miR-146a-5p induces intervertebral disc degeneration by targeting SOD2

Abstract

This study aimed to investigate the role and underlying mechanism of miR-146a-5p in inflammationinduced intervertebral disc degeneration (IDD).

We performed miRNA sequencing and bioinformatic analysis on IL-1β-treated rat primary nucleus pulposus cells (RPNPCs). The function of miR-146a-5p was validated using mimics and inhibitors in vitro, and its therapeutic effect was assessed in a needle-punctured rat disc organotypic explant model. Target gene prediction and validation, along with loss- and gain-of-function experiments for Sod2, were conducted.

miR-146a-5p was significantly upregulated in degenerative human discs and IL-1β-treated RPNPCs. Its overexpression promoted extracellular matrix catabolism and inhibited anabolism, and induced apoptosis in RPNPCs, while its inhibition exerted protective effects. Mechanistically, miR-146a-5p directly targeted and downregulated superoxide dismutase 2 (SOD2). Knockdown of Sod2 exacerbated degenerative phenotypes, whereas its overexpression mitigated IL-1β-induced damage. In the explant model, inhibition of miR-146a-5p restored Sod2 expression and ameliorated disc degeneration.

miR-146a-5p drives inflammation-induced IDD by suppressing Sod2, leading to mitochondrial oxidative stress and NP cell dysfunction. Targeting the miR-146a-5p/Sod2 axis represents a novel potential therapeutic strategy for IDD.

Graphical abstract

Keywords

IL-1β / intervertebral disc degeneration / miR-146a-5p / miRNA / Sod2

1 Introduction

Low back pain is a common musculoskeletal disease and a leading cause of disability, which occurs in almost everyone during their lifetime.^[1–^4] To date, numerous studies have confirmed a causative link between low back pain and intervertebral disc degeneration (IDD).^[5–^7] With population aging, the incidence and disability burden attributable to IDD are rising dramatically, severely impacting patients' quality of life and work capacity.^[8,^9] The intervertebral disc (IVD) consists of a central nucleus pulposus (NP), the peripheral annulus fibrosus (AF), and the covering cartilage endplates. As the central part of IVDs, NP tissues function as a cushioning pad between adjacent vertebrates, maintaining the stability and flexibility of the spine at the same time.^[10,^11] The NP tissue, which secretes proteoglycans and type Ⅱ collagen, is crucial for regulating the extracellular matrix (ECM) abundance and, consequently, the water content and disc height. The degeneration of the NP is considered a significant pathological progression in IDD.^[11,^12] To date, various factors have been reported to engage in the occurrence and development of IDD, including genetics, aging, and even daily actions, such as smoking, excessive loading, and even circadian disturbance. Although the precise pathogenesis of IDD remains elusive, inflammation is generally thought to be the most common cause of IDD.^[12–^14] Moreover, as the degeneration of IVDs aggravates, NP cells secrete more inflammatory mediators, such as interleukin (IL)-1 and tumor necrosis factor alpha (TNF-α), resulting in the secretion of matrix-degrading enzymes and thus leading to the degradation of ECM, and finally contributing to the progression of IDD.^[15–^17] However, the mechanism of inflammation-induced IDD has not been fully elucidated.

miRNA is a class of endogenous noncoding RNA, which widely exists in eukaryotic cells and plays an important role in cell differentiation, proliferation, and survival. miRNA regulates the transcription and translation of mRNA by targeting its 3′-untranslated region (3′-UTR), thus resulting in mRNA translational inhibition or degradation.^[18] Previous studies have confirmed that miRNAs are involved in the pathological processes of IDD, such as miR-141, miR-338-3p, miR-22-3p and hsa-let-7f-1-3p, which offer some potential therapeutic targets.^[19–^23] In addition, some essential functional regulatory genes, such as Sirt1, Sirt6, and Bmal1, have also been proved as the inhibitory targets of these miRNAs.^[20,²¹,²³–^25] However, it is still unclear which miRNA plays the most important role in the process of inflammation-induced IDD and which gene is subsequently inhibited by this miRNA. In this study, a miRNA sequencing strategy was applied to identify differentially expressed miRNAs in rat primary nucleus pulposus cells (RPNPC) treated with IL-1 or not, and we found that the expression of miR-146a-5p was significantly increased in IL-1β-treated RPNPC. Mechanically, Sod2, a gene encoding superoxide dismutase (SOD2), a matrix enzyme expressed in mitochondria and known as an important free radical scavenger, was the target gene of miR-146a-5p. Overexpression of miR-146a-5p by mimics decreased the expression of SOD2 and increased the inflammatory response, oxidative stress, and apoptosis of RPNPC, while inhibition of miR-146a-5p by its inhibitor significantly ameliorated IL-1β-induced IDD phenotypes. This study provides a new understanding of the role of miRNAs in inflammation-induced IDD and puts forward a potential therapeutic target for the prevention and treatment of IDD.

2 Materials and methods

2.1 Patient samples

NP specimens were obtained from 12 patients (5 males and 7 females; mean age = 49.9 ± 14.4 years) with degenerative disc disease or scoliosis. The degree of IDD was assessed by 3 other blinded orthopedic researchers according to the modified Pfirrmann grading system by magnetic resonance imaging. Grade Ⅱ (n = 3) and Ⅲ (n = 3) samples were combined into a moderate group (mean age = 44.7 ± 15.2), while Grade Ⅳ (n = 3) and Ⅴ (n = 3) samples were combined into a severe group (mean age = 55.2 ± 12.6 years). All the information of these samples is shown in Table S1, Supplemental Digital Content, https://links.lww.com/SPRES/A3. Ethics approval was obtained from the Institutional Review Board of Xijing Hospital of Fourth Military Medical University (KY20203146-1), and informed consent was obtained from each donor. The work presented in this article was performed according to The Code of Ethics of the World Medical Association (Declaration of Helsinki).

2.2 Isolation, culture, and in vitro IDD model of rat primary NP cells

In this study, RPNPC were isolated from male Sprague-Dawley (SD) rats (8 weeks). Rats were euthanized by inhaling excessive isoflurane. Under aseptic conditions, the rat tail was taken, the tail vein was separated, and the gel-like NP tissue was isolated from the AF under a microscope. For the isolation of RPNPC, the obtained NP tissue was digested in 0.4% pronase (10165921001, Roche Diagnostics, Germany) and 0.0125% collagenase P for 30 minutes (11213865001, Roche Diagnostics, Germany), and then the digested tissue was washed 3 times with phosphate buffer through a cell filter with a pore diameter of 100 μm. The isolated cells were stored in DF12 medium containing 10% fetal bovine serum (10099141C, Gibco), supplemented with 1% penicillin-streptomycin combination (15070063, Gibco), and cultured in 5% carbon dioxide incubator. The primary cells used in this study are the second generation. In vitro inflammation model, IL-1β (10 ng/24 h) was used to treat the second generation of primary cells.^[15,²⁶,^27]

2.3 miRNA sequencing

GENE DENOVO tested the quality and amount of miRNA and constructed and sequenced the miRNA library. Total RNA was extracted from the samples by the TRIzol method, and the bands in the range of 18–30 nt were selected by polyacrylamide gel electrophoresis (PAGE), and small RNA was recovered. The 3' and 5' connectors were connected, respectively, and then the small RNA connected to the 2 connectors was reverse transcribed and polymerase chain reaction (PCR) amplified. Finally, the PAGE gel was used to recover and purify the 140 bp bands, dissolve in EB solution, and complete the library construction. The constructed library uses Agilent2100 and ABI Step One Plus Real-Time PCR System (Life Technologies) to detect the quality and yield, and is sequenced on the computer. The information analysis part is mainly divided into 3 modules. First of all, the original sequencing data of each sample are processed to get the tag sequence of the small RNA. Secondly, the tag was annotated, the tag sequence composition of the small RNA in the sample was identified, and the miRNA was identified. Finally, the miRNA expression profile was obtained, and miRNA gene difference analysis, target gene prediction, and target gene enrichment analysis were obtained.

2.4 Organotypic tissue-explant of IVD

Under the approval of the Animal Experiment Administration Committee of the Air Force Military Medical University, tails were collected from 10.8-week SD rats under pathogen-free conditions, after they were euthanized. The muscles and tendons were dissected and removed by a scalpel and surgical scissors under aseptic conditions. Explants consist of the IVD and 2 adjacent vertebrae. The explants were randomly divided into 4 groups: uninjured group (Control), injured group (Needle-punctured), injured and mimics-treated group (Needle-punctured + mimics), and injured and inhibitor-treated group (Needle-punctured + inhibitor). AF was stabbed with a 20 G needle to enter the center of the NP, rotates 360 degrees, and stays for 1 minute. The explants were cultured in DF12 containing 10% fetal bovine serum (#10099141C, Gibco), supplemented with 1% penicillin-streptomycin combination (#15070063, Gibco) at 37 ℃ in a humidified atmosphere of 5% CO₂. The medium was replaced with fresh medium every other day. After 14 days, the explants of all groups were collected. No explant was excluded from the analysis.^[15]

2.5 In vitro siRNA, miRNA, and adenovirus transfection

Mimics and inhibitors targeting rat miR-146a-5p were designed and synthesized by Tsingke (Beijing, China) in vitro. The mimics and inhibitor sequences used in this study are listed in Table 2, Supplemental Digital Content, https://links.lww.com/SPRES/A3. The siRNA double strand for rat Sod2 was designed and synthesized by Hanbio (China). The siRNA sequences used in this study are listed in Table 2, Supplemental Digital Content, https://links.lww.com/SPRES/A3. According to the manufacturer's instructions, RNAfit reagent (Hanbio, China) was used for siRNA transfection. To put it simply, the RPNPC of the second passage were seeded in a 6-well plate. After adhesion, the cells were incubated with Optic-MEM medium (# 31985062 Thermo, USA) at 37 ℃ for 2 hours. Then, 100 nM siRNA or NC siRNA was transfected with RNAfit and incubated at 37 ℃ for 6 hours. Finally, the cells were washed with phosphate-buffered saline (PBS) and replaced with complete medium. After 24 hours, the cells were treated accordingly. For the overexpression of Sod2, an adenovirus vector overexpressing rat Sod2 was manufactured by Hanbio, and the transfection was performed according to the manufacturer's instructions.

2.6 Protein extraction and western blotting analysis

RPNPC were washed with PBS (Gibco) twice. Then the cells were lysed in radioimmune precipitation assay buffer (Beyotime, China) with a complete protease inhibitor cocktail (Roche, Germany). The total protein of RPNPC was collected by centrifuging at 12,500 rpm for 15 minutes at 4 ℃. The concentrations of protein were measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The total proteins were diluted by loading buffer, heated at 95 ℃ for 5 minutes, and then certain amounts of protein were separated in 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes (Millipore, Germany). Then the nitrocellulose membranes were blocked by 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 40 minutes at room temperature. These membranes were incubated overnight at 4 ℃ with primary antibodies, including the following: anti-SOD2 (1:1000), anti-Aggrecan (1:1000), anti-matrix metalloproteinase 3 (MMP3) (1:1000), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000). Then the membranes were incubated with the horseradish peroxidase-linked goat antirabbit IgG or horse antimouse IgG secondary antibody for 1 hour at room temperature. The nitrocellulose membranes were washed 3 times with tris-buffered saline containing 0.1% Tween 20 for 15 minutes after each step. Finally, the nitrocellulose membranes were visualized by Immobilon Western Chemiluminescent horseradish peroxidase Substrate (Millipore Corporation, Germany, #WBKLS0100), and the density of nitrocellulose membranes was quantified by Image J software (National Institutes of Health, Bethesda, MD, USA).^[15,^26] The detailed information of antibodies used in this study is listed in Table 3, Supplemental Digital Content, https://links.lww.com/SPRES/A3.

2.7 RNA extraction and quantitative reverse transcription-polymerase chain reaction analysis

Total RNA of RPNPC, human NP tissues, or rat coccygeal NP tissues was harvested using Mini BEST Universal RNA Extraction Kit (TaKaRa, China) according to the manufacturer's instructions. Reverse transcription was performed with Prime Script RT Master Mix (TaKaRa, China). Synthesized cDNA was then subjected to qPCR analysis using TB Green Premix Ex Taq II (TaKaRa, China). The reactions were performed with CFX96 (Bio-Rad, USA). Gene expression levels were reported as relative fold change, with Gapdh (Acan, Mmp3, Sod2) and U6 (miR-146a-5p) as an internal control. Primers sequences used in this study are shown in Table 4, Supplemental Digital Content, https://links.lww.com/SPRES/A3.

2.8 Apoptosis assay

For in vitro cell experiments, RPNPC were harvested in 0.25% trypsin and washed 3 times with cold PBS. The apoptosis of RPNPC was evaluated by Annexin V-PE/propidium iodide (PI) apoptosis detection kit (BD Biosciences, USA) by flow cytometry. For disc sections, terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed using In Situ Cell Death Detection Kit (#11684795910, Roche Diagnostics, Germany) according to manufacturer's instructions.^[26,^28]

2.9 In vitro immunofluorescence staining

RPNPC were fixed in freshly prepared 4% paraformaldehyde for 30 minutes, permeabilized by 0.1% Triton X-100 (Beyotime, China) for 30 minutes. Then the cells were blocked by 1% BSA in PBS for 1 hour, incubated with primary antibody at 4 ℃ overnight, with appropriate secondary antibodies at room temperature (RT) for 2 hours, and with 4', 6-diamidino-2-phenylindole (C1006, Beyotime, China) at RT for 10 minutes. Finally, the cells were analyzed under a fluorescence microscope (BX53, OLYMPUS, Japan).^[29,^30] The antibodies used for in vitro immunofluorescence (IF) staining are listed in Table 3, Supplemental Digital Content, https://links.lww.com/SPRES/A3.

2.10 Histology and IF staining for disc sections

Explants were fixed in 4% freshly prepared paraformaldehyde for 48 hours, decalcified for 8 weeks with 10% ethylenediaminetetraacetic acid at RT under gentle shaking, then dehydrated, paraffin-embedded, and sectioned at 5 µm. The sections were deparaffinized by xylene and rehydrated by ethanol. The hematoxylin-eosin (HE) staining kit (Solarbio, China) or safranin O-fast green staining kit (Solarbio, China) was used according to the manufactures' instructions, and the sections were graded by a previously published method. For the IF staining, citrate buffer (0.1 mol/L, pH 6.0) was used to perform antigen-retrieval on deparaffinized and dehydrated sections. After blocking in 10% normal goat serum (Solarbio, China) at RT for 1 hour, the sections were then incubated with primary antibody at 4 ℃ overnight, with appropriate secondary antibodies at RT for 2 hours and with 4', 6-diamidino-2-phenylindole (C1006, Beyotime, China) for 10 minutes. Finally, the sections were analyzed under a fluorescence microscope (BX53, OLYMPUS, Japan). Fluorescence intensity was quantified using ImageJ (National Institutes of Health, USA) software.^[30] The antibodies used for sections' IF staining in the study are listed in Table 3, Supplemental Digital Content, https://links.lww.com/SPRES/A3.

2.11 Statistical analysis

Data are presented as mean ± SD in all experiments. Whether the data present a normal distribution was tested by the Shapiro–Wilk test or D'Agostino test. The differences between 2 or multiple groups were analyzed by Student t test or one-way analysis of variance, followed by Tukey multiple-comparison post hoc test, respectively. All statistical analyses were performed with SPSS 22.0 and GraphPad Prism 9.0 software. Differences were considered statistically significant at p value < 0.05.

3 Results

3.1 The expression of miR-146a-5p was significantly upregulated during IDD

Given the central role of inflammation in IDD pathogenesis and the established regulatory functions of miRNAs in IVD homeostasis, we first comprehensively profiled miRNA expression in RPNPCs under inflammatory conditions. The RPNPC were subjected to IL-1β (10 ng/mL) for 24 hours to simulate an inflammatory microenvironment and then harvested, and the miRNA sequencing was carried out by GENE DENOVO. Compared with untreated controls, the IL-1β-treated group exhibited 297 upregulated and 198 downregulated miRNAs, and some significantly expressed candidate miRNAs were shown in the heat map (Fig. 1A). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed based on these differentially expressed miRNAs. The biological process and molecular function of all differentially expressed miRNAs, whether upregulated miRNAs or downregulated miRNAs, focused on the metabolic process and catalytic activity, indicating a coherent function of these miRNAs (Fig. 1B–E). Among these differentially expressed miRNAs, miR-146a-5p demonstrated the most consistent and pronounced upregulation and was therefore selected for further investigation (Fig. 1F, G). These findings identified miR-146a-5p as a potentially key disease-associated miRNA in IDD.

3.2 Overexpression of miR-146a-5p led to development of degenerative phenotype and apoptosis of RPNPC in vitro, and miR-146a-5p inhibition showed a therapeutic effect against treatment of IL-1β

To ascertain the role of miR-146a-5p during inflammation-induced IDD, overexpression of mimics or inhibition of miR-146a-5p was performed in cultured RPNPCs with or without IL-1β treatment. The aggrecan (ACAN) and catabolism (MMP3) of ECM were evaluated by immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The increased expression of MMP13 and reduced expression of Aggrecan were observed in the IL-1β-treated group, indicating a successful establishment of inflammation-induced IDD model. Similarly, the degradation of ECM was also seen in the miR-146a-5p mimics-treated group, indicating a deleterious effect of miR-146a-5p. On the contrary, the inhibitor of miR-146a-5p slightly promoted the anabolism and inhibited the catabolism of ECM, indicating a protective effect (Fig. 2A–M). Given the established link between IDD progression and NP cell apoptosis. We also evaluated the apoptosis of RPNPC in these groups by Annexin V-PE/PI detection. The results of flow cytometry showed that both IL-1β treatment and miR-146a-5p mimics significantly promoted the apoptosis of RPNPC, and the miR-146a-5p inhibitors partially rescued the apoptosis of RPNPC induced by IL-1β treatment (Fig. 2N, O). Collectively, these results demonstrated that miR-146a-5p critically drives IDD pathogenesis, and its inhibition mitigated inflammation-induced degenerative phenotypes in NP cells.

3.3 miR-146a-5p promoted the development of IDD by targeting Sod2

miRNAs primarily exert their biological functions by inhibiting target gene transcripts. To investigate miR-146a-5p's role in IDD pathogenesis, bioinformatic analysis was performed to predict the downstream target genes of miR-146a-5p. To this end, public databases like RNAhybrid, miRanda, and TargetScan were included in our analysis. GO and KEGG enrichment analysis was performed based on downregulated genes caused by miR-146a-5p. The bubble diagram of KEGG enrichment analysis showed that most of the miR-146a-5p target genes concentrate upon the metabolic pathways, proteoglycan synthesis, cholesterol and glycerolipid metabolism, oxidative phosphorylation, and other metabolic pathways. And the bar diagram of GO enrichment analysis showed metabolism process, biological regulation, and response to stimulus as the top catalogues of biological process (Fig. 3A, B). Coincident portion of the Venn analysis identified Sod2 as a consensus target (Fig. 3C). 3′-UTR sequence of Sod2 also showed a binding site of miR-146a-5p according to the public databases (Fig. 3D). In addition, increased miR-146a-5p expression as well as decreased Sod2 expression were confirmed in human pfirrmann Ⅳ-Ⅴ IVDs compared with the pfirrmann Ⅱ-Ⅲ IVDs, further confirming the involvement of miR-146a-5p and its target gene Sod2 in the development of IDD (Fig. 3E, F). To verify the effect of miR-146a-5p on Sod2, overexpression of mimics or inhibition of miR-146a-5p was performed in cultured RPNPCs with or without IL-1β treatment, and the expression level of Sod2 was evaluated by qRT-PCR, western blot analysis, and immunofluorescence staining. Both mimics treatment and IL-1β treatment significantly inhibited the expression of Sod2, while the inhibitor of miR-146a-5p partially recovered the decreased expression of Sod2 under the inflammatory environment (Fig. 3G–N). Collectively, these results established Sod2 as a direct functional target of miR-146a-5p in IDD.

3.4 Loss of Sod2 led to a dysfunction of RPNPC, and overexpression of Sod2 protected RPNPC against Inflammatory attack

Building on the finding that miR-146a-5p directly targets Sod2, we next investigated whether Sod2 exerts protective effects against IDD pathogenesis. Loss and gain of function of Sod2 was achieved by the delivery of siRNA targeting Sod2 or adenovirus overexpressing Sod2, respectively. The knockdown efficiency was validated by western blot analysis, and siRNA1 showed the highest efficiency (Fig. 4A, B). The immunofluorescence staining results showed a decreased ACAN and increased catabolism (MMP3) of ECM after the treatment of siSod2 (Fig. 4C–E). Moreover, loss of Sod2 further exacerbated the degenerative phenotype of the RPNPC under the treatment of IL-1β, and overexpression of Sod2 showed a robust rescuing effect against IL-1β-induced degeneration, which was confirmed by immunofluorescence staining, western blot, and apoptosis assay (Fig. 4F–O). These results demonstrate that Sod2 deficiency accelerated IDD progression, whereas its overexpression conferred therapeutic protection against disc degeneration.

3.5 Inhibition of miR-146a-5p recovered Sod2 expression and ameliorated IDD in vivo

To investigate the therapeutic effect of miR-146a-5p inhibitor in IDD and elucidate the underlying molecular mechanisms involved, we used needle-punctured organotypic tissue-explants of rat IVD to establish an IDD model, followed by injection of miR-146a-5p mimics or inhibitors, respectively. The delivery of miR-146a-5p mimics developed an IDD phenotype, including a degradation of ECM and increased apoptosis of IVD cells, which were confirmed by immunofluorescence staining (ACAN and MMP3) and terminal deoxynucleotidyl transferase dUTP nick end labeling staining (Fig. 5A–D). Furthermore, the injection of miR-146a-5p mimics in needle-punctured IVDs further aggravated the degenerative phenotype. Conversely, local delivery of miR-146a-5p inhibitor significantly protected cultured IVDs against puncture-induced IDD phenotypes (Fig. 5A–C). Moreover, Sod2 expression level was also detected by immunofluorescence staining, and the result showed that miR-146a-5p mimics inhibited the expression of Sod2 and miR-146a-5p inhibitor promoted the expression of Sod2 (Fig. 5A–E). The morphology of IVDs reflected by HE and SO staining further confirmed the protective effect of miR-146a-5p inhibitor (Fig. 5A–F). These results validated the therapeutic effect of the miR-146a-5p inhibitor in IDD and Sod2 as a direct target of miR-146a-5p.

4 Discussion

Accumulative evidence indicates that miRNAs participate in the pathological process of IDD and modulate inflammatory responses across multiple tissues.^[31] Although many miRNAs have been proved to be involved in the occurrence and development of IDD, such as miR-660,^[32] miR-145,^[33] and miR-34a,^[34] key mediators of inflammation-induced disc degeneration remain incompletely characterized.^[34,^35] In this study, we first found that miR-146a-5p expression is significantly upregulated in samples acquired from severe IDD patients and IL-1β-treated cells, indicating a core role of miR-146a-5p in inflammation-induced IDD. This observed upregulation prompts consideration of the upstream drivers of miR-146a-5p expression. miR-146a-5p is a well-established inflammation-responsive miRNA, often induced via the nuclear factor Kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway.^[36,^37] Pro-inflammatory cytokines like IL-1β and TNF-α, central to IDD pathogenesis,^[1,^2] are potent activators of NF-κB. Indeed, studies in other cell types, including chondrocytes and macrophages, have demonstrated that IL-1β/TNF-α/NF-κB signaling directly transactivates the miR146a gene.^[36–^38] Given the elevated levels of IL-1β and TNF-α in degenerative discs,^[10] it is highly plausible that this canonical NF-κB-mediated pathway is a primary upstream regulator responsible for miR-146a-5p overexpression in our IDD patient samples and IL-1β-treated NP cells. Future research should explicitly investigate NF-κB binding to the miR146a promoter and the effects of NF-κB inhibition on miR-146a-5p levels in disc cells. Furthermore, we demonstrated that miR-146a-5p targets Sod2 mRNA and inhibits the expression of SOD2, thus leading to the degradation of ECM and apoptosis of NP cells. By delineating this miR-146a-5p/Sod2 axis, our findings establish miR-146a-5p as a critical mediator of inflammation-induced IDD pathogenesis, revealing a promising therapeutic target for disc degeneration intervention.

SODs constitute a critical class of antioxidant enzymes that neutralize reactive oxygen species (ROS) generated under cellular stress. SOD2 is one of the most important members, which is located within the mitochondrial matrix, the main site of free radical production from the electron transport chain.^[39] SOD2 catalyzes the reaction of superoxide (O₂⁻) to the less reactive hydrogen peroxide (H₂O₂) (which is not considered a free radical) at diffusion-limited rates, before it can oxidize macromolecules such as DNA, proteins, or lipids.^[39–^41] This conversion to hydrogen peroxide also facilitates a passive diffusion of hydrogen peroxide away from the mitochondrial matrix, preventing a high accumulation of superoxide close to the site of ATP production.^[41,^42] In this study, the bioinformatic analysis revealed that miR-146a-5p targeted the 3′-UTR of Sod2, and the KEGG enrichment results showed that the oxidative stress pathway was significantly enriched in the IL-1β-treated RPNPC. This finding aligns with established SOD2 biology. Domingues CC demonstrated that overexpression of Sod2 within human adipose-derived mesenchymal stem cells could significantly reduce oxidative stress and showed a considerable therapeutic effect on systemic inflammation.^[33,⁴¹–^43] Our findings resonate with recent work highlighting oxidative stress as a key driver of IDD.^[44] A recent study has also established a causative link between Sod2, Cat, and oxidative stress in the disc, as SOD enzymes can promote the dismutation of superoxide to produce hydrogen peroxide (H₂O₂), which will be converted to water and oxygen by Catalase.^[40] There were also studies that showed that SIRT3 primarily regulated mROS clearance by altering the acetylation of SOD2.^[45] More importantly, SIRT3 directly binds and deacetylates SOD2, which increases SOD2 activity and leads to a significant effect on mROS homeostasis and autophagic flux.^[46,^47] The identification of miR-146a-5p as a direct negative regulator of SOD2 in NP cells adds a novel layer of post-transcriptional control to the established importance of mitochondrial antioxidant defense in disc health.^[48] This mechanism parallels findings in osteoarthritis where miR-146a-5p contributes to chondrocyte dysfunction under inflammatory stress,^[49] underscoring a potential common pathogenic miRNA pathway in joint and disc degeneration involving impaired antioxidant responses.

While miR-146a-5p's role in IDD remained undefined, this study employed an integrated approach: in vitro modeling followed by comprehensive miRNA sequencing to characterize dysregulated miRNAs in disc degeneration. Previous studies showed that, compared with the control group, miR-146a-5p in osteoarthritis samples was significantly upregulated. Some studies have also shown that miR-146a-5p promotes chondrocyte apoptosis induced by IL-1β through the TRAF6-mediated NF-κB signal pathway.^[50] However, the role of miR-146a-5p in IDD was unexplored. In our study, it was found that the expression of miR-146a-5p was upregulated in degenerative RPNPC, which was consistent with the results of miRNA sequencing of RPNPC induced by inflammation, suggesting that miR-146a-5p played a key role in IDD. Therefore, we further instigated the detailed mechanism by which miR-146a-5p regulates the function and fate of RPNPC under an in vitro IDD model.

To explore the regulation of miR-146a-5p NPC, we used qRT-PCR to detect the effects of overexpression and silencing of miR-146a-5p on anabolism and catabolism. Overexpression of miR-146a-5p significantly decreased the production of ACAN and increased the production of MMP3 in RPNPC induced by inflammatory cytokine IL-1β. Inhibition of miR-146a-5p yielded an opposite result. After overexpression and silencing of miR-146a-5p, apoptosis was detected by flow cytometry. The results show that miR-146a-5p significantly promoted the apoptosis of RPNPC under inflammatory conditions. Subsequent mechanistic investigation identified miR-146a-5p targeted oxidative stress-related molecule Sod2 during the development and progression of IDD. Critically, silencing of miR-146a-5p attenuated the dysfunction of RPNPC induced by inflammation in vitro and puncture-induced degeneration of rat NP tissue in organic tissue-explant culture.

In a word, this study established for the first time that miR-146a-5p is significantly upregulated in IDD. The presence of miR-146a-5p in RPNPC led to a decrease in the expression of its target gene Sod2, which in turn resulted in an increase of cell catabolism, a decrease of anabolism, an increase of apoptosis, and a degradation of ECM. Inhibition of miR-146a-5p or overexpression of Sod2 successfully ameliorated the progression of disc degeneration (Fig. 6). These findings position the miR-146a-5p/SOD2 axis as a crucial link between inflammation, oxidative stress, and NP cell dysfunction in IDD, offering a novel and mechanistically defined therapeutic target.

5 Conclusion

This study extends the relationship between miRNA and IVD homeostasis and provides a potential therapeutic method for the prevention and recovery of IDD by targeting miR-146a-5p.

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