MANF overexpression ameliorates oxidative stress-induced apoptosis of human nucleus pulposus cells by facilitating mitophagy through promoting MFN2 expression

Abstract

Intervertebral disc degeneration (IDD) is a degenerative condition associated with impaired mitophagy. MANF has been shown to promote mitophagy in murine kidneys; however, its role in IDD remains unexplored. This study aimed to elucidate the mechanism by which MANF influences IDD development through the regulation of mitophagy. Human nucleus pulposus (NP) cells were exposed to tert-butyl hydroperoxide (TBHP) to establish an oxidative stress-induced cellular model. The expression levels of MANF in NP cells were quantified using quantitative real-time PCR (qPCR) and Western blotting. The impact of MANF on TBHP-induced NP cells was evaluated by assessing cell viability, apoptosis, and the levels of mitophagy-related proteins. The underlying mechanisms were further investigated using RNA-binding protein immunoprecipitation (RIP), dual-luciferase reporter assays, qPCR, and Western blotting. Results indicated that MANF expression was significantly downregulated in both IDD patients and TBHP-induced NP cells. Overexpression of MANF inhibited apoptosis, enhanced cell viability, and promoted mitophagy in TBHP-treated NP cells. MFN2 was identified as a downstream target of MANF, and MANF overexpression upregulated MFN2 expression in NP cells, whereas TBHP markedly suppressed MFN2 expression. Furthermore, knockdown of MFN2 partially reversed the effects of MANF overexpression on apoptosis, cell viability, and mitophagy in TBHP-treated NP cells. Collectively, these findings demonstrate that MANF overexpression enhances mitophagy by upregulating MFN2 expression, thereby mitigating oxidative stress-induced apoptosis in NP cells. These results provide novel insights into the pathogenesis of IDD.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-84167-9.

Introduction

Intervertebral disc degeneration (IDD) is a prevalent degenerative condition that represents the most common cause of low back pain and serves as a primary initiator of spinal degenerative diseases^1,2. IDD is characterized by a reduction in the extracellular matrix, a decrease in chondrocyte-like cells within the nucleus pulposus, disruption of the structural integrity of the intervertebral disc, and vascular proliferation^3,4. According to previous studies, cellular oxidative stress, mitochondrial dysfunction, and apoptosis have been demonstrated to play significant roles in the pathogenesis of IDD^5,6. In addition, the development of IDD is associated with age, physical injury, genetic susceptibility, microenvironmental changes, and inflammation⁷. IDD has a significant negative impact on human health and can lead to the loss of working ability in patients. Current primary treatments for IDD include medications and surgery, which can provide temporary relief of pain symptoms. However, these methods do not effectively control the progression of IDD⁸. Therefore, it is crucial to conduct in-depth studies on the mechanisms of IDD development and to develop new IDD-targeted therapies.

Mitophagy refers to the selective engulfment and degradation of damaged mitochondria via the autophagy mechanism, maintaining the stability of mitochondrial quantity and quality, and thus preserving normal cellular functions⁹. Damaged mitochondria not only lose their ability to produce ATP but also release higher levels of reactive oxygen species (ROS). If ROS are not promptly cleared and accumulate within cells, they can lead to apoptosis¹⁰. Mitochondrial dysfunction in the nucleus pulposus (NP) is considered a critical feature of IDD¹¹. With aging, damaged mitochondria progressively accumulate in the NP, potentially inducing oxidative stress and inflammatory responses, thereby accelerating the progression of IDD^12,13. Normal mitophagy can eliminate damaged mitochondria, reduce oxidative stress, and thereby protect NP cells from further damage. However, mitophagy is often dysfunctional in patients with IDD, leading to the accumulation of damaged mitochondria and promoting the progression of IDD. In recent years, multiple studies have elucidated the mechanisms related to mitophagy in IDD and have identified several targets involved in mitophagy dysregulation, including DJ-1, SIRT1, EGR1, and Parkin^14–17. In addition, Song et al.¹⁸ demonstrated that regulation of mitophagy can alleviate IDD, indicating the therapeutic potential of mitophagy in the treatment of IDD. However, the mechanisms of mitophagy in IDD have not been fully elucidated. Therefore, it is necessary to further investigate the role and mechanisms of mitophagy in the development of IDD.

In recent years, scientists have focused on the role of RNA binding proteins (RBPs) in IDD. RBPs primarily function by binding to RNA and can play diverse roles in transcription and post-transcriptional regulation, affecting mRNA production, modification, and stabilization¹⁹. Some studies have shown that RBPs play important roles in IDD. For example, Shao et al. demonstrated that RBP-HuR inhibits inflammation and extracellular matrix degradation in NP cells by stabilizing NKRF and inhibiting the NF-κB signaling pathway²⁰. In diabetic IDD, RBP-HuR inhibits the aging of nucleus pulposus (NP) cells through Atg7-mediated autophagy activation²¹. However, the functions of most RBPs in IDD remain unknown.

MANF is an endoplasmic reticulum (ER) stress response protein whose expression is induced by ER stress. MANF has been identified as a therapeutic target in various conditions, including immune checkpoint inhibitor-induced myocarditis, tubulointerstitial kidney disease, hepatic fibrosis, and colitis^22–25. MANF has been demonstrated to reduce inflammation, ER stress, and apoptosis by binding to neuroplastin in various cell types²⁶. Upregulation of MANF can promote autophagy conversion and protect pancreatic β-cells from ER stress²⁷. Moreover, MANF inhibits oxidative stress and reverses MPP⁺-induced loss of mitochondrial membrane potential, thereby improving mitochondrial function²⁸. Notably, oxidative stress can inhibit mitophagy. Induction of mitophagy can alleviate mitochondrial dysfunction under oxidative stress, reduce NP cell apoptosis and extracellular matrix degradation, thereby improving IDD²⁹. Notably, studies have found that MANF can stimulate mitophagy in renal tubules and cancer cells, which can protect renal function and promote tumor growth^23,30. However, the role of MANF in IDD remains unclear.

In this study, we investigated the mechanism by which MANF mediates IDD through mitophagy and identified its target. These results may provide novel insights for the treatment of IDD. This study establishes a novel theoretical foundation for the mechanisms underlying mitophagy-mediated IDD, potentially offering a new therapeutic target for the treatment of IDD.

Methods

Sample collection and ethical approval

The study protocol was approved by the Ethics Committee of The Sixth Affiliated Hospital of Xinjiang Medical University. Inform consents were obtained from all participants. Fasting venous blood samples were collected from patients with IDD (30 cases) and patients with spinal cord injury due to external forces (30 cases) and stored at -80 °C for subsequent use. The exclusion criteria of patients with IDD were as follows: (1) history of spinal trauma; (2) patients diagnosed with scoliosis or other spinal deformities during adolescence; (3) metabolic disorders; (4) spinal infections; (5) history of tumor. The exclusion criteria of patients with spinal cord injury were as follows: (1) tumor or immune disease; (2) spine malformation; (3) history of spinal disease.

Bioinformatic analysis

The GSE167199 dataset was obtained from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) to analyze differentially expressed genes (DEGs) in patients with IDD and patients with spinal cord injury. Data were analyzed using GEO2R. The differentially expressed genes (DEGs) were defined as P < 0.05 and |log (fold change)| > 2. The enrichment analysis of DEGs was performed by Gene Ontology (GO).

Cell culture and treatment

Human intervertebral disc NP cells were provided by Procell (Cat. No. CP-H097, Wuhan, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 15% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin at 37 °C in a 5% CO₂ incubator. The cells used in this study were at passage three. During the cell culture process, we characterized the NP cells to confirm their purity (positive rate of approximately 95%), as shown in Figure S1. To establish an oxidative stress-induced model, NP cells were treated with 100 µmol/L tert-butyl hydroperoxide (TBHP; Sigma-Aldrich, St. Louis, MO, USA) for 4 h.

Cell transfection

Cells in the logarithmic growth phase were inoculated in six-well plates at a density of 2 × 10⁵ cells/well for cell transfection. Short hairpin RNA targeting MFN2 (shMFN2), shRNA negative control (shNC), MANF overexpressing plasmid (pcDNA3.1-MANF), and empty vector (pcDNA3.1) (GenePharma, Shanghai, China) were transfected into NP cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The transfected cells were harvested 48 h post-transfection.

Quantitative real-time PCR (qPCR)

Total RNA was isolated from NP cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA templates were synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). qPCR was performed on the QuantStudio™ 6 Flex system (Thermo Scientific) using SYBR Green Master Mix (Thermo Scientific). Relative mRNA expression levels were calculated using the 2^−ΔΔCt method and normalized to GAPDH. The primers for qPCR were as follows: MANF, 5’-TTTACCAGGACCTCAAAGACAGA-3’ (sense) and 5’-TTGCTTCCCGGCAGAACTTTA-3’ (antisense); MFN2, 5’-CTCTCGATGCAACTCTATCGTC-3’ (sense) and 5’-TCCTGTACGTGTCTTCAAGGAA-3’ (antisense).

Western blot

Total protein was isolated from NP cells using RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) and quantified using a BCA kit (Beyotime, Shanghai, China). Protein samples were loaded onto 10% SDS-PAGE gels for electrophoretic separation and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-MANF (1:1000, ab316935, Abcam, Cambridge, UK), anti-MFN2 (1:1000, ab124773, Abcam), anti-LC3-II/I (1:1000, ab62721, Abcam), anti-P62 (1:10000, ab109012, Abcam), anti-Beclin-1 (1:2000, ab207612, Abcam), anti-PINK1 (1:1000, ab216144, Abcam), anti-Parkin (1:1000, ab73015, Abcam), and anti-GAPDH (1:10000, ab181602, Abcam). The next day, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1:10000, ab205718, Abcam) for 2 h at room temperature. Finally, the membranes were washed with 1× TBST and visualized using the ECL reagent (Thermo Scientific).

Cell viability

Cell viability of NP cells was measured using a Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China). Cells were seeded in a 96-well plate at a density of 100 µL/well and incubated with 10 µL of CCK-8 solution for 1 h. The absorbance was measured using a microplate reader at 450 nm.

Detection of apoptosis

Apoptosis of NP cells was evaluated using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China). Cells were resuspended in Annexin V-FITC binding buffer and mixed with Annexin V-FITC. They were then stained with propidium iodide staining solution for 20 min in the dark. Finally, apoptosis was assessed by flow cytometry using a CytoFLEX instrument (Beckman Coulter, Brea, CA, USA).

RNA sequencing (RNA-seq)

RNA-seq was performed to identify DEGs in NP cells transfected with MANF overexpression plasmids or empty vector, conducted by Novogene (Beijing, China). DEGs were defined as those with a p-value < 0.05 and an absolute log2(fold change) > 2. Upregulated DEGs were used for Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (www.kegg.jp/kegg/kegg1.html) to predict enriched pathways. A p-value < 0.05 was considered the threshold for enrichment.

RNA-binding protein immunoprecipitation (RIP) assay

The interaction between MANF and MFN2 was identified using a RIP assay with the Imprint RIP Kit (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed in 100% RIP lysis buffer containing protease and RNase inhibitors. The lysate was then incubated with magnetic protein A/G beads coated with anti-MFN2 antibody or anti-IgG antibody overnight at 4 °C. After incubation, the beads were purified, and MFN2 was isolated using Trizol reagent. The expression of MFN2 was measured by qPCR.

Dual luciferase reports

To determine the interaction between MFN2 and MANF, wild-type (wt)-MFN2 and mutant (mut)-MFN2 luciferase reporter plasmids were constructed. NP cells were co-transfected with these plasmids along with pcDNA3.1 or pcDNA3.1-MANF using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) for 24 h. After transfection, luciferase activity was measured using a Dual-Luciferase Reporter Assay System (Promega, San Luis Obispo, CA, USA). The intensity of the firefly luciferase signal was normalized to the Renilla luciferase signal intensity.

Statistical analysis

All data were analyzed and processed using SPSS 22.0 software. Results are expressed as the mean ± standard deviation of at least three independent replicates. Comparisons between two groups were performed using Student’s t-test, while comparisons among three or more groups were analyzed using one-way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.

Results

MANF expression is downregulated in patients with IDD

To investigate the mechanism of IDD, we identified DEGs in patients with IDD and patients with spinal cord injury using microarray analysis. As shown in Fig. 1A, we identified 831 downregulated genes and 899 upregulated genes. Moreover, a heatmap indicated that IDD samples and control samples were well separated (Fig. 1B). GO analysis revealed that the upregulated and downregulated genes were enriched in distinct biological pathways (Fig. 1C). Subsequently, we analyzed the differentially expressed RBPs in IDD and found that MANF expression was significantly decreased in patients with IDD (Fig. 1D and E). Additionally, we detected MANF expression in clinical samples, and qPCR results confirmed that MANF expression was significantly downregulated in the blood of patients with IDD (Fig. 1F).

Fig. 1.

MANF expression was downregulated in patients with IDD. The DEGs were presented in (A) a volcano map and (B) a heatmap. (C) GO pathway analysis of genes significantly upregulated and downregulated in IDD (top 10). (D) Co-expression network of MANF and variable splicing genes in IDD. (E) MANF expression was significantly downregulated in IDD. ***P < 0.001 vs. the control group. (F) The expression of MANF in blood of patients with IDD and patients with spinal cord injury due to external forces was measured by qPCR.

MANF expression is decreased in TBHP-induced NP cells

To explore the role of MANF in IDD, NP cells were treated with TBHP to simulate the IDD condition in vitro. Next, we measured the expression of MANF in NP cells. Compared with the control group, MANF expression was significantly decreased in TBHP-induced NP cells (Fig. 2A). Moreover, we detected the protein levels of MANF in NP cells and found that they were downregulated by TBHP treatment (Fig. 2B).

Fig. 2.

MANF expression was decreased in TBHP-induced NP cells. (A) The expression of MANF in NP cells was measured by qPCR. (B) The protein levels of MANF in NP cells were detected by western blot. ***P < 0.001 vs. the control group.

MANF overexpression inhibits apoptosis through mitophagy in TBHP-treated NP cells

Next, we investigated the mechanism of MANF in TBHP-treated NP cells. MANF overexpressing plasmids were transfected into NP cells, resulting in a significant increase in MANF expression (Fig. 3A). Additionally, the protein level of MANF was also upregulated by MANF overexpression (Fig. 3B). We then measured the cell viability of NP cells in different groups. The results indicated that TBHP treatment significantly inhibited the viability of NP cells, which was partially reversed by MANF overexpression (Fig. 3C). PI staining showed that apoptosis of NP cells was markedly promoted by TBHP treatment, and this effect was restored by MANF overexpression (Fig. 3D and E). Finally, we measured the protein levels of several mitophagy-related proteins. The results showed that TBHP downregulated the protein levels of Beclin-1, PINK1, and Parkin, and decreased the ratio of LC3-II/I, while upregulating the protein level of P62. These effects were partially reversed by MANF overexpression (Fig. 3F). These findings confirm that TBHP inhibits mitophagy in NP cells, and this inhibition is restored by MANF overexpression.

Fig. 3.

MANF overexpression inhibited apoptosis through mitophagy in TBHP-treated NP cells. (A) The expression of MANF in NP cells was measured by qPCR. (B) The protein levels of MANF in NP cells were detected by western blot. (C) The cell viability of NP cells in different groups was measured using a CCK8 kit. (D and E) Apoptosis of NP cells in different groups was assessed by flow cytometry. (F) The protein levels of mitophagy-related proteins in NP cells in different groups were detected by western blot. **P < 0.01 and ***P < 0.001 vs. the control group. ##P < 0.01 and ###P < 0.001 vs. the TBHP group.

MANF overexpression promotes MFN2 expression in NP cells

Subsequently, we identified DEGs in TBHP-treated NP cells with or without MANF overexpression. The top 20 upregulated and downregulated genes are shown in Fig. 4A. KEGG pathway enrichment analysis was performed on the upregulated genes, and we found that they were enriched in the mitophagy-related pathway (Fig. 4B). MFN2 has been demonstrated to regulate IDD progression through mitophagy³¹. Therefore, we measured the expression of MFN2 in NP cells with MANF overexpression. Results showed that MANF overexpression increased both the mRNA expression and protein levels of MFN2 (Fig. 4C and D). RIP assays revealed the interaction between MANF and MFN2 (Fig. 4E). Additionally, dual-luciferase reporter assays suggested that MANF overexpression only increased the luciferase activity of the MFN2-wt construct, but did not affect the luciferase activity of the MFN2-mut construct (Fig. 4F). We also assessed the effect of TBHP on MFN2 expression in NP cells. Results showed that both the mRNA expression and protein levels of MFN2 were suppressed in TBHP-treated NP cells, indicating that oxidative stress may inhibit MFN2 expression (Fig. 4G and H).

Fig. 4.

MANF overexpression promoted MFN2 expression in NP cells. (A) The DEGs in TBHP-treated NP cells with or without MANF overexpression. (B) Bubble diagram of KEGG pathway enrichment analysis. (C) The expression of MFN2 in NP cells was measured by qPCR. (D) The protein level of MFN2 in NP cells were detected by western blot. (E) The interaction between MFN2 and MANF was assessed by RIP. (F) Dual luciferase reports were used to evaluate the luciferase activity of MFN2-wt and MFN2-mut in NP cells transfected with pcDNA3.1 and pcDNA3.1-MANF. (G) MFN2 expression in NP cells was measured by qPCR. (H) The protein levels of MFN2 in NP cells were detected by western blot. **P < 0.01 and ***P < 0.001 vs. the control group or the IgG group.

MFN2 knockdown promotes apoptosis in TBHP-treated NP cells with MANF overexpression through suppressing mitophagy

Finally, the role of MFN2 was confirmed through rescue experiments. NP cells were transfected with shMFN2 to inhibit MFN2 expression (Fig. 5A). The protein level of MFN2 was significantly downregulated by MFN2 knockdown (Fig. 5B). We found that the increase in cell viability induced by MANF overexpression in TBHP-treated cells was significantly inhibited by MFN2 knockdown (Fig. 5C). Moreover, MFN2 knockdown markedly restored the apoptosis of TBHP-treated NP cells that was suppressed by MANF overexpression (Fig. 5D and E). Additionally, the upregulated protein levels of Beclin-1, PINK1, and Parkin, as well as the increased ratio of LC3-II/I and the downregulated protein level of P62 in TBHP-treated NP cells with MANF overexpression, were partially reversed by MFN2 knockdown (Fig. 5F).

Fig. 5.

MFN2 knockdown promoted TBHP-treated NP cells with MANF overexpression apoptosis through suppressing mitophagy. (A) The expression of MFN2 in NP cells was measured by qPCR. (B) The protein levels of MFN2 in NP cells were detected by western blot. (C) The cell viability of NP cells in different groups was measured using a CCK8 kit. (D and E) Apoptosis of NP cells in different groups was assessed by PI staining. (F) The protein levels of mitophagy-related proteins in NP cells in different groups were detected by western blot. ***P < 0.001 vs. the control group. #P < 0.05 and ##P < 0.01 vs. the MANF overexpression group.

Discussion

This study has demonstrated that MANF expression is downregulated in patients with IDD and in TBHP-induced NP cells. Overexpression of MANF promoted mitophagy in NP cells, which was suppressed by TBHP treatment. Furthermore, MFN2 was identified as a target of MANF, and knockdown of MANF inhibited mitophagy in TBHP-treated NP cells, even when MANF was overexpressed.

There is evidence that dysfunction of NP cells may be one of the main causes of the development and progression of IDD³². Interestingly, mitophagy dysfunction promotes the development of IDD through the impairment of NP cells. Damage to mitophagy leads to the incremental accumulation of defective mitochondria, resulting in increased apoptosis and ECM degradation in NP cells, thereby contributing to the progression of IDD⁶. Additionally, growing evidence suggests that mitophagy is involved in the progression of IDD. Lin et al.¹⁷ found that LRRK2 has been identified as a target in IDD. Knockdown of LRRK2 significantly suppressed oxidative stress-induced mitochondria-dependent apoptosis in NP cells and promoted mitophagy. Moreover, Hu et al.³³ demonstrated that Optineurin relieves senescence and matrix degeneration in NP cells caused by oxidative stress by promoting mitophagy to scavenge damaged mitochondria and excess reactive oxygen species, thereby slowing the progression of IDD. Recently, Lin et al.¹⁴ identified a novel target of IDD, DJ-1, and demonstrated that DJ-1 promotes HK2-mediated mitophagy under oxidative stress conditions to inhibit mitochondria-dependent apoptosis in NP cells. In our study, we demonstrated that TBHP treatment promoted apoptosis and suppressed cell viability through the inhibition of mitophagy, suggesting that oxidative stress suppresses mitophagy in NP cells.

Moreover, we demonstrated that MANF expression is downregulated in IDD. MANF has been shown to be involved in the progression of several degenerative diseases. For example, Chen et al.³⁴ confirmed that MANF expression is increased in Aβ1-42-treated neuronal cells, and MANF overexpression partially protected against Aβ1-42-induced neuronal cell death. These findings suggest that MANF may exert neuroprotective effects against Aβ-induced neurotoxicity, making it a potential therapeutic candidate for Alzheimer’s disease. Hao et al.³⁵ demonstrated that MANF supplementation in the striatum of a rat Parkinson’s disease model produces long-term neuroprotective and neuroregenerative effects in DA neurons and improves behavioral outcomes. Moreover, a study has demonstrated that MANF can mediate mitophagy in autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD). Specifically, renal tubular upregulation of MANF promotes mitophagy and improves mitochondrial biogenesis in the ADTKD-UMOD mouse model²³. However, whether MANF mediates mitophagy in IDD remains uncertain. In the present study, NP cells were treated with TBHP to simulate an IDD model of oxidative stress in vitro. Our results demonstrated that MANF overexpression enhanced cell viability and mitophagy while inhibiting apoptosis in TBHP-treated NP cells. These findings indicate that MANF overexpression inhibits apoptosis through enhanced mitophagy in an in vitro oxidative stress cell model.

Additionally, MFN2 was identified as a target of MANF in NP cells. MFN2 was initially described for its role in mitochondrial fusion, and its dysregulation has been associated with a variety of diseases, including cardiovascular disease and cancer^36,37. Recently, MFN2 has been shown to be involved in a variety of diseases by mediating mitophagy. For example, Yang et al.³⁸ revealed that morinda officinalis oligosaccharides have been shown to upregulate MFN2 expression in astrocytes, thereby activating mitophagy and resisting mitochondrial damage. This mechanism helps alleviate depression-like behaviors in hypertensive rats. Glytsou et al.³⁹ demonstrated that MFN2 expression is positively correlated with drug resistance to BH3 mimetics in patients with AML. MFN2 overexpression promotes drug resistance by eliminating mitochondrial damage through increased mitophagy. Notably, Chen et al.³¹ first reported that MFN2 is low expressed in IDD, and that overexpression of MFN2 ameliorates the development of IDD in rats through promoting mitophagy. However, whether MFN2 is mediated by MANF in IDD has not been previously reported. In this study, we identified that MFN2 interacts with MANF, and MANF overexpression increased MFN2 expression in NP cells. Moreover, MFN2 expression was decreased by TBHP treatment in NP cells, and MFN2 knockdown suppressed mitophagy in TBHP-treated NP cells, even when MANF was overexpressed.

In conclusion, these results confirm that MANF overexpression ameliorates TBHP-induced apoptosis of NP cells by inducing mitophagy through the promotion of MFN2 expression. This study explores the pathogenesis of IDD through in vitro experiments, which may provide a new therapeutic target for the treatment of IDD.

Electronic supplementary material

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Author contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. L M drafted the work and revised it critically for important intellectual content; X M, T A, Y L and J G were responsible for the acquisition, analysis and interpretation of data for the work; W S made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

Funding

The work was supported by Scientific Research Special Fund of The Sixth Clinical Medical School of Xinjiang Medical University (Sixth Affiliated Hospital)-Special Fund for Studying Doctoral Students under grant number LFYKYZX2023-18.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

This study was approved by the Ethics Committee of The Sixth Affiliated Hospital of Xinjiang Medical University. Informed consent was obtained from all individual participants included in the study. This study was performed in line with the principles of the Declaration of Helsinki. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.