The protective up-regulation of metallothionein-2A in intervertebral disc degeneration inhibits nucleus pulposus cell ferroptosis through activation of the PI3K/AKT/mTOR pathway

Abstract

Intervertebral disc degeneration (IVDD) is a major contributor to low back pain, influenced by various factors including cellular senescence, apoptosis, oxidative stress, and inflammation. Metallothionein-2A (MT2A), due to its unique metal-binding and antioxidant capacity, plays a critical role in various diseases. This research sought to clarify how MT2A inhibits the progression of IVDD. Single-cell sequencing analysis revealed that ferroptosis was involved in IVDD, and MT2A was significantly upregulated in the degenerated nucleus pulposus tissue. In vitro, Tert-Butyl Hydroperoxide (TBHP) treatment induced MT2A expression. Knockdown of MT2A exacerbated TBHP-induced ferroptosis, whereas MT2A overexpression or treatment with ferrostatin-1 reversed ferroptosis, lipid peroxidation, and mitochondrial damage. In vivo, AAV-mediated MT2A overexpression significantly alleviated puncture-induced IVDD in rats. Mechanistically, MT2A overexpression activated PI3K/AKT/mTOR pathway, and this protective effect was significantly attenuated upon treatment with specific pathway inhibitors. In Conclusion, our findings demonstrate that MT2A is protectively upregulated in IVDD and mitigates ferroptosis of NP cells and IVDD progression through activation of the PI3K/AKT/mTOR pathway, which designates MT2A as a promising target for therapy in IVDD.

Introduction

According to the 2021 Global Burden of Disease statistics, low back pain (LBP) ranks among the leading contributors to disability around the world [1]. Approximately 11% to 42% of the global population experience LBP, with over 500 million people affected annually, imposing a significant socioeconomic burden [2]. Although multiple causes and risk factors can result in LBP, intervertebral disc degeneration (IVDD) is deemed a significant underlying cause [3]. The intervertebral disc is a physiological avascular tissue composed mainly of the centrally located, water-rich gelatinous nucleus pulposus (NP), the surrounding annulus fibrosus (AF), and cartilage endplates (CEP) [4]. Degeneration and reduction in NP cell number, as well as an imbalance between extracellular matrix (ECM) synthesis and degradation, are important pathophysiological mechanisms in IVDD [5, 6]. Therefore, investigating molecular mechanisms underlying NP cell death may have important clinical implications for elucidating the pathogenesis of IVDD.

Ferroptosis is a type of programmed cell death driven by iron accumulation and lipid peroxidation [7]. This process involves a buildup of intracellular free iron, which sparks an overproduction of reactive oxygen species (ROS). ROS subsequently cause oxidative damage to cell membranes, resulting in membrane rupture. Key regulators of ferroptosis include disrupted metabolic pathways, glutathione deficiency, and the suppression of GPX4—an essential antioxidant enzyme. When these systems fail, cells lose their ability to function and ultimately perish [8]. Ferroptosis is intricately linked to metabolic stability, oxidative stress, and inflammatory signaling. Its impact spans a range of diseases, including cancer, neurodegenerative disorders, osteoporosis, and macular degeneration [9]. Recent research also highlights its significant involvement in IVDD, further underscoring its broad biological relevance [10].

Metallothioneins (MTs) are a type of low-molecular-weight protein, rich in cysteine. Their unique structure endows them with antioxidant properties and metal ion-binding capabilities [11]. Metallothionein-2A (MT2A) is the main subtype expressed in humans, which consists of 61 amino acids [12]. MT2A functions include the regulation of zinc homeostasis, metal detoxification, protection against oxidative stress, and modulation of inflammatory responses [13]. Recent studies have also shown its association with cell proliferation and differentiation, as well as angiogenesis [14, 15]. A recent study has revealed that MT2A enhances cells’ ability to combat oxidative stress and facilitates the elimination of harmful iron ions by activating the ERK1/2 and JNK MAPK signaling pathways [16]. These findings underscore MT2A’s possible involvement in modulating ferroptosis. However, current research on MT2A’s regulation of ferroptosis is limited, and the exact molecular mechanisms are still not fully understood, necessitating additional research.

Consequently, this study aimed to investigate whether MT2A can regulate ferroptosis in NP cells and impact the development of IVDD. We first reviewed a single-cell sequencing (scRNA-seq) dataset of IVDD from GEO database and found that ferroptosis is connected to IVDD, with MT2A identified as one of the most markedly upregulated genes. Given its demonstrated antioxidant stress and metal-binding capabilities in other diseases, we hypothesized that the upregulation of MT2A in IVDD may represent a compensatory protective response to oxidative stress or ferroptosis. Subsequent in vitro and in vivo experiments supported this hypothesis. Further investigation revealed that MT2A inhibits NP cell ferroptosis and IVDD progression through the PI3K/AKT/mTOR pathway. In conclusion, this study represents the first exploration of the role and mechanism of MT2A in IVDD, potentially offering a novel therapeutic target for the disease.

Result

Bioinformatics analysis suggests involvement of MT2A and ferroptosis in IVDD progression

To investigate changes in gene expression of NP cells during IVDD, we performed scRNA-seq analysis on dataset GSE199866. UMAP analysis grouped NP cells into 10 distinct clusters (Fig. 1A). Clusters 0 and 1 represented the major NP cell populations, while cluster 3 cells were almost exclusively present in healthy NP tissue (Fig. 1B, C). All clusters expressed NP cell markers such as aggrecan (ACAN), MIA SH3 Domain Containing (MIA), SRY-box transcription factor 9 (SOX9), and Fibromodulin (FMOD) (Fig. 1D). Based on their highly expressed genes, enrichment analyses (Fig. S1A–E), and previously published single-cell studies, these clusters were categorized into four subpopulations (Fig. 1G, H): (1) Homeostatic NPCs (Hom-NPCs, clusters 1 and 2) expressing mRNAs related to ribosome biogenesis and protein translation, such as RPS29 and RPS21 [17, 18]. (2) Regulatory NPCs (Reg-NPCs, clusters 0, 5, 6, 7) expressing genes involved in inflammation and signaling regulation, such as BMP2, CHI3L1, or CHI3L2 [17, 19]. (3) Extracellular matrix NPCs (ECM-NPCs clusters 3, 4, 9) expressing extracellular matrix-related mRNAs such as COL3A1, COL6A1, COL1A1, and COL2A1. (4) Progenitor NPCs (Pro-NPCs, cluster 8) expressing cell cycle and proliferation markers, such as UBE2C and TOP2A [20]. We performed differential expression gene (DEGs) analysis between healthy NP (HNP) and degenerated NP (DNP) cells to identify IVDD-related genes. MT2A was highly expressed in Reg-NPCs and Pro-NPCs (Fig. 1G). Volcano and UMAP plots showed that MT2A expression was elevated in DNP tissue (Fig. 1E, F). To further elucidate the potential mechanisms of NP cell degeneration during IVDD, KEGG pathway enrichment analysis was performed, highlighting ferroptosis and mineral absorption pathways as involved in IVDD (Fig. 1I). A heatmap showed expression levels of ECM genes and genes from ferroptosis and mineral absorption KEGG pathways across the NP cell clusters. MT2A expression was increased in nearly every cluster of DNP cells, with the highest expression in cluster 7 (Fig. 1J).

Fig. 1. Bioinformatics analysis suggests involvement of MT2A and ferroptosis in IVDD progression.

A UMAP plot showing 10 cell clusters in human NP tissue; each point represents a single cell. B, C UMAP plot and bar graph showing distribution of the 10 clusters in HNP and DNP tissue. D UMAP clustering displaying expression distribution of chondrocyte markers ACAN, MIA, SOX9, and FMOD across the 10 cell clusters. E Volcano plot showing significantly upregulated (orange) and downregulated (blue) genes in DNP compared to HNP. F UMAP clustering showing MT2A expression in HNP and DNP. G Dot plot showing marker gene expression in each NP cell subpopulation. H UMAP visualization of 4 distinct NP cell subpopulations after reclustering. I KEGG enrichment analysis of DEGs between HNP and DNP. J Heatmap showing expression of ECM genes and genes from ferroptosis and mineral absorption KEGG pathways across the 10 clusters.

Increased MT2A expression in human IVDD

To ascertain how ferroptosis and MT2A function in IVDD, we collected NP tissues from patients with mild degeneration and severe degeneration (Fig. 2A). IHC analysis was performed to evaluate MT2A level in degenerative NP tissue. As expected, IHC result demonstrated that the severely degenerated discs had much higher levels of MT2A expression than mildly degenerated discs (Fig. 2B). Furthermore, we observed a downregulation of COL2 and GPX4 levels with increasing degeneration grade, indicating ECM degradation and the activation of ferroptosis in IVDD. Western blot (WB) and real-time quantitative PCR (RT-qPCR) results further confirmed the upregulation of MT2A in NP tissues (Fig. 2C–E).

Fig. 2. The upregulation of MT2A and activation of ferroptosis in IVDD.

A Representative MRI images of IVDD patients with different Pfirrmann grade. B IHC of NP tissue from IVDD patients with Pfirrmann grade showing expression of MT2A, COL2 and GPX4. C, D WB and quantification analysis of MT2A in NP tissue from MDD and SDD patients. E RT-qPCR analysis of MT2A in NP tissue from MDD and SDD patients. F Representative MRI and CT images of control and IVDD rat models. G Representative images of H&E staining and Safranin-O staining in rat intervertebral disc tissue. H–L Representative IHC images of MT2A, COL2, GPX4, ACSL4 and PTGS2 in rat NP tissue. Data are presented as mean ± SD (n = 3); ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Increased MT2A expression in a rat model

To further investigate the roles of MT2A and ferroptosis in IVDD, we examined the expression of MT2A and ferroptosis-related indicators in an IVDD rat model. MRI results showed that T2-weighted signals significantly decreased in the intervertebral discs of the IVDD group, while CT imaging indicated a reduction in intervertebral disc height (Fig. 2F). Hematoxylin and eosin (H&E) staining, along with Safranin-O staining, revealed that NP and AF tissues exhibited a well-organized structure with clear boundaries in control group. In contrast, the IVDD group exhibited disorganized disc structures with unclear boundaries between the NP and AF (Fig. 2G), confirming the successful establishment of the IVDD rat model. IHC results indicated an upregulation of MT2A expression and a downregulation of COL2 in the IVDD group (Fig. 2H, I). Furthermore, we observed significant alterations in ferroptosis markers in the IVDD group, characterized by a significant downregulation of GPX4 and concurrent upregulation of ACSL4 and PTGS2 (Fig. 2J–L). Collectively, these results suggest that MT2A expression is elevated in the degenerative NP tissue of both human and animal models, accompanied by activation of ferroptosis.

Oxidative stress induces upregulation of MT2A and triggers ferroptosis in NP cells

TBHP is a commonly used oxidative stress inducer, and its ability to induce ferroptosis in NP cells through oxidative stress has been demonstrated in several studies [21]. To simulate oxidative stress during IVDD progression, NP cells were treated with varying concentrations of TBHP (25, 50, 100, 200 μM; 4 h) and different durations (100 μM; 1, 2, 4, 6, 12 h). CCK-8 assays indicated a decrease in NP cell viability with higher TBHP concentrations or longer treatment times (Fig. 3A, B). WB and immunofluorescence (IF) analyses revealed a dose-dependent increase in MT2A expression correlating with TBHP concentration (Fig. 3C–E). Further examination of ferroptosis and ECM genes showed that with increasing TBHP concentrations, the expression of GPX4, FTH1, ACAN, and COL2 gradually decreased, while the expression of ACSL4, PTGS2, MMP3, and ADAMTS5 increased (Fig. 3F–O), indicating TBHP induces NP cell ferroptosis and IVDD in a dose-dependent manner.

Fig. 3. Oxidative stress induces upregulation of MT2A and triggers ferroptosis in NP cells.

A, B NP cell viability after treatment with varying concentrations of TBHP (25, 50, 100, 200 μM; 4 h) and different exposure times (100 μM; 1, 2, 4, 6, 12 h). C Representative IF images of MT2A expression in NP cells treated with different concentrations of TBHP; scale bar = 100 μm. D, E WB and quantitative analysis of MT2A expression in NP cells following TBHP treatment at different concentrations. F–J WB and quantitative analysis of ferroptosis genes in TBHP-treated NP cells at varying concentrations. K–O WB and quantitative analysis of ECM genes in TBHP-treated NP cells at varying concentrations. Data are presented as mean ± SD (n = 3); ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

MT2A deficiency exacerbates oxidative stress-induced ferroptosis in NP cells

We hypothesized that the upregulation of MT2A represents a compensatory protective response against increased oxidative stress in NP cells. To further investigate its function, we reduced the expression of MT2A through siRNA. As predicted, WB analysis showed that MT2A knockdown aggravated TBHP-induced downregulation of GPX4 and FTH1, as well as upregulation of ACSL4 and PTGS2 (Fig. 4A–E). Real-Time Quantitative PCR(RT-qPCR) results further confirmed these changes (Fig. 4F). IF staining revealed a similar trend in GPX4 expression (Fig. 4G). ELISA assays demonstrated that TBHP treatment decreased the antioxidant GSH levels and increased lipid peroxidation marker MDA in NP cells, while MT2A knockdown further intensified these effects (Fig. 4H, I). Using FerroOrange and DCFH-DA staining, intracellular Fe²⁺ and ROS levels were measured, respectively. TBHP increased both Fe²⁺ and ROS levels, and MT2A knockdown further elevated these intracellular levels (Fig. 4J–M). Transmission electron microscopy (TEM) analysis showed that MT2A deficiency exacerbated TBHP-induced mitochondrial damage, characterized by significant mitochondrial shrinkage or swelling and loss of mitochondrial cristae and membrane integrity (Fig. 4N). In summary, MT2A knockdown enhances oxidative stress-induced ferroptosis in NP cells in vitro.

Fig. 4. MT2A deficiency exacerbates oxidative stress-induced ferroptosis in NP cells.

NP cells were treated with TBHP (100 μM, 4 h) and/or si-MT2A. A–E WB and quantitative analysis of ferroptosis-related genes in NP cells from each group. F RT-qPCR analysis of ferroptosis-related genes in NP cells from each group. G Representative IF images of GPX4 expression in NP cells from each group. H, I ELISA measurements of intracellular GSH and MDA levels. J, L FerroOrange staining and quantification of intracellular Fe²⁺ levels of NP cells in each group. K, M DCFH-DA staining and quantification of ROS levels of NP cells in each group. N Representative TEM images illustrating mitochondrial morphology of NP cells in each group. Data are presented as mean ± SD (n = 3); ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

MT2A overexpression alleviates oxidative stress-induced ferroptosis in NP cells

To further validate the role of MT2A in ferroptosis, MT2A was overexpressed in NP cells using a plasmid. As an effective and selective ferroptosis inhibitor, Ferrostatin-1 (Fer-1) functions via a reductive mechanism to protect lipid membranes from damage [22]. WB and RT-qPCR analyses showed that both MT2A overexpression and Fer-1 treatment reversed the TBHP-induced changes in ferroptosis proteins and mRNA (Fig. 5A–F). IF results demonstrated that GPX4 expression was significantly increased after MT2A overexpression or Fer-1 treatment (Fig. 5G). Additionally, MT2A overexpression and Fer-1 markedly reduced intracellular Fe²⁺, ROS, and MDA levels, while increasing GSH levels in NP cells (Fig. 5H–M). TEM analysis showed that MT2A overexpression and Fer-1 rescued TBHP-induced mitochondrial morphological alterations (Fig. 5N). In summary, MT2A overexpression alleviates oxidative stress-induced ferroptosis in NP cells.

Fig. 5. MT2A overexpression alleviates oxidative stress-induced ferroptosis in NP cells.

NP cells were treated with TBHP following transfection with MT2A overexpression plasmid (oe-MT2A) or pretreated with Fer-1. A–E WB and quantitative analysis of ferroptosis-related genes in NP cells from each group. F RT-qPCR analysis of ferroptosis-related genes in NP cells from each group. G Representative IF images of GPX4 expression in NP cells from each group. H, I ELISA measurements of intracellular GSH and MDA levels. J, L FerroOrange staining and quantification of intracellular Fe2+ levels of NP cells in each group. K, M DCFH-DA staining and quantification of ROS levels of NP cells in each group. N Representative TEM images illustrating mitochondrial morphology of NP cells in each group. Data are reported as mean ± SD (n = 3), with significance levels as follows: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.

MT2A alleviates oxidative stress-induced ferroptosis and IVDD in NP cells by activating the PI3K/AKT/mTOR pathway

To further investigate the role of MT2A in IVDD, we performed RNA-seq on normal and MT2A-knockdown human NP cells (Fig. 6A, B). KEGG pathway analysis revealed significant alterations in genes related to the PI3K/AKT pathway following MT2A knockdown (Fig. 6C). Recent studies have demonstrated the vital role of the PI3K/AKT/mTOR signaling pathway in regulating ferroptosis [23]. We modulated MT2A expression in NP cells using siRNA and overexpression plasmids and assessed changes in the PI3K/AKT/mTOR pathway by WB (Fig. 6D, E). The results showed that MT2A knockdown decreased the levels of phosphorylated PI3K (p-PI3K/PI3K), AKT (p-AKT/AKT), and mTOR (p-mTOR/mTOR), whereas MT2A overexpression increased their phosphorylation levels, indicating that MT2A regulates the PI3K/AKT/mTOR pathway. To determine whether MT2A regulates oxidative stress-induced ferroptosis and IVDD through this pathway, we applied PI3K inhibitor Pictilisib, AKT inhibitor MK-2206, and mTOR inhibitor Temsirolimus. WB results showed that these inhibitors blocked the protective effects of MT2A overexpression on ferroptosis and ECM protein markers (Fig. 6F–H). IF analysis revealed a similar trend for GPX4 expression (Fig. 6I). As expected, MT2A overexpression decreased intracellular Fe²⁺, ROS, and MDA levels while increasing GSH levels in NP cells, and these effects were reversed by PI3K/AKT/mTOR pathway inhibitors (Fig. 6J–N). TEM also demonstrated that the inhibitors reversed the rescue effect of MT2A overexpression on mitochondrial morphology (Fig. 6O). In conclusion, MT2A likely alleviates oxidative stress-induced ferroptosis and IVDD in NP cells by activating the PI3K/AKT/mTOR pathway.

Fig. 6. MT2A alleviates oxidative stress-induced ferroptosis and IVDD in NP cells by activating the PI3K/AKT/mTOR pathway.

A, B Volcano plot and heatmap showing significantly altered genes in NP cells treated with si-MT2A. C KEGG pathway enrichment analysis of DEGs between control and si-MT2A treated cells. D, E WB and quantitative analysis of PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR in NP cells treated with si-MT2A or oe-MT2A. F–H WB and quantitative analysis of ferroptosis and ECM proteins in NP cells treated with TBHP, oe-MT2A, and PI3K/AKT/mTOR pathway inhibitors. I Representative IF images of GPX4 expression in NP cells after various treatments. J–L FerroOrange and DCFH-DA staining with quantification of intracellular Fe²⁺ and ROS levels in treated NP cells. M, N ELISA measurements of intracellular GSH and MDA levels. O Representative TEM images of mitochondrial morphology in NP cells under different treatments. Data are presented as mean ± SD (n = 3); ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

MT2A overexpression mitigates IVDD progression in a rat model

In order to confirm the therapeutic potential of MT2A overexpression in vivo, we constructed a rat model of puncture-induced IVDD and locally injected AAV-MT2A or the corresponding control. MRI examinations revealed that both the IVDD group and AAV-Ctrl group exhibited decreased T2-weighted signals compared to the AAV-MT2A treatment group (Fig. 7A, B). CT results indicated significant collapse of disc height in IVDD and AAV-Ctrl groups after the puncture surgery, while the AAV-MT2A group showed partial recovery of disc height (Fig. 7A). The analysis of the Disc Height Index (DHI) further confirms this result (Fig. 7C). Safranin-O and H&E staining showed that boundaries of NP and AF tissues in IVDD group and AAV-Ctrl group were blurred, while treatment with AAV-MT2A reduced this alteration (Fig. 7D). Subsequently, we assessed the degradation of ECM and the activation of ferroptosis in the intervertebral disc tissue through IHC. The IHC results showed that IVDD induced a decline of COL2 and GPX4 expression in NP tissue, whereas AAV-MT2A reversed this change (Fig. 7E–H). These results indicate that MT2A overexpression can inhibit ECM degradation and ferroptosis in IVDD. In summary, these results demonstrate that overexpression of MT2A can suppress ECM degradation and inhibit ferroptosis in intervertebral disc tissue, ultimately alleviating IVDD occurrence and progression.

Fig. 7. MT2A overexpression mitigates IVDD progression in a rat model.

A Representative T2-weighted MRI images and CT images of rat intervertebral discs at 8 weeks after surgery. B Pfirrmann grade score analysis of rat intervertebral discs. C Disc height index (DHI) analysis of rat intervertebral discs. D Representative H&E and Safranin-O staining of intervertebral disc samples from each groups at 8 weeks after surgery. E–H IHC detection and quantitative analysis of COL2 and GPX4 expression in rat intervertebral discs. Data are presented as mean ± SD (n = 5); ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

Discussion

Currently, LBP has become an urgent global public health issue [24]. As one of the main culprits of LBP, IVDD is still primarily treated with symptomatic treatments and surgery [25]. Although there has been increasing research on the pathogenesis of IVDD in recent years, the specific mechanisms underlying its occurrence and progression remain unclear. In consequence, studying the precise pathogenesis of IVDD and developing new treatment methods based on this knowledge is of significant importance.

The molecular pathological mechanisms of IVDD are multifactorial, with various factors such as cellular senescence, apoptosis, oxidative stress, and inflammation believed to be involved in the process of IVDD [26]. Recent studies have shown that oxidative stress exacerbates IVDD progression by inducing ferroptosis in NP cells [21, 27]. Ferroptosis is a unique form of iron-dependent cell death, and its occurrence is associated with intracellular oxidative stress homeostasis, iron ion levels, mitochondrial activity, lipid metabolism, and multiple signaling pathways. Recent evidence further implicates ferroptosis in the pathogenesis of IVDD [19]. Our results provide additional support for this viewpoint. KEGG pathway analysis of dataset GSE199866 revealed that ferroptosis is partly involved in IVDD. Furthermore, we observed downregulation of ferroptosis-related negative markers GPX4 in degenerated NP tissues from both humans and rats. In vitro, treatment of NP cells with the oxidative stress inducer TBHP triggered ferroptosis, as evidenced by altered ferroptosis markers, lipid peroxidation, and mitochondrial damage. These changes were effectively suppressed by Fer-1, confirming the critical role of ferroptosis in IVDD.

MTs are small proteins rich in cysteine that play crucial roles in maintaining metal homeostasis, regulation of cell proliferation and differentiation, and protection against heavy metal toxicity and oxidative stress [28]. Recent investigations also indicate the potential of MTs in regulating ferroptosis [16, 29, 30]. Grignano et al. found that MTs contribute to glutathione recycling and Oxidation-Reduction Balance, thereby counteracting ferroptosis triggered by dihydroartemisinin [31]. Our investigation using scRNA-seq uncovered a pronounced elevation in MTs expression within degenerated NP cells, with MT2A emerging as the most prominently upregulated subtype. Both cytological and animal studies further confirmed the upregulation of MT2A in IVDD. As expected, siRNA-mediated knockdown of MT2A markedly exacerbated TBHP-induced ferroptosis, lipid peroxidation, and mitochondrial damage. Conversely, MT2A overexpression ameliorated ferroptosis and IVDD progression in NP cells and IVDD models. Therefore, we suggest that upregulation of MT2A in IVDD represents a protective response. Upregulation of MT2A has also been reported in other degenerative diseases [32–34]. However, the exact mechanisms underlying MT2A upregulation in IVDD remain unclear. We successfully induced MT2A upregulation in NP cells in vitro using TBHP, suggesting that oxidative stress stimulation may be one contributing factor to MT2A elevation in NP tissues.

To investigate the underlying mechanisms of MT2A, we conducted RNA-seq analysis. KEGG pathway enrichment revealed MT2A’s potential involvement in modulating the PI3K-AKT signaling cascade. This fundamental intracellular pathway serves as a master regulator of numerous biological processes, ranging from cell growth and proliferation to motility, metabolic homeostasis, and cell survival. In recent years, several studies have emphasized the critical regulatory function of PI3K/AKT pathway in IVDD [35, 36]. Contemporary research further demonstrates that PI3K/AKT/mTOR pathway activation can suppress ferroptosis [37, 38]. Supporting this, Cheng et al. reported that Semaphorin 5A inhibits ferroptosis in rheumatoid arthritis synovial fibroblasts by activating the PI3K/AKT/mTOR pathway to enhance GPX4 expression and SREBP1/SCD-1-mediated lipogenesis [39]. Conversely, Huang et al. found that Salidroside promotes NCOA4-mediated ferroptosis by inhibiting the PI3K/AKT/mTOR pathway [40]. These studies suggest a protective role of the PI3K/AKT/mTOR pathway against ferroptosis. In this study, we report for the first time the regulatory role of MT2A on the PI3K/AKT/mTOR pathway. Overexpression of MT2A increased pathway activity, while pathway inhibitors abolished its protective effects against ferroptosis and IVDD progression.

This research investigates how MT2A influences ferroptosis in NP cells and the development of IVDD. However, some limitations should be noted. First, MT2A can regulate NP cell metabolism via multiple signaling pathways, while this study focused only on the PI3K/AKT/mTOR pathway. Second, the precise mechanisms underlying the upregulation of MT2A during IVDD remain unclear. Further investigation is required to fully clarify the precise mechanisms through which MT2A operates in IVDD.

Conclusion

In summary, our research shows that MT2A is protectively upregulated in IVDD. Local overexpression of MT2A in a rat puncture-induced IVDD model significantly alleviates ferroptosis and IVDD progression. Further mechanistic investigation revealed that MT2A suppresses ferroptosis in NP cells and slows IVDD progression by activating PI3K/AKT/mTOR pathway. As the first investigation into MT2A’s role and mechanism in IVDD, this research suggests a promising therapeutic target for IVDD treatment.

Method

Bioinformatics analysis

Single-cell RNA sequencing dataset GSE199866 was obtained from GEO database (https://www.ncbi.nlm.nih.gov/geo/). Data analysis was performed using the Seurat package in R software [41]. After quality control, data normalization and scaling were conducted using the NormalizeData and ScaleData functions, respectively. Principal component analysis was then performed for dimensionality reduction. The FindNeighbors and FindClusters functions were used for cell clustering, and the clustering results were visualized in two-dimensional space using the UMAP algorithm. Differentially expressed genes between clusters or groups were identified using the FindMarkers function in Seurat. Using the ClusterProfiler package, we conducted enrichment analyses for differential genes in Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) [42]. The NP cell subpopulations were functionally classified based on their highly expressed genes, KEGG and GO enrichment results, and alignment with previously published single-cell RNA sequencing studies on the intervertebral disc.

Human nucleus pulposus samples collection

This study received approval from the Ethics Committee of Xinhua Hospital affiliated with Shanghai Jiao Tong University School of Medicine (XHEC-NSFC-2022-085) and was conducted in adherence to the Declaration of Helsinki. Informed consent was acquired from all patients. NP tissues were categorized into two groups according to preoperative magnetic resonance imaging (MRI) utilizing the Pfirrmann grading system: mild degeneration group (MDD, Grade I and II) and severe degeneration group (SDD, Grade III, IV, and V).

Primary human nucleus pulposus cell culture

Human nucleus pulposus cells were extracted from NP tissues of patients graded as Pfirrmann II. After surgical separation, NP tissues were immediately stored in phosphate-buffered saline (PBS, Gibco, USA) and transported to a biosafety cabinet. The NP tissues were washed three times with sterile PBS, minced into small pieces, and then digested at 37 °C for 3–4 h with 1 mg/mL type II collagenase (40508ES60, Yeasen, China; ≥125 μ/mg solid). After complete digestion, the cell suspension was centrifuged at 300 × g for 5 min, and the fragmented NP tissue was resuspended in complete DMEM/F12 medium (Gibco, USA) supplemented with 1% penicillin-streptomycin (Gibco, USA) and 10% fetal bovine serum (FSD500, ExCell Bio, China). Cells were cultured in a humidified incubator at 37 °C with 5% CO₂. Cells were subcultured upon reaching 80% confluence. NP cells from passages 2 to 4 were used for all experiments in this study. The following reagents were used to treat cells: Pictilisib (HY-50094; MedChemExpress, USA); MK-2206 (HY-108232; MedChemExpress, USA); Temsirolimus (HY-50910; MedChemExpress, USA); Fer-1 (HY-100579; MedChemExpress, USA).

Western blot

Total protein was extracted from NP tissue samples or primary cells using RIPA (P0013B, Beyotime, China) containing phenylmethylsulfonyl fluoride (PMSF). Proteins were separated by 10% or 12.5% SDS-PAGE and transferred onto PVDF membranes (ISEQ00010, Millipore, USA). Membranes were blocked at room temperature for 1 h with Blocking Buffer (PS108P, Epizyme, China). The membranes were then incubated overnight at 4 °C with specific primary antibodies. After washing three times with TBST, membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 2 h. Immunoreactive bands were visualized with a chemiluminescent substrate kit (WBKLS0100, Millipore, USA). Band intensities were quantified using ImageJ software. Primary antibodies used were: MT2A (Abcam, ab192385, 1:1000); GPX4 (Proteintech, 30388-1-AP, 1:1000); FTH1 (Cell Signaling Technology, 4393, 1:1000); ACSL4 (Abcam, ab155282, 1:1000); PTGS2 (Proteintech, 27308-1-AP, 1:1000); ACAN (Affinity, DF7561, 1:1000); ADAMTS5 (Abcam, ab41037, 1:1000); COL2 (Abcam, ab188570, 1:1000); MMP3 (Abcam, ab52915, 1:1000); PI3K (Cell Signaling Technology, 4257, 1:1000); p-PI3K (Cell Signaling Technology, 4228, 1:1000); AKT (Cell Signaling Technology, 9272, 1:1000); p-AKT (Cell Signaling Technology, 4060, 1:1000); mTOR (Cell Signaling Technology, 2983, 1:1000); p-mTOR (Cell Signaling Technology, 5536, 1:1000).

Real-time quantitative PCR

Total RNA was extracted from NP cells or tissue samples using TRIzol reagent (Invitrogen, USA). After measuring the RNA concentration and purity, cDNA was synthesized using a reverse transcription kit (RR036A, Takara, Japan). The cDNA, SYBR Green (11202ES08, Yeason, China), and specific primers were mixed in appropriate ratios and analyzed using a real-time fluorescence quantitative PCR machine. The primers used were as follows: Human MT2A (F: 5′-TCCTGCAAATGCAAAGAGTGC-3′, R: 5′-GTTTGTGGAAGTCGCGTTCT-3′); Human GPX4 (F: 5′-TGAAGATCCAACCCAAGGGC-3′, R: 5′-GACGGTGTCCAAACTTGGTG-3′); Human FTH1 (F: 5′-AGCTCTACGCCTCCTACGTT-3′, R: 5′-CCTGAAGGAAGATTCGGCCA-3′); Human ACSL4 (F: 5′-GGAATGACAGGCCAGTGTGA-3′, R: 5′-TAGCACATGAGCCAAAGGCA-3′); Human PTGS2 (F: 5′-CCCTTCTGCCTGACACCTTT-3′, R: 5′-TTCTGTACTGCGGGTGGAAC-3′); Human GAPDH (F: 5′-CAAATTCCATGGCACCGTCAA-3′, R: 5′-AGCATCGCCCCACTTGATTT-3′).

Immunohistochemistry

Human and rat NP tissue sections were deparaffinized with xylene and rehydrated using a graded ethanol series. Antigen retrieval was performed by microwave heating in citrate buffer. A 5% bovine serum albumin (BSA) solution was used to block the sections. Subsequently, specific primary antibodies were applied overnight at 4 °C. The next day, following three PBS washes, the sections were incubated with HRP-conjugated secondary antibodies for 1 h. Then, substrate color development was performed using diaminobenzidine (DAB, Beyotime, China), and nuclei were counterstained with hematoxylin. The sections were examined and photographed using an optical microscope.

Cell viability assay

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; C0037, Beyotime, China). Cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and cultured until the logarithmic growth phase. Cells were exposed to different TBHP doses. Subsequently, CCK-8 reagent (C0037, Beyotime, China) was introduced to each well, followed by incubation of the plates at 37 °C for 1 h. Absorbance was recorded at 450 nm using a microplate reader.

Immunofluorescence

Cells were seeded on sterile 20 mm glass coverslips placed in 12‑well plates and cultured until reaching the logarithmic growth phase. The cell‑covered coverslips were then washed three times with PBS to remove the residual culture medium. Cells were fixed using 4% paraformaldehyde for 15 min. The samples were then subjected to three consecutive washes using PBS. The samples were subsequently treated with 0.2% Triton X-100 to increase their permeability. A 5% BSA solution was used to block the cells. After blocking at room temperature for 1 h, a specific primary antibody was added and incubated overnight at 4 °C. On the following day, fluorescent-labeled secondary antibodies were added after removing the primary antibody and washing with PBS. After 1 h incubation, samples stained and mounted with antifade mounting medium with DAPI (Beyotime, China). Fluorescence was imaged under a fluorescence microscope.

Transmission electron microscopy

Cell samples were fixed with 2.5% glutaraldehyde overnight at 4 °C and washed with PBS. The following day, 1% osmium tetroxide was used for further fixation of the cell samples. After fixation for 2 h, the samples were washed again. Dehydration was performed using a graded ethanol series, followed by acetone replacement. Samples were infiltrated with embedding medium in varying ratios, polymerized at 65 °C, and trimmed into a trapezoidal shape. Finally, ultrathin sections of 70 nm were cut and stained with uranyl acetate and lead citrate for observation.

Reactive oxygen species and detection

Cells at the logarithmic growth phase were rinsed 1–2 times gently with PBS after removing the culture medium. Cells were then incubated with culture medium containing DCFH-DA (S0033S, Beyotime, China) at 37 °C in the dark for 30 min. Fluorescence was imaged under a fluorescence microscope.

FerroOrange staining

Cells at the logarithmic growth phase were gently washed 1–2 times with PBS after removing the culture medium. Then, 1 μmol/L FerroOrange (F374, Dojindo, Japan) working solution was added, and cells were incubated at 37 °C in the dark for 1 h. Fluorescence was imaged under a fluorescence microscope.

Glutathione and malondialdehyde assays

According to the kit instructions, intracellular reduced glutathione (GSH) content was measured using the GSH/GSSG detection kit (S0053, Beyotime, China). Simultaneously, malondialdehyde (MDA) levels, an indicator of lipid peroxidation, were determined following the instructions of the lipid peroxidation MDA assay kit (S0131S, Beyotime, China).

Small interfering RNA and plasmid transfection

Small interfering RNA (siRNA) targeting human MT2A (sequence: CGUUUGCUAUAUUCCUUUU) and overexpression plasmids were constructed by Genomeditech (Shanghai, China) to regulate MT2A expression. When human NP cells reached 60–70% confluence, siRNA or plasmids were mixed with Lipofectamine 3000 (Invitrogen, USA) and incubated at room temperature to form transfection complexes. Cells were incubated with the complexes for 6 h at 37 °C with 5% CO₂, then the medium was replaced with fresh complete culture medium. Cells were further cultured for 48 h before subsequent experimental assays. Control groups were transfected with blank siRNA or plasmid.

RNA sequencing

Human NP cells were divided into two groups based on treatment with si-Ctrl or si-MT2A, each group containing three replicates. After 2 days of treatment, total RNA was extracted for subsequent analysis. Sangon Biotech (Shanghai) executed RNA sequencing (RNA-seq) and data analysis.

Animal experiments

Animal experiments for this study complied with the International Guiding Principles for Biomedical Research Involving Animals and were approved by the Experimental Animal Center of Xinhua Hospital, associated with Shanghai Jiao Tong University School of Medicine. Adeno-associated virus (AAV)-mediated MT2A overexpression was used to evaluate the therapeutic effect of MT2A in rat model. The AAV vectors were constructed by Genomeditech (Shanghai, China). Male Sprague-Dawley (SD) rats, 6 weeks old, were randomly divided into four groups (n = 5 each): (1) Control group (2) IVDD group; (3) IVDD + AAV-Ctrl group; (4) IVDD + AAV-MT2A group. After anesthesia, rats were placed supine and fixed on the surgical table; the tail was positioned, and the target intervertebral disc was marked. A 21-gauge needle was punctured into the marked point to a depth of 4 mm. The puncture needle was then rotated 360 degrees horizontally and held in place for 30 s to establish a puncture-induced rat intervertebral disc degeneration model. Injections were performed as follows: IVDD group rats received 10 μL PBS; IVDD + AAV-Ctrl group rats received 10 μL AAV-Ctrl; IVDD + AAV-MT2A group rats received 10 μL AAV-MT2A.

Statistical analysis

All experiments in this study were independently performed at least three times. Statistical analysis was conducted using SPSS 21.0 and GraphPad Prism 9.0 software. Comparisons between two groups were performed using Student’s t-test, and one-way ANOVA was used for multiple group comparisons. P < 0.05 was considered statistically significant.

Author contributions

HC: Conceptualization, Methodology, Investigation, Original Draft; H-lZ: Conceptualization, Funding acquisition; Q-zC: Methodology; S-kS: Software; B-yY: Formal analysis; YW: Investigation; HD: Resources; Z-yL: Software; P-bC: Methodology; Q-yX: Resources; MM: Investigation; BL: Project administration; X-fZ: Review & Editing; L-sJ: Review & Editing; S-dJ: Funding acquisition, Supervision, Review & Editing.

Funding

This study was supported by the Shanghai Sailing Program (23YF1425900), National Natural Science Foundation of China (82272535), National Natural Science Foundation of China (82302741).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41420-026-02972-9.