Visual abstract

Introduction

Intervertebral disc degeneration (IDD) is a leading contributor to chronic back pain, affects a large number of individuals globally, and imposes a substantial socioeconomic impact [1,2]. At the core of IDD pathology is the degeneration of the nucleus pulposus (NP). The NP is a gellike core within the intervertebral disc which is essential for the maintenance of disc structure and biomechanical function through the production of extracellular matrix (ECM) components, including aggrecan and Type 2 collagen alpha 1 (COL2A1) [3,4].

While the precise cause underlying NP degradation remains unclear, it is established that during the progression of IDD, NP cells undergo significant changes driven by various factors, including mechanical overload, inflammatory cytokines, and oxidative stress [5]. Among these, oxidative stress plays a pivotal role. It results from an imbalance between the generation of reactive oxygen species (ROS) and the cellular antioxidant defense systems that neutralize them or repair associated damage caused [6,7]. Elevated ROS levels contribute to the deterioration of the disc microenvironment by promoting ECM breakdown, induction of NP cell apoptosis, and acceleration of overall disc degeneration [8]. As the number of NP cells is progressively reduced, disc height diminishes, mechanical properties are compromised, and this manifests the clinical features of IDD [9]. Oxidative stress amplifies these effects when ROS levels in NP cells increase, this activates catabolic enzymes such as matrix metalloproteinases (MMPs) and aggrecanases like A Disintegrin, and Metalloproteinase with Thrombospondin motifs (ADAMTS)-4, resulting in ECM degradation [10,11]. Given the central role of oxidative stress in driving both cellular loss and ECM breakdown [12], modulating oxidative stress pathways present a promising strategy for therapeutic intervention in IDD.

Apamin (APM), a bioactive peptide derived from bee venom, has gained attention for its neuroprotective, anti-inflammatory, and antioxidant properties [13–16]. It has been shown to mitigate oxidative stress by restoring hepatic glutathione levels and upregulating antioxidant enzymes in acetaminophen-induced hepatotoxicity in a murine model [17]. As the 3^rd largest peptide component in bee venom, APM is less toxic than melittin, and phospholipase A2 [14]. This underscores its potential applicability in therapeutic settings, notably in the context of neurological disorders.

In this study, we investigated the potential protective effects of APM on NP cells under oxidative stress conditions. We hypothesized that APM could reduce oxidative damage in NP cells through the upregulation of the nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) regulatory pathway, thereby lowering ROS levels and preserving ECM integrity. Nrf2, a multifunctional regulator, is known for its cytoprotective role, regulation of genes involved in antioxidant, anti-inflammatory, and detoxifying processes, while also preventing oxidative stress-induced apoptosis [18,19]. Nrf2 activity is controlled by genes such as HO-1, which not only detoxifies heme but also produces protective molecules like biliverdin and carbon monoxide [20,21]. The Nrf2/HO-1 pathway is essential in the mitigation of oxidative stress and inhibition of cell apoptosis [22,23]. Additionally, we explored whether APM could influence the expression of key disc degeneration markers, such as MMP3 and ADAMTS-4. Understanding how APM protects NP cells from oxidative stress may offer valuable insights into new therapeutic strategies for treating IDD.

Materials and Methods

1. Human NP cell culture

Human NP cells [ScienCell Research Laboratories (SCRL; Carlsbad, California, USA)] were cultured in NP cell-specific medium (SCRL) containing 2% fetal bovine serum, 1% NP cell growth supplement, and 1% penicillin-streptomycin. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Subculturing was performed at 2-day intervals. Cells were rinsed twice with prewarmed phosphate-buffered saline (PBS) and subsequently detached with 0.05% trypsin solution.

2. Sequential treatment of NP cells with APM and H₂O₂

NP cells were exposed to APM 30 minutes prior to 200 μM H₂O₂ (Sigma-Aldrich, St. Louis, MO, USA) treatment to induce oxidative stress. Cells were incubated for 24 hours after which analysis was conducted.

3. Human NP cell viability assay

To evaluate the effect of APM on human NP cell survival, cells were exposed to increasing concentrations of APM (1, 10, 25, 50, and 100 μg/mL), and incubated either alone or in combination with 200 μM H₂O₂ for 24 hours. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan), and absorbance was measured at 450 nm with a microplate reader (BioTek, Winooski, VT, USA).

4. Flow cytometry

Intracellular ROS generation was quantified to assess oxidative stress and the potential antioxidant activity of APM. To detect ROS, following cell detachment, NP cells were incubated with the fluorogenic probe 2′,7′-dichlorofluorescin diacetate, for 20 minutes at 37°C in a CO₂-enriched environment. Cells were then washed and fixed using a commercial fixation buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 10 minutes. Fluorescence activity was measured using an Accuri C6 Plus flow cytometer (BD Biosciences), and data were expressed as a percentage relative to untreated controls.

5. Immunohistochemistry

NP cells were permeabilized with 0.2% Triton X-100 in PBS for 5 minutes and then blocked with 10% normal goat serum for 1 hour. Cells were incubated overnight at 4°C with primary antibodies against: aggrecan (1:100; Proteintech, IL, USA), COL2A1 (1:100; Invitrogen, Waltham, MA, USA), MMP3 (1:50; Abcam, Cambridge, UK), MMP13 (1:100; Abcam), ADAMTS-4 (1:100; Abcam), ADAMTS-5 (1:100; Invitrogen), Nrf2 (1:200; Abcam), and HO-1 (1:200; Enzo Biochem, NY, USA). The cells were washed with PBS for 5 minutes, 3 times, and then the cells were treated with a secondary antibody (1:300; Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours at 25 ± 1°C, followed by 3, 5-minute PBS washes. Nuclear staining of the cells with 4′,6-diamidino-2-phenylindole (DAPI; TCI, Tokyo, Japan) was performed, after which the cells were mounted on a slide with a mounting solution (Dako, Glostrup, DK). The stained cell imaging was performed using a confocal microscope (Eclipse C2 Plus, Nikon, Tokyo, Japan) at ×100 magnification.

6. Real-time polymerase chain reaction

Total ribonucleic acid was extracted from the NP cells using Trizol reagent (Thermo Fisher Scientific). Complementary deoxyribonucleic acid was synthesized using oligo deoxythymidine primers and AccuPower RT PreMix (Bioneer, Daejeon, Korea). Quantitative real-time polymerase chain reaction was conducted in triplicate using iQ SYBR Green Supermix on CFX Connect real-time polymerase chain reaction detection system (Bio-Rad, Hercules, CA, USA). Gene sequences used for amplification are listed in Table 1. Target gene expression levels were normalized to β-actin expression and presented as fold change relative to the untreated control group.

7. Statistical analysis

Results were expressed as mean ± standard error of the mean. Statistical significance was assessed via one-way or 2-way analysis of variance with Tukey’s post hoc test using GraphPad Prism 8.0 (GraphPad, Inc., La Jolla, CA, USA). A p < 0.05 was considered significant. Significance indicators: #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. blank group, and *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. the untreated control group.

Results

1. APM protects cell viability and suppressed ROS accumulation in oxidatively stressed NP cells

NP cells were treated with various concentrations of APM (0.1, 1, 5, 10, 25, 50, and 100 μg/mL) for 24 hours. APM concentrations between 10 and 50 μg/mL led to a significantly higher cell viability, compared with the remaining concentrations of APM, which demonstrated no statistically significant difference with the blank (Figure 1A). Under oxidative stress, induced by 200 μM H₂O₂, APM pretreatment protected viability in a dose-dependent manner starting from 5 μg/mL to 50 μg/mL (Figure 1B). Based on these findings, 10 and 50 μg/mL APM were selected for subsequent experiments. Notably, using 2′,7′-dichlorofluorescin diacetate staining, APM pretreatment (10 and 50 μg/mL) led to a significantly lower level of H₂O₂-induced ROS production in NP cells compared with the control (Figures 1C and 1D).

2. APM preserved ECM components in NP cells under oxidative stress

Immunostaining of ECM molecules indicated that H₂O₂ exposure diminished aggrecan and COL2A1 expression. APM pretreatment, prior to H₂O₂ exposure, led to a higher level of aggrecan and COL2A1 expression, which was observed in a concentration-dependent manner compared with the control (Figures 2A–2D). These findings suggest that APM protects NP cells from oxidative stress by mitigating the degradation of critical ECM components.

3. APM mitigated H₂O₂-induced ECM degradation by inhibiting MMPs and ADAMTS in NP cells

H₂O₂-treated NP cells exhibited marked upregulation of matrix-degrading enzymes including MMP3 and MMP13. However, pretreatment with APM at concentrations of 10 and 50 μg/mL substantially reduced their expression, compared with the control, in a dose-dependent manner (Figures 3A, 3B, 3E, and 3F). In the investigation of the effect of APM on ADAMTS enzymes in NP cells, which are the key mediators of ECM degradation, H₂O₂ exposure significantly upregulated the expression of these enzymes, further contributing to ECM breakdown. However, APM pretreatment at 10 and 50 μg/mL resulted in significantly lower expression, as compared with the untreated control, of both ADAMTS-4 and ADAMTS-5 in a dose-dependent manner (Figures 3C, 3D, 3G, and 3H). These results implied effective suppression of the oxidative stress-induced upregulation of ADAMTS enzymes by APM, supporting its protective role in maintaining ECM integrity and slowing disc degeneration.

4. APM activated the Nrf2/HO-1 pathway to enhance antioxidant defense in NP cells from oxidative damage

To further elucidate the molecular mechanisms of APM’s antioxidant effects, protein, and messenger ribonucleic acid levels of Nrf2 and HO-1 pathway were examined. Immunocytochemistry revealed that H₂O₂ treatment significantly reduced Nrf2 and HO-1 expression, compared with the blank group, indicating a disruption of the antioxidant defense system, but APM pretreatment at 10 and 50 μg/mL markedly protected both expressions at the protein (Figures 4A–4C) and transcript levels (Figures 4D and 4E), supporting activation of the Nrf2 and HO-1antioxidant response.

Discussion

APM, a component of bee venom, exerts significant protective effects on NP cells under oxidative stress by protecting cell viability, preserving ECM integrity, inhibiting matrix-degrading enzymes, and activating the Nrf2/HO-1 antioxidant pathway. Despite traditionally being regarded as a neurotoxic peptide in bee venom, these findings align with previous reports suggesting that APM possesses neuroprotective and anti-inflammatory properties [13–17]. The neuroprotective roles of APM, particularly in the context of central nervous system diseases, have garnered significant attention. APM regulates synaptic plasticity, learning, and memory by selectively inhibiting small-conductance calcium-activated potassium channels, which are present in various neuronal populations, including midbrain dopaminergic neurons [13,14,16]. This current study extends the understanding of APM’s protective effects on intervertebral disc cells, where it mitigates oxidative stress-induced damage. This broadens the potential applications of APM beyond neuroprotection, highlighting its role in cellular defense against oxidative stress across different tissue types. Our findings demonstrated that APM had a significantly higher level of Nrf2 and HO-1 expression, as compared with the control, which is relevant because Nrf2 and HO-1 are key regulators of cellular antioxidant defenses. APM’s neuroprotective effects have also been attributed to this mechanism, where it reduces oxidative damage by upregulating antioxidant enzymes. In NP cells, APM pretreatment inhibited H₂O₂ -induced downregulation of Nrf2 and HO-1, suggesting that APM enhanced the antioxidant capacity of these cells, which resulted in lower ROS levels and prevention of ECM degradation. Given that oxidative stress is a key driver of IDD, APM is a promising agent for delaying or preventing IDD progression. In addition, APM treated cells had a significantly lower level of expression of matrix-degrading enzymes, such as MMP3, MMP13, ADAMTS-4, and ADAMTS-5, compared with the control, and these enzymes contribute to ECM breakdown in IDD. This low level of expression of catabolic enzymes is consistent with APM’s anti-inflammatory actions through the modulation of key signaling pathways. The protective effect of APM preserves ECM integrity, which is crucial for maintaining the structural and functional properties of the intervertebral disc. In addition to its effects on NP cells, APM has been shown to promote axonal regeneration and neurite outgrowth in neuronal injury models [14]. This is particularly important for understanding its broader therapeutic applications. This study has reported that APM enhances F-actin content in the growth cones of regenerating axons, improving neurite extension and axonal growth. Furthermore, APM upregulates the expression of regeneration-associated genes, such as NF200 and GAP43, as well as neurotrophic factors, such as BDNF and NGF. Although this current study focused on NP cells, these regenerative properties further support the potential of APM in treating degenerative conditions involving oxidative stress and cellular damage.

However, this study has some limitations that must be acknowledged. Firstly, this study was performed in vitro using NP cells, which does not represent the complexity of the in vivo environment of the intervertebral disc. Future investigations into the effects of APM in animal models of disc degeneration are needed to determine its therapeutic efficacy. In addition, although the level of expression of Nrf2 and HO-1 was significantly higher in the APM treated group, as compared with the control NP cells, the precise molecular mechanisms by which APM activates these pathways requires exploration. Finally, while no adverse effects were observed at the concentrations tested in vitro in this study, higher doses may cause toxicity, and the optimal therapeutic dose of APM for animal experiments needs to be determined.

Conclusion

APM offers promising protective effects for NP cells by enhancing antioxidant defenses, preserving ECM integrity, and inhibiting matrix-degrading enzymes. These protective mechanisms, along with APM’s neuroprotective and regenerative properties, position it as a potential therapeutic agent for treating IDD and other oxidative stress-related conditions. These preliminary findings need to be validated in vivo in animal models, and in clinical trials, and the broader therapeutic potential of APM in degenerative diseases should be explored.

Article information

Author Contributions

Conceptualization: HK and HB. Methodology: HK. Validation: HK. Formal analysis: HK. Investigation: HK and HSC. Data curation: HK and HSC. Writing - original draft: HK. Writing - review & editing: HSC and HB. Visualization: HK. Supervision: HB. Project administration: HB

Conflicts of Interest

The authors declare no conflicts of interest.

Author Use of AI Tools Statement

Artificial intelligence tools were not involved in any part of the scientific research, including data interpretation or result formulation.

Ethical Statement

Not applicable

Data Availability

Figure 1

Protective effect of APM in the viability of H2O2-treated NP cells. (A) NP cells (5 × 10³/well in 96-well plates) were treated with various concentrations of APM for 24-hour, and cell viability was assessed by CCK-8 assay (n = 6). (B) Cells were pretreated with APM for 30 minutes, followed by H₂O₂ exposure and 24-hour incubation. Viability was measured using CCK-8 (5 × 10³ cells/well, n = 6). (C) ROS levels were evaluated by DCFDA-FITC staining and flow cytometry after 24-hour co-treatment with APM and H₂O₂ (2 × 10⁵ cells/well in 6-well plates, n = 4). (D) Quantification of DCFDA⁺ cells from flow cytometry analysis in each group.

Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test.

^####p < 0.0001 (compared with the blank group).

*p < 0.05 (compared with the control group).

**p < 0.01 (compared with the control group).

***p < 0.001 (compared with the control group).

****p < 0.0001 (compared with the control group).

ANOVA = analysis of variance; APM = apamin; CCK-8 = cell counting kit-8; DCFDA = 2′;7′-dichlorofluorescin diacetate; FITC = fluorescein isothiocyanate; H₂O₂ = hydrogen peroxide; NP = nucleus pulposus; ROS = reactive oxygen species.

Figure 2

Protective effect of APM on ECM components in H₂O₂-treated NP cells. (A,B) Representative confocal immunofluorescence images of aggrecan (ACAN) and collagen type 2 alpha 1 (Col2a1) expression in NP cells after 24-hour treatment. Yellow scale bar = 200 μm; white scale bar = 50 μm. (C,D) Quantification of fluorescence intensity for each marker (2 × 10⁴ cells/well in 24-well plates, n = 6).

Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test.

^####p < 0.0001 (compared with the blank group).

****p < 0.0001 (compared with the control group).

ANOVA = analysis of variance; APM = apamin; ECM = extracellular matrix; NP = nucleus pulposus.

Figure 3

Protective effect of APM on the expression of degenerative enzyme expression in H₂O₂-treated NP cells. (A–D) Representative confocal immunofluorescence images showing the expression of MMP3, MMP13, ADAMTS-4, and ADAMTS-5 in NP cells after 24-hour treatment with APM and H2O2. Yellow scale bar = 200 μm; white scale bar = 50 μm. (E–H) Quantification of fluorescence intensity for each marker (2 × 10⁴ cells/well in 24-well plates, n = 6).

Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test.

^####p < 0.0001 (compared with the blank group).

*p < 0.05 (compared with the control group).

**p < 0.01 (compared with the control group).

***p < 0.001 (compared with the control group).

****p < 0.0001 (compared with the control group).

ADAMTS = a disintegrin and metalloproteinase with thrombospondin motifs; ANOVA = analysis of variance; APM = apamin; H2O2: hydrogen peroxide; MMP = matrix metalloproteinase.

Figure 4

Protective effect of APM on the expression of Nrf2 and HO-1 in H₂O₂-treated NP cells. (A) Representative confocal immunofluorescence images of Nrf2 and HO-1 expression in NP cells after 24-hour treatment with APM and H₂O₂. Yellow scale bar = 200 μm; white scale bar = 50 μm. (B, C) Quantification of fluorescence intensity for each marker (2 × 10⁴ cells/well in 24-well plates, n = 6). (D, E) mRNA expression levels of Nrf2 and HO-1 analyzed by quantitative real-time PCR (2 × 10⁵ cells/well in 6-well plates, n = 5).

Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test.

^#p < 0.05 (compared with the blank group).

^##p < 0.01 (compared with the blank group).

^###p < 0.001 (compared with the blank group).

*p < 0.05 (compared with the control group).

**p < 0.01 (compared with the control group).

****p < 0.0001 (compared with the control group).

ANOVA = analysis of variance; APM = apamin; H₂O₂ = hydrogen peroxide; HO-1 = heme oxygenase-1; mRNA = messenger ribonucleic acid; Nrf2 = Nuclear factor erythroid-2-related factor 2; PCR = polymerase chain reaction.

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