in osteoclasts, promoting osteoporosis [97]. Additionally, the cystine/glutamate antiporter system Xc-, which supplies cysteine in GSH production pathway, was found to be impaired in diabetic bone loss, further compromising antioxidant defenses and facilitating ferroptosis [98]. Contrarily, Nrf2/HO-1 axis has been shown to protect against osteoblast ferroptosis in diabetic osteoporosis by enhancing antioxidant capacity [98]. Lastly, transferrin receptor 2 (Tfr2), which regulates iron uptake, has been found to control bone mass by modulating the BMP-p38 MAPK-Wnt signaling axis in osteoblasts [99]. Together, these studies delineated the complex interplay between iron metabolism, oxidative stress, and ferroptosis in the pathogenesis of diabetic bone loss, with various ferroptosis-related proteins serving as potential therapeutic targets.

Osteoarthritis (OA)

Ferroptosis in chondrocytes has been recognized as a detrimental event in osteoarthritis development. Several lines of evidence suggest the dysregulation of key ferroptosis regulators in OA cartilage and chondrocytes. Transcriptomics profiling along with various other studies have shown a significant decrease in GPX4 expression within OA cartilage [100], [101], [102]. Additionally, it has been documented that the levels of solute carrier family 3 member 2 (SLC3A2) are decreased in OA cartilage [103]. On the other hand, inhibitors of ferroptosis, such as ferrostatin-1 (Fer-1) and desferrioxamine (DFO), have demonstrated the ability to suppress the ferroptosis of chondrocytes and mitigate OA [104]. Yan et al. identified that the ferroptosis activator Erastin instigated chondrocyte ferroptosis while suppressing type II collagen expression; conversely, Fer-1 was able to counteract this process and activate the Nrf2/GPX4 and Nrf2/HO-1 signaling [105]. Excessive mechanical stress also accelerates the progression of OA. Piezo1, a key mechanosensing ion channel, has been implicated in regulating iron homeostasis [106]. Mechanical disruptions activate Piezo1 protein, resulting in an increased inflow of calcium ions. This activation results in a reduction of GSH and inhibition of GPX4, which promotes chondrocyte ferroptosis and subsequently OA [107]. The above evidence highlights chondrocyte ferroptosis as a key player in OA. Nonetheless, the precise underlying mechanisms remain to be elucidated. One potential therapeutic approach for OA is the targeted suppression of chondrocyte ferroptosis, particularly focusing on the GSH-GPX4 pathway.

Rheumatoid arthritis (RA)

Rheumatoid arthritis (RA) is a prevalent autoimmune condition marked by immune cell infiltration and the proliferation of synovial fibroblasts, culminating in cartilage and bone destruction and resulting in aggressive arthritis [108]. The role of ferroptosis is significantly associated with the development of RA. When compared to healthy individuals, patients with RA and osteoarthritis (OA), particularly those with RA, exhibit elevated synovial iron levels [109]. Fibroblast-like synoviocytes (FLSs) are a crucial cell type involved in RA, which promote RA progression by secreting cytokines, supporting neovascularization, and degrading the matrix [110]. Studies have shown that elevated levels of iron and ROS can promote the differentiation of osteoclasts, enhance bone breakdown, induce ferroptotic death in chondrocytes, and lead to the degradation of type II collagen in cartilage [111], 112]. FLSs, however, maintain normal survival and proliferation by resisting ferroptosis. Despite increased ROS levels in RA FLSs, several anti-ferroptosis proteins become upregulated while ACSL4 is reduced, resulting in balanced lipid ROS generation and detoxification through improved antioxidant capacity [113]. Research conducted by Wu and colleagues found that tumor necrosis factor boosts the expression of SLC7A11 along with increased GSH production in FLSs during the process of collagen-induced arthritis, thereby increasing resistance to ferroptosis [114]. Additionally, macrophages demonstrated a protective role against ferroptosis in FLSs upon RSL3 treatment. Another study by Cheng and team uncovered that raised levels of Semaphorin 5A (SEMA5A) synthesized by synovial macrophages enhanced the expression of GPX4 and stearoyl-CoA desaturase-1 (SCD1) through the PI3K/Akt/mTOR pathway. This enhancement inhibits lipid peroxidation and ferroptosis in FLSs [115]. The fact that FLSs are resistant to ferroptosis suggests that providing iron supplements to RA patients suffering from iron deficiency could potentially worsen RA symptoms [116]. Besides, Kong et al. found that exosomes derived from osteoarthritic FLS contain increased levels of microRNA-19b-3p, which targets and downregulates SLC7A11. This leads to a suppression of GSH synthesis and increased oxidative stress in chondrocytes, ultimately promoting ferroptosis and cartilage damage [117]. Recent efforts have been devoted to identifying potential ferroptosis-related genes implicated in RA progression [118]. Given the crucial role of FLSs in RA progression, therapeutic strategies targeting FLSs are needed to restore synovial homeostasis and improve disease outcomes in RA patients [119].

Osteoporosis

Osteoporosis is characterized by a disproportion between the bone resorption activities of osteoclasts and the bone-forming roles of osteoblasts. This condition causes a reduction in bone mass and strength, which elevates the risk of fragility fractures and impairs the healing process [120], 121]. Recent evidence suggests that ferroptosis induction in MSCs, osteoblasts and osteoclasts may be implicated in the pathogenesis of osteoporosis. In bone mesenchymal stem cells (BMSCs), Fanconi anemia complementation group D2 (FANCD2) was found to suppress erastin-induced ferroptosis by reducing iron overload and lipid peroxidation [122], indicating an anti-ferroptosis role of FANCD2 during differentiation of BMSCs under certain circumstances. In a rat model of type 2 diabetes osteoporosis (T2DOP), the overexpression of FtMt reduces oxidative stress and inhibits ferroptosis in osteoblasts. Conversely, knocking down FtMt triggers mitophagy and exacerbating cell death [86], implying that FtMt could be a significant therapeutic target for T2DOP. Furthermore, high glucose levels result in the induction of ferroptosis in the osteoblasts of T2DOP rats through the enhancement of ROS, lipid peroxidation, and GSH consumption. In contrast, melatonin mitigates ferroptosis by stimulating the Nrf2/HO-1 pathway, thereby boosting the osteogenic capacity of MC3T3-E1 cells [85]. Moreover, high doses of steroids can lead to osteoporosis and ferroptosis in osteoblasts, likely through the downregulation of GPX4 and the cystine/glutamate antiporter system Xc-. Interestingly, extracellular vesicles derived from endothelial progenitor cells can prevent osteoblastic ferroptosis by restoring the levels of GPX4 and system Xc-, thus slowing the progression of osteoporosis [88]. Endothelial cell-secreted exosomes could also reverse the detrimental impact of glucocorticoids on osteoblasts by repressing ferritinophagy-dependent ferroptosis [87].

Furthermore, RANKL stimulation has been reported to increase ferroptosis markers, such as malondialdehyde (MDA), PTGS2 and labile iron levels, in bone marrow-derived macrophages undergoing osteoclast differentiation, suggesting ferroptosis may occur during osteoclast differentiation [92]. However, this can be antagonized by hypoxia through the inhibition of RANKL-induced ferritinophagy, indicating an interplay of hypoxia signaling and the survival of osteoclasts. In ovariectomized mice, administration of an HIF-1α inhibitor prevented bone loss by inducing ferroptosis in osteoclasts by targeting HIF-1α and ferritin [92], providing a potential alternative treatment for postmenopausal osteoporosis. Moreover, the deficiency in estrogen levels after menopause can lead to a diminished inhibitory effect of estrogen on HIF-1α, resulting in enhanced osteoclast activity. This mechanism may contribute to the sustained activity of osteoclasts and the development of postmenopausal osteoporosis [123].

Regarding osteocytes, a recent study demonstrated that iron overload induced by hepcidin deficiency led to increased osteocyte cell death and bone degeneration in Hamp−/− mice. The bone loss was linked to heightened expression in osteocyte-derived proteins (sclerostin and RANKL), which inhibits osteogenesis and promotes bone resorption, respectively [124]. Nonetheless, limited research has been conducted to study the potential engagement of ferroptosis in osteocytes within osteoporosis. In summary, ferroptosis appears to occur in both osteoclasts and osteoblasts under various pathological conditions related to osteoporosis, including diabetes, glucocorticoid treatment, and estrogen deficiency. Key players such as GPX4, system Xc-, and iron homeostasis regulators play important roles. Modulating these pathways may serve as new intervention options for managing osteoporosis. Future research is required to fully decipher the underlying mechanisms.

Lumbar disc degeneration

Lumbar disc herniation is a widespread cause of persistent low back pain, impacting 70%–85 % of the global population. The main reason for lumbar disc herniation is intervertebral disc degeneration, influenced by multiple factors including age [125]. The causes of intervertebral disc degeneration encompass AF rupture, oxidative stress in NPC, and cartilage endplate degeneration [126]. Recent studies propose a potential role of ferroptosis in intervertebral disc degeneration. In individuals with intervertebral disc degeneration, the levels of GPX4 and FTH in disc tissue were decreased compared to those without the condition, while the levels of the ferroptosis marker PTGS2 were elevated, indicating the presence of ferroptosis in degenerative disc tissues [127]. Exposure to tert-butyl hydroperoxide (TBHP, a chemical that induces oxidative stress) led to an increase in PTGS2 and ACSL4 levels, accelerated lipid peroxidation, and reduced the expression of FTH and GPX4 in both AF cells and NPCs. The impact of these changes was counteracted by ferroptosis inhibitors Fer-1 and DFO [127], 128]. Additionally, inducing iron overload experimentally was found to exacerbate the deterioration of intervertebral discs and cartilage endplates in a manner dependent on the dosage [128]. Treatment with DFO delayed the progression of disc degeneration by suppressing ferroptosis, indicating that ferroptosis is involved in the pathophysiological process of intervertebral disc degeneration. Furthermore, the levels of hemoglobin signal and iron granules were found to be significantly elevated in herniated NPs compared to nonherniated NPs [129]. Higher expression of HO-1 was observed in the NP of herniated discs, and heme-mediated ferroptosis was associated with alterations in the Notch signaling pathway, which could be mitigated by co-treatment with DFO [129].

Single-cell RNA sequencing identified that various ferroptosis-associated genes exhibited differential expression in the nucleus pulposus cells (NPCs) of individuals with intervertebral disc degeneration (IDD) when compared to healthy controls [130]. This discovery suggests an alteration in the propensity for ferroptosis. Dysregulation of FPN has been implicated in causing intercellular iron accumulation in cases of IDD. When FPN expression was silenced, there was an elevation in iron levels within NPCs. Conversely, overexpression of FPN mitigated iron accumulation and reduced ferroptosis [131]. Additionally, nuclear translocation of the metal-regulated transcription factor 1 (MTF1) enhanced FPN transcription, thereby restoring its capacity to alleviate iron overload and safeguarding NPCs from ferroptosis. The deregulation of non-coding RNAs also plays a role in influencing ferroptosis in intervertebral disc cartilage. In comparison to normal tissue, the expression of miR-10a-5p was found to be diminished in degenerative intervertebral disc cartilage, whereas its target gene, IL-6R, was upregulated [132]. IL-6 induced chondrocyte ferroptosis by suppressing GPX4 expression in diseased cartilage, suggesting the involvement of IL-6-mediated inflammatory axis in chondrocyte loss during intervertebral disc degeneration [132]. Overall, recent studies have unveiled ferroptotic features in NPCs and the cartilage tissues of herniated intervertebral disc, and experimental evidence indicates the beneficial effect of targeting oxidative stress, iron overload and ferroptosis. This opens an avenue to mitigate the detrimental progression of lumbar disc degeneration.

Melatonin

Under high glucose conditions, melatonin triggers the pathways Nrf2/HO-1/GPX4 and SLC7A11 in osteoblasts. This action decreases ROS levels and lipid peroxidation, ultimately preventing ferroptosis. Consequently, osteoblasts exhibit enhanced osteogenic capability, alleviating osteoporosis induced by type 2 diabetes [85].

Qing’e pill

This traditional Chinese medicine preparation reduces DMT1 and TFR1 levels while promoting FPN1 expression in osteoblasts, reducing intracellular iron accumulation. It also upregulates GPX4 expression and inhibits ROS production, thus suppressing ferroptosis in osteoblasts and attenuating diabetic osteoporosis [133].

Artesunate (ART)

ART, a drug for malaria prevention, hinders the differentiation of osteoclasts and bone resorption in elevated glucose environments by controlling the iron levels and production of ROS inside cells. By reducing the activity of genes specific to osteoclasts such as TRAP, CTR, and CTSK, it helps mitigate diabetic osteoporosis [134].

Ferrostatin-1 and liproxstatin-1

These are ferroptosis inhibitors that reduce iron overload and lipid ROS levels in osteoblasts and osteocytes under diabetic conditions. They inhibit ROS production, preserve mitochondrial function, and prevent cell death, thereby maintaining bone homeostasis and preventing diabetic bone loss [98], 135]. These inhibitors also decrease lipid peroxidation and ferroptosis induced by IL-1β and ferric ammonium citrate in chondrocytes by enhancing Nrf2 system activation, reducing the advancement of osteoarthritis [104].

Deferoxamine (DFO)

DFO acts as an iron chelator to reduce intracellular iron levels and ROS production in osteoblasts and MSCs under high glucose conditions. It promotes osteogenic differentiation of MSCs and enhances osteoblast function, thus attenuating diabetic osteoporosis [35], 136]. Furthermore, DFO improves osteoarthritis by blocking chondrocyte ferroptosis by activating the Nrf2 pathway [94]. DFO has also been reported to delay disc deterioration by mitigating ferroptosis in annulus fibrosus cells and nucleus pulposus cells, demonstrating the therapeutic potential of iron chelation in intervertebral disc degeneration [127], 128].

Mitochondrial ferritin (FtMt) overexpression

Overexpression of FtMt in osteoblasts reduces high glucose induced oxidative stress by storing excess iron in mitochondria. This inhibits ferroptosis in osteoblasts and prevents bone loss in type 2 diabetic osteoporosis. Conversely, FtMt knockdown induces mitochondrial autophagy via the ROS/PINK1/Parkin pathway and promotes ferroptosis in osteoblasts [86].

2-Methoxyestradiol (2ME2)

The HIF-1α-specific inhibitor 2ME2 was found in in vivo experiments to protect against bone loss in ovariectomized mice, mimicking postmenopausal osteoporosis. By focusing on HIF-1α and ferritin, 2ME2 triggers ferroptosis in osteoclasts. The researchers suggest that triggering ferroptosis in osteoclasts via the targeting of HIF-1α and ferritin may offer a new approach to treating osteoporosis [92].

Endothelial cell-derived extracellular vesicles (EPC-EVs)

EPC-EVs hinder the initiation of the ferroptosis pathway in bone cells by reinstating GPX4 and system Xc-levels. The administration of EPC-EVs reverted alterations triggered by dexamethasone in cysteine and indicators of oxidative stress (MDA, GSH, GSSG) and enhanced bone characteristics in rodents. These findings propose that EPC-EVs have the potential to prevent glucocorticoid-triggered osteoporosis through the inhibition of ferroptotic pathway in osteoblasts [88].

Carbonyl cyanide-m-chlorophenyl-hydrazone (CCCP)

CCCP is a mitochondrial uncoupling agent that activates mitophagy. Treatment of osteoblasts with CCCP increased ferroptosis and disrupted cellular functions, as evidenced by reduced levels of GPX4, osteocalcin, alkaline phosphatase, and osteoprotegerin, impaired formation of mineralized nodules, and elevated production of ROS and lipid peroxidation. Ferroptosis inhibitors rescued these effects, suggesting that mitochondrial dysfunction and ferroptosis in osteoblasts may contribute to osteoporosis [86].

GPX4 activation

GPX4 expression was reported to be downregulated in the cartilage tissue of osteoarthritis patients. Upregulating GPX4 decreases the susceptibility of chondrocytes to oxidative damage, reduces cartilage degeneration, and slows osteoarthritis progression [100].

D-Mannose, stigmasterol, and icariin

By activating the GPX4 pathway or reducing lipid peroxidation, natural products with antioxidant or anti-inflammatory properties can slow down cartilage degradation and inhibit ferroptosis in chondrocytes. This, in turn, helps delay the development of osteoarthritis [95], 137], 138].

BAPTA acetoxymethyl ester

This chelator of calcium prevents the import of iron into chondrocytes, which hinders the production of ROS and the dysfunction of mitochondria caused by an excess of iron, thus potentially attenuating ferroptosis in osteoarthritis [139].

Rosiglitazone

This PPAR-γ activator inhibits the production of lipid ROS and ferroptosis in chondrocytes by suppressing ACSL4 activity, suggesting a promising beneficial impact on osteoarthritis [140], 141].

Ferroportin (FPN) regulation

FPN dysregulation causes an over-accumulation of intercellular iron in intervertebral disc degeneration. FPN overexpression alleviated iron overload and ferroptosis in nucleus pulposus cells [131].

Metal-regulated transcription factor 1 (MTF1)

Increased nuclear translocation of MTF1 promotes FPN transcription and restored its activity to expel excessive iron, preventing nucleus pulposus cells against ferroptosis [131].

miR-10a-5p/IL-6R axis

MiR-10a-5p levels were decreased in degenerative intervertebral disc cartilage, which causes an increase in IL-6R expression. The activation of IL-6 led to chondrocyte ferroptosis, implying a potential link between this pathway and intervertebral disc degeneration [132].

Together, these examples highlight different approaches to regulating iron metabolism and ferroptosis in key cell types involved in diabetic bone loss, osteoporosis, osteoarthritis and intervertebral disc degeneration, including MSCs, osteoblasts, osteoclasts, chondrocytes, annulus fibrosus cells, and nucleus pulposus cells. These interventions target crucial molecules and pathways such as GPX4, Nrf2, ACSL4, FPN, and MTF1. By modulating these targets, the primary goal is to inhibit excessive iron accumulation, lipid ROS generation, and ferroptosis, thereby maintaining joint and intervertebral disc homeostasis and preventing disease progression.