Molecular regulatory mechanism of the SIRT2/NF-κB p65 signaling pathway in ferroptosis-promoted spinal cord injury repair

Abstract

Background

Spinal cord injury (SCI) is a debilitating neurological condition that often results in long-term disability and functional impairment. Recent studies have identified ferroptosis as a significant pathological mechanism in SCI. SIRT2, a deacetylase enzyme, is closely associated with inflammatory responses and apoptosis, playing a crucial role in the pathogenesis of various neurological disorders. This research aims to elucidate the specific mechanisms by which SIRT2 overexpression inhibits ferroptosis and promotes SCI repair through the deacetylation of nuclear factor-κB (NF-κB) p65.

Results

Utilizing cell models and a rat SCI model, we discovered that SIRT2 overexpression promotes NF-κB p65 deacetylation, subsequently inhibiting ferroptosis and oxidative stress. Conversely, the use of AK-7 elevated NF-κB p65 acetylation levels, exacerbating ferroptosis and oxidative stress. In SCI rats, intrathecal injection of SIRT2-overexpressing recombinant adenovirus successfully inhibited NF-κB p65 acetylation and ferroptosis in the dorsal root ganglia, thereby reducing neuronal apoptosis and enhancing motor function recovery.

Conclusion

In summary, these findings indicate that SIRT2 overexpression can suppress ferroptosis through NF-κB p65 deacetylation, facilitating SCI repair. Therefore, a deeper understanding of the interaction between SIRT2 and NF-κB p65 and their roles in the regulation of ferroptosis is of paramount importance for developing novel therapeutic approaches for spinal cord injury.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12883-025-04164-x.

Introduction

Spinal cord injury (SCI) is a severe traumatic disorder of the central nervous system, typically resulting from vertebral fractures that cause vertebral protrusion into the spinal canal, damaging the spinal cord and spinal nerves [1]. SCI can be categorized into primary and secondary injuries [2]. Primary injury is induced by mechanical forces, leading to irreversible compression and contusion of the spinal cord [3]. Secondary injury arises from a series of physiological responses triggered by the primary injury, including vascular disturbances, electrolyte imbalances, increased lipid peroxidation, edema, and apoptosis [4]. In recent years, the incidence of SCI in China has been rising, imposing a substantial economic burden on patients and their families. Consequently, the prevention, treatment, and rehabilitation of spinal cord injuries have become critically important [5].

Ferroptosis is an iron-dependent form of programmed cell death, typically regulated by intracellular iron overload and the accumulation of lipid-derived reactive oxygen species (ROS) [6]. This unique mechanism of cell death occurs in the pathological processes of various central nervous system diseases and injuries, including secondary SCI [7]. Excessive accumulation of iron ions exacerbates oxidative stress within cells, leading to the production of a large amount of lipid peroxides, which in turn damages cellular structure and function [8]. In this process, glutathione peroxidase 4 (GPX4) plays a pivotal role; this key protein utilizes glutathione to inhibit lipid peroxidation associated with ferroptosis [9]. Conversely, 4-hydroxynonenal (4HNE), a major byproduct of lipid peroxidation, serves as a reliable indicator of the severity of ferroptosis [10]. In our study, GPX4 and 4HNE were employed as crucial biomarkers for assessing ferroptosis following SCI, providing a more comprehensive understanding of the role of this cell death mechanism in the injury process [11]. Therefore, in-depth research into strategies to inhibit ferroptosis is of significant importance for promoting recovery from spinal cord injuries.

Sirtuin 2 (SIRT2) is a vital member of the Sirtuin family, belonging to the class of NAD+-dependent deacetylases [12]. SIRT2 plays a crucial role in cellular metabolic regulation and various life processes, including cell proliferation, aging, apoptosis, and inflammatory responses [13]. Furthermore, studies suggest that SIRT2, by modulating protein acetylation states, may influence neuronal survival and function, thereby exhibiting neuroprotective effects [14]. Protein acetylation refers to the process wherein acetyl groups are added to proteins under the catalysis of acetyltransferases, a modification that aids in regulating gene expression and protein activity [15]. Deacetylation, involving the removal of acetyl groups from proteins, consequently impacts their function [16]. The regulation of deacetylation is essential for controlling key physiological processes such as gene transcription, cell cycle progression, and oxidative stress, all of which are critical for maintaining cellular homeostasis and function [17]. Abnormal protein acetylation is closely associated with the pathogenesis of various diseases, including cancer and neurodegenerative disorders [18]. Recent research has found that deacetylation of the NF-κB p65 protein can inhibit ferroptosis and suppress the expression of proteins involved in ferroptosis-related signaling pathways [19]. Therefore, protein acetylation holds a significant position in our research.

A growing body of research indicates that SIRT2 is involved in regulating nervous system functions and the process of ferroptosis. Nevertheless, it remains unclear whether SIRT2 contributes to ferroptosis and SCI repair through the deacetylation of NF-κB p65. This study aims to investigate the potential mechanism by which SIRT2, via the deacetylation of NF-κB p65, inhibits ferroptosis and promotes SCI repair. Our findings reveal that in a rat model of SCI, the overexpression of SIRT2 suppresses the acetylation of the NF-κB p65 protein, upregulates the expression of GPX4, and downregulates the expression of 4HNE, thereby facilitating the repair of spinal cord injuries. In summary, these results suggest that SIRT2 promotes SCI repair by inhibiting ferroptosis through the deacetylation of NF-κB p65. Therefore, SIRT2 may represent a potential therapeutic target for the treatment of spinal cord injuries.

Materials and methods

Recombinant adenovirus construction

Target gene sequences obtained from the National Center for Biotechnology Information (NCBI) GenBank were cloned into the pAdTrack-CMV-3flag vector to construct the target gene plasmid. This plasmid was then recombined with the adenoviral backbone plasmid pAdeasy-1, resulting in the generation of the final overexpression recombinant adenovirus. Both the empty control adenovirus and the SIRT2 overexpression adenovirus were constructed and sequenced by Hanbio Biotechnology (Shanghai) Co., Ltd.

The efficacy evaluation of the SIRT2 overexpression Recombinant adenovirus

PC12 cells were utilized for validation experiments. Wild-type cells served as the negative control, while cells transfected with the empty control adenovirus acted as the virus-only control. The experimental group consisted of cells transfected with the SIRT2 overexpression adenovirus. Transfection was performed using a semi-infection method when the cells reached 50% confluency. Briefly, after removing half of the culture medium, fresh medium containing the adenovirus was added. Subsequently, the medium was restored to its original volume after 4 h, and then completely replaced with fresh medium after 6 h. Forty-eight hours post-transfection, fluorescence intensity was measured using a fluorescence microscope, and target gene expression was quantified using quantitative real-time PCR (qRT-PCR) to assess transfection efficiency.

Cell culture and model

PC12 is a cell line derived from rat adrenal pheochromocytoma cells, known for its high similarity to neurons and neuro-like physiological characteristics. It is frequently used in research related to the central nervous system, making it a significant model in this field. The PC12 cells used in our experiments were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured under the following conditions: 37 °C, 5% CO2, in DMEM/F12 medium supplemented with 10% horse serum, 5% fetal bovine serum, and 1% penicillin-streptomycin solution. Cells were passaged every 2–3 days. For in vitro drug treatment experiments, RAS-selective-lethal-3 (RSL3, CSN17581, CSNpharm), a classic ferroptosis inducer, was used to induce intracellular ferroptosis in PC12 cells. The experiment utilized a concentration of 5 µM RSL3 to induce ferroptosis in the PC12 cells. In the PC12 cell experiments, RSL3 was added after cell adherence and attainment of appropriate density, with a 24-hour treatment duration to induce ferroptosis. AK-7, a cell-permeable and blood-brain barrier-penetrating SIRT2 inhibitor, exerts its effect by inhibiting the deacetylase activity of SIRT2, thereby increasing acetylation levels of target proteins. In PC12 cell experiments, AK-7 (at a concentration of 10 µM) was administered 48 h after SIRT2-overexpressing adenovirus transfection.

Cell counting Kit-8 (CCK-8) assay

To assess cell viability, the CCK-8 assay was employed. Initially, cell suspensions were evenly distributed into 96-well plates at a density of 1.5 × 104 cells per well, with five replicates per group (n = 5), and incubated for 24 h. Following the relevant treatments, 10 µL of CCK-8 solution was added to each well. The plates were then incubated in the dark at 37 °C for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader (Synergy HT, Bio-Tek, United States).

Detection of intracellular ROS, SOD, and MPO

According to the instructions provided by the manufacturer, the activities of reactive oxygen species (ROS), superoxide dismutase (SOD), myeloperoxidase (MPO) and 4-hydroxynonenal (4HNE) in PC12 cells were measured using the corresponding kits from Solarbio (Beijing, China). The level of 4-hydroxynonenol (Cat# YPJ1152, UpingBio) was determined by enzyme-linked immunosorbent assay (ELISA).

Experimental animals

Female rats are preferred for SCI studies due to their shorter and wider urethras, which facilitate easier post-injury bladder management. In this study, we selected 20 adult female Sprague-Dawley rats (weighing 220 ± 20 g) obtained from the Experimental Animal Center of Dalian Medical University. The rats were housed individually in a controlled environment with a temperature of 22 ± 1 °C and a humidity of 50–60%, under a 12/12-hour light/dark cycle. They had free access to food and water. This experimental protocol was approved by the Ethics Committee of the Affiliated Hospital of Zhongshan Hospital (Approval Number: DW2024-063-01).

SCI rat model

SCI was induced through a hemisection procedure at the T9-T10 spinal segments. Forty rats were randomly divided into four groups: the Sham group, the SCI group, the SCI + Ad-control group, and the SCI + Ad-SIRT2 group, with ten rats in each group. Initially, the rats were acclimatized to the experimental environment for one week. Anesthesia was induced with 3% isoflurane (R510-22-10, RWD Life Science, Shenzhen, China) and maintained with 2% isoflurane (2–3 L/min). A midline incision was made on the dorsal surface of the rats, the muscles were dissected to expose the vertebral column, and a laminectomy was performed at the T10 vertebra to expose the spinal cord. A precision impactor device (68099II, RWD Life Science) was used to induce the SCI with the following parameters: velocity of 2 m/s, depth of 1 mm, and dwell time of 0.4 s, resulting in a moderate contusion (60 kdyn) as per the manufacturer’s guidelines. Immediately following the injury, hemorrhage and swelling of the spinal cord were observed. The success of the SCI model was confirmed by observing involuntary spasms in the hind limbs and tail twisting. Upon recovery from anesthesia, loss of muscle strength and paralysis were also noted. The wound was then irrigated with saline and sutured. For the Sham group, the same surgical procedures were performed except for the contusion injury. During surgery, the rats’ body temperature was maintained at 37 ± 0.3 °C using a heating pad. After recovering from anesthesia, the rats exhibited flaccid paralysis of the hind limbs and tail, indicating successful establishment of the SCI model. Post-surgery, all rats were manually voided twice daily until bladder function recovered. All surgical procedures were conducted under anesthesia with efforts made to minimize pain. Following the experiment, the rats were euthanized via carbon dioxide inhalation, a method noted for its safety, cost-effectiveness, and capacity to euthanize multiple animals simultaneously. This method allows operators to euthanize animals with minimal risk while preserving their anatomical integrity.CO2 is injected into the euthanasia box at a rate of 10–30% per minute, effectively replacing the box’s volume. The flow rate in this system, set at 20% (equivalent to 5.8 L/min), is regulated by rotating the left knob. This process ensures the animal’s immobility, cessation of breathing, and dilation of pupils. Subsequently, the CO2 cylinder switch is turned off, and a further observation period of 5 min is conducted to confirm the cessation of vital signs in the animals. The animal experimental protocols adhered strictly to the relevant regulations on animal use and care under Chinese legislation concerning animal welfare.

Behavioral tests

Motor function was quantified using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale, which ranges from 0 to 21 points. This scale assesses joint movement, weighted gait, gait coordination, and tail movement. Each rat was placed in an open field for 5 min and allowed to explore freely to acclimate to the environment before the assessment. BBB scores were recorded at baseline (time 0) and subsequently on post-operative days 1, 3, 7, 14, 21, and 28. The evaluations were conducted by two independent observers who were blinded to the group assignments. The inclined plane test was conducted using the Rivlin method. The instrument used allows for free adjustment of the inclination. A piece of cardboard was placed on the instrument, and a 6 mm thick rubber pad was laid on the surface of the cardboard. Then, the rat was positioned on the rubber pad with its body length aligned with the long axis. The angle between the inclined plane and the horizontal line was slowly increased until the rat could stably stand on the plane for 5 s. At this point, the inclination angle was measured. Each rat was measured three times, and the average value was calculated. Evaluations were conducted on the 1st, 3rd, 7th, 14th, and 21st days post-surgery.

Nissl staining

First, the tissue sections were deparaffinized by placing them in xylene for 5–10 min, followed by another 5–10 min in fresh xylene. The sections were then sequentially treated in absolute ethanol for 5 min, followed by another 5 min in fresh absolute ethanol, then 5 min in 75% ethanol, and finally rinsed in water for 2 min. The prepared samples were stained with Nissl staining solution for 2–5 min, with the time adjusted based on staining requirements. After staining, the sections were rinsed with water until the rinse water ran clear. Next, the excess water was removed by drying the sections, which were then placed in a 65 °C oven and baked for 4 h or until completely dry. Once dried, the sections were immersed in xylene for 10 min to make them transparent, or alternatively, the slides were allowed to cool in xylene before being mounted with neutral balsam for coverslipping.

Hematoxylin and eosin (H&E) staining

On the 28th day after SCI, the tissue is collected, paraffin-embedded, and made into serial sections. The tissue Sect. (4 μm) are placed on water at 46 °C, then baked in a 72 °C oven for 2 h. After cooling the sections for 10 min, immerse them sequentially in the following solutions: Xylene I (10 min), Xylene II (10 min), absolute ethanol I (5 min), absolute ethanol II (5 min), 90% ethanol (2 min), 80% ethanol (2 min), 70% ethanol (2 min), tap water (5 min), hematoxylin (5–10 min), tap water (5 min), hydrochloric acid-ethanol (2–3 s), tap water (5 min), lithium carbonate (10 min), tap water (10 min), eosin (2 min), tap water (5 min), 80% ethanol (2 min), 90% ethanol (2 min), absolute ethanol (2 min), and xylene (2 min). Finally, mount the sections with neutral balsam and observe under a light microscope.

Measurement of iron, glutathione, and malondialdehyde

First, the contusion site and approximately 5 mm of surrounding tissue were homogenized and then diluted tenfold in phosphate-buffered saline (PBS) (C0221A, Beyotime). Subsequently, enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of iron (Cat#ab83366, Abcam), glutathione (GSH, Cat#S0053, Beyotime), and malondialdehyde (MDA, Cat#S0131S, Beyotime). The assays were performed following the manufacturer’s instructions.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Using the StradyPure Universal RNA Extraction Kit (StradyPure, China), total RNA was extracted separately from cells and spinal cord tissues. Subsequently, reverse transcription was performed using either the PrimeScript™ RT reagent kit (TaKaRa, Tokyo, Japan) or the miRCURY LNA Universal cDNA Synthesis Kit (Exiqon, Vedbæk, Denmark), following the manufacturer’s protocols. Gene expression levels were then detected using the LightCycler 480 Fluorescent Quantitative PCR Instrument (Roche Diagnostics, Indianapolis, USA), adhering strictly to the instructions of the fluorescent quantitative PCR kit (SYBR Green Mix, Roche Diagnostics). The thermal cycling parameters were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s, extension at 72 °C for 10 s, and a final extension at 72 °C for 5 min. Each quantitative PCR reaction was set up with three replicates. In the experiments, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 was used as an internal reference, and the data were analyzed using the 2^-ΔΔCt method: ΔΔCt = (Ct target gene - Ct internal reference) in the experimental group - (Ct target gene - Ct internal reference) in the control group. The relevant primers were synthesized by Sangon Biotech (Shanghai, China) (see Table 1 for detailed information).

Table 1.

Gene primer sequences used for qRT-PCR

Gene name	Primer sequences
SIRT2	F: 5’-ATGGCGACCTGGCTTCTG-3’
	R: 5’-TCAGTGCTGCTGTCCTTCC-3’
Acetly-NF-κB p65	F: 5’-TACCTTGGAGCAGGTTGCAG-3’
	R: 5’-GAGGTGTTGGTGGCAACTCT-3’
Total NF-κB p65	F: 5’-ATGGAGGACCATGACCTG-3’
	R: 5’-TTAGGCTTCTCCATGGTC-3’
GPX4	F: 5’-ATGGCTGCCCAAGGCTTAC-3’
	R: 5’-TTAGCCATCGGTCAGCATCC-3’

Western blot analysis

First, the samples need to be processed. For cells, perform conventional trypsin digestion, then centrifuge at 1000 rpm for 5 min, and transfer the cells to a 1.5 ml centrifuge tube. Wash the cells with PBS three times, then add lysis buffer at a specific ratio, pipette the cells, and mix well. Place the mixture on ice for 30 min. After lysis, centrifuge at 12,000 rpm for 30 min at 4 °C, and collect the supernatant for later use. Next, perform a preliminary quantification of the protein samples. Prepare BCA working solution at a specific ratio, dilute the standards, and add them to a 96-well plate along with the corresponding volume of samples. Add BCA working solution to each EP tube, incubate under specific conditions, and measure the absorbance at 450 nm using a microplate reader. Calculate the protein concentration based on the standard curve. Proceed with SDS-PAGE electrophoresis by cleaning the glass plates, pouring the gel, and loading the samples. Run the electrophoresis at a certain voltage until the appropriate position is reached. Then perform protein transfer by pre-treating the PVDF membrane, forming the transfer stack in a specific order, and placing it in the transfer cassette. Conduct the transfer at a certain current and low temperature based on the molecular weight of the protein. Finally, perform immunoblotting by washing and blocking the membrane, then incubating it with the primary antibody overnight at 4 °C. After multiple washes, incubate with the secondary antibody, place the membrane on a transparent plastic sheet, and add chemiluminescent substrate evenly for reaction. Complete the process by performing chemiluminescence. Use ImageJ analysis software (NIH, Bethesda, MD, USA) to quantify the gray levels of the protein bands in the Western blot image, with GAPDH as a loading control. Each experiment is repeated three times.Use the following primary antibodies at specified dilutions: SIRT2 Polyclonal antibody (19655-1-AP, Proteintech, USA, dilution 1:5000)Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade (ab218533, Abcam, UK, dilution 1:1000)Anti-NF-kB p65 antibody [E379] (ab32536, Abcam, UK, dilution 1:1000)GPX4 Monoclonal antibody (67763-1-Ig, Proteintech, USA, dilution 1:5000)Anti-4 Hydroxynonenal antibody [HNEJ-2] (ab48506, Abcam, UK, dilution 1:1000)GAPDH (ab8245, Abcam, UK, dilution 1:2000).

Transmission electron microscopy

Take fresh tissues from rats, quickly determine the sampling site, and obtain tissue blocks of about 1 mm³ within 1–3 min to minimize mechanical damage. First, put the tissue blocks into a petri dish filled with electron microscope fixative. After cutting, transfer them to a new EP tube containing electron microscope fixative. Fix, store, and transport the samples at 4 °C. Rinse the samples with 0.1 M phosphate buffer (PB, pH 7.4) three times, 15 min each time. Subsequently, fix the samples with 1% osmium tetroxide prepared with 0.1 M PB (pH 7.4) in the dark at room temperature for 2 h. Then, rinse the samples with PB of the same concentration three times, 15 min each time. Next, perform an ascending dehydration process with alcohol concentrations ranging from 30 to 100% successively, 20 min each time, and treat the samples with 100% acetone twice, 15 min each time. Conduct permeation treatment with different ratios of acetone to 812 embedding agent successively, pour in pure 812 embedding agent, insert the samples, and keep them overnight at 37 °C. Then, polymerize the samples at 60 °C for 48 h to obtain resin blocks. Cut the resin blocks into 60–80 nm thin slices using an ultramicrotome, and pick up the slices with 150-mesh copper grids. Stain the copper grids successively with uranyl acetate and lead citrate solutions, wash them with ultrapure water, and dry them at room temperature. Finally, observe and analyze the images under a transmission electron microscope.

Statistical analysis

Data are presented as mean ± standard deviation (Mean ± SD). All statistical analyses were performed using SPSS 18.0 and GraphPad software. For continuous variables, one-way analysis of variance (ANOVA) was used to assess intergroup differences. If significant overall differences were detected (P < 0.05), Dunnett’s test was further applied for multiple comparisons with the blank control group as the reference. For BBB score and inclined plane test data, two-way ANOVA was conducted to examine both time effects and intergroup interactions, followed by Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical significance was defined as a two-tailed P value < 0.05, with all tests performed at the α = 0.05 significance level.

Results

Overexpression of SIRT2 can inhibit oxidative stress in PC12 cells following ferroptosis

To accurately evaluate the efficiency of SIRT2 overexpression, we transfected PC12 cells with wild-type adenovirus (as control), control overexpression adenovirus, and SIRT2 overexpression adenovirus. Using qRT-PCR analysis, we found that the expression level of SIRT2 mRNA in the SIRT2 overexpression cells was significantly higher than in the control group. Additionally, fluorescence microscopy confirmed successful transfection in both the control and SIRT2 overexpression groups, as evidenced by the expression of enhanced green fluorescent protein (EGFP) (Fig. 1A).

Fig. 1.

Overexpression of SIRT2 inhibits oxidative stress in PC12 cells. (A): qRT-PCR analysis of SIRT2 expression levels and EGFP fluorescence microscopy. (B): Measurement of intracellular ROS, MPO, and SOD levels. (*P < 0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs.SCI group, ***P<0.001 vs.0 µmol/L group)

To assess the impact of SIRT2 overexpression on oxidative stress in PC12 cells, we divided the cells into five groups: Sham group, SCI group, SCI + Ad-control group, SCI + Ad-SIRT2 group, and SCI + Ad-SIRT2 + AK-7 group. Ferroptosis was induced in PC12 cells by treatment with 5 µM RSL3 to simulate neuronal ferroptosis following SCI. Using ELISA, we measured the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA). Compared to the SCI group, the SCI + Ad-SIRT2 group showed a significant increase in SOD levels, while ROS and MDA levels were significantly decreased (Fig. 1B). In the SCI + Ad-SIRT2 + AK-7 group, the addition of AK-7 inhibited the expression of SIRT2. These results indicate that overexpression of SIRT2 can mitigate oxidative stress in PC12 cells following ferroptosis.

SIRT2 overexpression inhibits ferroptosis in PC12 cells via deacetylation of NF-κB p65

To evaluate whether the overexpression of SIRT2 inhibits ferroptosis in PC12 cells by promoting the deacetylation of NF-κB p65, we conducted a series of experiments. Initially, we assessed the expression levels of acetylated NF-κB p65 and proteins associated with ferroptosis. NF-κB p65 plays a pivotal role in the onset and progression of SCI, acting as a key transcription factor in apoptosis regulation. Our qRT-PCR results revealed that, compared to the Sham group, the SCI group exhibited a significant increase in acetylated NF-κB p65 and total NF-κB p65, alongside a notable decrease in SIRT2 levels. Conversely, in the SCI + Ad-SIRT2 group, the acetylation levels of NF-κB p65 were markedly reduced compared to the SCI group, while the SCI + Ad-control group showed no significant difference. Furthermore, the SCI + Ad-SIRT2 + AK-7 group exhibited a pronounced increase in NF-κB p65 acetylation (Fig. 2A). These findings suggest that SIRT2 overexpression suppresses NF-κB signaling by promoting the deacetylation of NF-κB p65.Additionally, we evaluated the expression of ferroptosis-related proteins GPX4 and 4HNE. Compared to the Sham group, the SCI group demonstrated lower GPX4 levels and higher 4HNE levels. The SCI + Ad-SIRT2 group showed significantly elevated GPX4 levels and reduced 4HNE levels compared to the SCI + Ad-control group (Fig. 2B). Based on these results, we hypothesize that the overexpression of SIRT2 inhibits ferroptosis by promoting the deacetylation of NF-κB p65, a hypothesis further supported by the findings in the SCI + Ad-SIRT2 + AK-7 group.

Fig. 2.

Overexpression of SIRT2 inhibits terroptosis by promoting NF-κB p65 deacetylation. (A): The mRNA expression levels of SIRT2, Acetly-NF-κB p65 and Total NF-κB p65. (B): Determination of the mRNA expression level of GPX4 and the intracellular level of 4HNE. (C): Western blot analysis showing protein bands for SIRT2, acetylated NF-κB p65, total NF-κB p65, GPX4, 4HNE, and GAPDH. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. SCI group)

We further validated the interactions between these pathways through Western blot experiments. Compared to the Sham group, the SCI group exhibited significant increases in acetylated NF-κB p65, total NF-κB p65, and 4HNE, while SIRT2 and GPX4 levels were markedly decreased. In contrast, the SCI + SIRT2 group showed a downregulation of acetylated NF-κB p65, total NF-κB p65, and 4HNE, with an upregulation of SIRT2 and GPX4 compared to the SCI group. The SCI + Ad-control group displayed trends opposite to those observed in the SCI + SIRT2 group (Fig. 2C). These results are consistent with the previous qRT-PCR findings and further corroborate that SIRT2 overexpression might inhibit ferroptosis in PC12 cells by promoting the deacetylation of NF-κB p65.

The administration of an adenovirus overexpressing SIRT2 can inhibit ferroptosis in rats with SCI

To investigate the specific mechanisms by which adenoviral overexpression of SIRT2 inhibits ferroptosis in rats with SCI, we divided 40 SCI rats into four groups: Sham, SCI, SCI + Ad-control, and SCI + Ad-SIRT2. In the SCI + Ad-SIRT2 group, we increased SIRT2 expression in the spinal cord via intrathecal injection of Ad-SIRT2, with Ad-control injected intrathecally as a comparison. PCR analysis revealed that SIRT2 overexpression regulates the expression of ferroptosis-related genes through the deacetylation of NF-κB p65, thereby inhibiting the occurrence and progression of ferroptosis (Fig. 3A). Western blot experiments further confirmed the interactions between these pathways. Compared to the SCI group, the SCI + Ad-SIRT2 group exhibited significantly reduced acetylation levels of NF-κB p65, decreased expression of 4HNE, and increased expression of GPX4. These findings were consistent with our in vitro results (Fig. 3B).

Fig. 3.

Overexpression of SIRT2 can promote NF-κB p65 deacetylation inhibits ferroptosis. (A): Determination of the mRNA expression levels of SIRT2, Acetly-NF-κB p65, Total NF-κB p65 and GPX4 as well as the level of 4HNE. (B): Western blot analysis showing protein bands for SIRT2, Acetylated NF-κB p65, Total NF-κB p65, GPX4, 4HNE, and GAPDH. (C): ELISA measurements of Iron, GSH, and MDA concentrations in spinal cord tissue. (N = 10; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. SCI group)

To quantitatively analyze the process of ferroptosis in spinal cord tissue, we measured the concentrations of iron, GSH, and MDA using ELISA. Compared to the Sham group, the SCI group exhibited significantly increased levels of iron and MDA, and significantly decreased levels of GSH. In contrast, the SCI + Ad-SIRT2 group showed significantly reduced concentrations of iron and MDA, and significantly elevated levels of GSH compared to the SCI group (Fig. 3C). Through in vivo experiments, we further confirmed that SIRT2 overexpression inhibits ferroptosis in SCI rats by promoting the deacetylation of NF-κB p65.

Overexpression of SIRT2 inhibits neuronal degeneration and promotes motor function recovery in SCI rats

To further validate the effects of SIRT2 overexpression on inhibiting neuronal degeneration and promoting motor function recovery in SCI rats, we conducted experiments involving BBB scoring, inclined plane testing, Nissl staining, and HE staining. In the motor function tests, we evaluated the impact of SIRT2 on motor recovery by measuring BBB scores at 3, 7, 14, 21, and 28 days post-spinal cord injury. All rats had a pre-injury BBB score of 21, which dropped to 0 immediately after injury, indicating acute SCI-induced paralysis of the hind limbs. The data analysis revealed an extremely significant main effect of drug treatment (F [3, 8] = 71451, P < 0.0001), accounting for 91.61% of the total variance, indicating it served as the core determinant of the dependent variable. A significant time main effect was also observed (F(5, 40) = 473.5, P < 0.0001), contributing 4.775% to the total variance, demonstrating significant temporal variations in the dependent variable. The drug × time interaction reached extreme significance (F(15, 40) = 116.6, P < 0.0001), explaining 3.529% of the total variance, suggesting significantly divergent temporal trajectories across treatment groups. Notably, inter-individual variability (SD rats) showed no significant impact (F(8, 40) = 0.2119, P = 0.9870), contributing merely 0.003419% to the total variance.Post-hoc analysis using Tukey’s multiple comparisons demonstrated that the SCI + Ad-SIRT2 group exhibited significantly elevated BBB scores compared to the spinal cord injury group at postoperative days 14, 21, and 28.On days 14, 21, and 28, the BBB scores of the SCI + Ad-SIRT2 group were significantly higher than those of the SCI group.In the inclined plane test, statistical results similarly showed an exceptionally strong drug main effect (F [3, 8] = 7588, P < 0.0001), confirming substantial inter-group differences in therapeutic efficacy. The time main effect remained significant (F(5, 40) = 885.0, P < 0.0001), indicating measurable temporal progression of outcomes. The drug × time interaction maintained extreme significance (F(15, 40) = 117.7, P < 0.0001), reaffirming differential temporal patterns across treatment regimens. Individual variability (SD rats) again showed no statistical significance (F(8, 40) = 1.006, P = 0.4471).Tukey’s post hoc multiple comparisons revealed that at postoperative day 28, SCI + Ad-SIRT2-treated rats maintained posture at significantly greater angles on the inclined plane compared to the spinal cord injury group. Notably, the SCI + Ad-control group demonstrated significantly lower postural stability than the SCI + Ad-SIRT2 group (Fig. 4A). Nissl staining results revealed a reduction in the number of neurons in the SCI group compared to the Sham group; however, the SCI + Ad-SIRT2 group had an increased number of neurons compared to the SCI + Ad-control group (Fig. 4B). HE staining results showed severe spinal cord damage in the SCI and SCI + Ad-control groups compared to the Sham group, while the SCI + Ad-SIRT2 group exhibited less severe spinal cord damage than the SCI group (Fig. 4C).Transmission electron microscopy results demonstrated that, in contrast to the Sham group, the SCI group and the SCI + Ad-control group suffered from relatively severe damage. The organelles manifested alterations typical of ferroptotic injury, featuring a greater number of pyknosis and size reduction, a concentrated intramembrane matrix, elevated electron density, as well as diminished cristae.Conversely, the SCI + Ad-SIRT2 group significantly mitigated these pathological alterations. In this group, the mitochondrial structure approximated normalcy, with intact mitochondrial membranes, a homogeneously distributed matrix, and parallel-arranged cristae. (Fig. 4D).

Fig. 4.

Overexpression of SIRT2 promotes neuronal repair and motor function recovery in SCI rats. (A): BBB scores and inclined plane test results at 1, 3, 7, 14, 21, and 28 days post-injury. (B): Nissl staining images of the spinal cord. Scale bar: 50 μm. (C): HE staining images of the spinal cord. Scale bar: 50 μm. (D): Transmission electron microscope images. Scale bar:1 μm (upper figure) and 500 nm (lower figure).(n = 10)(*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. SCI group)

We found that SIRT2 overexpression can attenuate neuronal pathological changes and reduce the severity of SCI. Behavioral test results indicate that SIRT2 overexpression significantly improves motor function impairment.

Discussion

In recent years, the incidence of SCI has shown a significant upward trend, posing a substantial challenge in the prognosis of SCI patients. The mechanisms underlying the onset and progression of SCI, as well as the complexities of neuronal repair and motor function recovery, remain inadequately understood. In this study, our findings demonstrate that the overexpression of SIRT2 promotes the deacetylation of the NF-κB p65 protein, thereby inhibiting NF-κB pathway-mediated ferroptosis and oxidative stress. This, in turn, facilitates neuronal repair and motor function recovery in rats with SCI. Our research builds on studies related to protein modification and ferroptosis. These crucial discoveries suggest that SIRT2 overexpression, through its deacetylation effect on the NF-κB p65 protein, mitigates oxidative stress induced by ferroptosis and plays a vital role in neuronal repair and motor function recovery following SCI.

SCI is a profoundly severe traumatic disorder of the central nervous system. The death of spinal neurons and the interruption of nerve fibers responsible for signal transmission result in impairments in sensory, motor, and autonomic functions [20]. Current treatment strategies predominantly involve surgical interventions; however, there remains a significant lack of effective clinical measures to address the secondary cascade reactions following the initial injury. The secondary damage associated with SCI is a critical focus of clinical treatment, involving multiple forms of programmed cell death, with ferroptosis receiving considerable attention [21]. Recent experimental evidence increasingly suggests a close association between ferroptosis and SCI. Following SCI, a series of ionic imbalances occur, often accompanied by neuroinflammation, lipid peroxidation, oxidative stress, and disrupted iron homeostasis [22]. These damaging mechanisms trigger various forms of programmed cell death, such as apoptosis, autophagy, cuproptosis, and ferroptosis. Iron ions play a pivotal role in neural activities, and iron overload has been shown to correlate positively with the severity of SCI, potentially leading to an imbalance between reactive oxygen species (ROS) and the antioxidant system [23]. During ferroptosis, the accumulation of intracellular lipid peroxides and a diminished capacity to scavenge ROS exacerbate SCI [24]. Inhibiting neuronal ferroptosis can suppress the Fenton reaction, reduce ROS production, and consequently mitigate oxidative damage, thereby alleviating SCI [25]. Recent experiments have shown that iron-dependent lipid peroxidation, a distinctive form of non-apoptotic oxidative cell death, plays a significant role in various neurological disorders, including traumatic central nervous system injuries like SCI [26]. Neurons are particularly susceptible to oxidative stress due to the high content of polyunsaturated fatty acids in their phospholipid membranes, which impairs their ability to eliminate ROS [27]. Therefore, ferroptosis is a critical factor in the progression of SCI. Ge et al. reported that the overexpression of HO-1 inhibits SCI-induced ferroptosis via the Nrf2/SLC7A11/HO-1 pathway [28]. Similarly, Yao et al. demonstrated that bone marrow mesenchymal stem cells mitigate SCI by inhibiting mitochondrial quality control-related neuronal ferroptosis [29]. These studies highlight that modulating neuronal ferroptosis could be a promising therapeutic approach for promoting neuronal repair following SCI.

SIRT2 can modulate intracellular metabolic pathways and reduce oxidative stress, thereby protecting neurons from damage [30]. An increasing body of research indicates that SIRT2 is involved in various neurological disorders. Zhang et al. reported that SIRT2 overexpression alleviates neuropathic pain and neuroinflammation through the deacetylation of the transcription factor nuclear factor-Κb [31]. Yuan et al. found that SIRT2 inhibition exacerbates blood-brain barrier disruption and traumatic brain injury in mouse models [19]. Similarly, Zhao et al. demonstrated that SIRT2 in the spinal cord regulates chronic neuropathic pain in rats via the Nrf2-mediated oxidative stress pathway [32]. In our study, we utilized a recombinant adenovirus to overexpress SIRT2 in PC12 cells. Comparative analysis with other groups revealed that SIRT2 overexpression significantly inhibits Rsl3-induced iron overload and oxidative stress in PC12 cells. Histopathological staining of rat spinal cord tissue indicated that SIRT2 markedly reduces tissue lesions and neuronal damage in rats with SCI. Furthermore, functional motor tests showed that SIRT2 overexpression significantly promotes the recovery of motor function in these rats. Our research is the first to demonstrate, both in vitro and in vivo, that SIRT2 possesses the capability to inhibit neuronal ferroptosis following SCI, thereby mitigating the extent of the injury.

Protein modification refers to the chemical or enzymatic alteration of proteins, changing their structure, function, or state through processes such as phosphorylation, methylation, acetylation, ubiquitination, and glycosylation. Among these, acetylation research holds significant importance [24]. It has been reported that SIRT2 regulates NF-κB signaling and pro-inflammatory gene expression through the deacetylation of the NF-κB p65 protein [33]. Acetylation of NF-κB p65 increases its transcriptional activity, thereby promoting the expression of pro-inflammatory cytokines [34]. SIRT2 overexpression inhibits the inflammatory response in collagen-induced arthritis by enhancing the deacetylation of the NF-κB p65 protein [35]. Recent studies have highlighted a close association between the acetylation of the NF-κB p65 protein and ferroptosis. Yao et al. reported that targeting the LIFR − NF-κB − LCN2 axis can control liver tumorigenesis and vulnerability to ferroptosis [36]. Xu et al. found that during ulcerative colitis, NF-κB p65 plays a crucial role in inhibiting ferroptosis [37]. Wu et al. demonstrated that USP24 upregulates NF-κB expression, exacerbating ferroptosis in diabetic cardiomyopathy [38]. In our study, we further confirmed that the deacetylation of the NF-κB p65 protein can inhibit ferroptosis and oxidative stress. This finding underscores the pivotal role of NF-κB p65 deacetylation in modulating ferroptosis and highlights the potential therapeutic implications of targeting this pathway in diseases characterized by oxidative stress and cell death.

In conclusion, our study elucidates the relationship between ferroptosis and SCI, demonstrating that the overexpression of SIRT2 promotes the deacetylation of the NF-κB p65 protein, thereby inhibiting NF-κB-mediated ferroptosis and facilitating the recovery from SCI. Our research, grounded in the interplay between SIRT2, protein modification, and ferroptosis, employs both in vivo and in vitro experiments to deeply explore the mechanisms by which SIRT2 contributes to neuronal repair and motor function recovery in rats with SCI. These findings provide innovative insights and strategies for the treatment of SCI.

Conclusion

SIRT2 is a potential intervention for treating SCI. It can mitigate spinal cord injury by modulating the acetylation signaling pathway of NF-κB p65, reducing iron-mediated neuronal death post-injury, thereby alleviating SCI and promoting motor function recovery in SCI rats.

Electronic supplementary material

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Abbreviations

SCI

Spinal cord injury

SIRT2

Sirtuin 2

NF

κB-Nuclear factor-κB

GPX4

Glutathione peroxidase 4

4HNE

4-hydroxynonenal

ROS

Reactive oxygen species

SOD

Superoxide dismutase

MPO

Myeloperoxidase

BBB

Basso, Beattie, and Bresnahan

H&E

Hematoxylin and Eosin

GSH

Glutathione

MDA

Malondialdehyde

WB

Western blot

Author contributions

SS, ZZ and HX conceived the idea for the study, led study design and data collection, conducted the statistical analyses, interpreted the data, and drafting of manuscript; GC and ZZ performed the experiments and analyzed data；WT and ZY revised the article for important intellectual content; ZM, GJ and SP collected the data and helped in the statistical analyses. YX supervised the overall study, secured funding, provided critical guidance for search direction, and rigorously revised the manuscript for intellectual content.All authors read and approved the final manuscript and agreed on its submission.

Funding

The authors disclose receipt of the following financial or material support for the research, authorship.and/or publication of this article: This study was supported by Dalian Science and Technology Innovation Fund (2021JJ13SN68).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethical approval

This experimental protocol was approved by the Ethics Committee of the Affiliated Hospital of Zhongshan Hospital (Approval Number: DW2024-063-01).

Competing interests

The authors declare no competing interests.