Resveratrol alleviates neuropathic pain after spinal cord injury by suppressing δ-catenin palmitoylation-mediated membrane accumulation of Nav1.6 in the spinal dorsal horn

Abstract

Neuropathic pain is a severe adverse complication of spinal cord injury (SCI) for which effective therapies are currently lacking. Resveratrol, a natural phytoalexin found in many plants and fruits, has been shown to relieve chemotherapy- and nerve injury-induced neuropathic pain and to promote functional recovery after SCI. In this study, we found that intraperitoneal administration of resveratrol increased the expression and activity of silent information regulator 1 (SIRT1) in the rat spinal dorsal horn and markedly attenuated neuropathic pain after SCI while improving motor recovery. Building on our previous finding that palmitoylation of δ-catenin promotes the membrane trafficking of the voltage-gated sodium channel Nav1.6 in dorsal root ganglion neurons and contributes to oxaliplatin- and nerve injury-induced neuropathic pain, we further examined whether resveratrol produces analgesia by inhibiting δ-catenin palmitoylation-mediated membrane accumulation of Nav1.6 in the spinal dorsal horn after SCI. Our results showed that SCI markedly increased both Nav1.6 membrane localization and δ-catenin palmitoylation in the dorsal horn and intrathecal delivery of Nav1.6 shRNA or pharmacological inhibition of palmitoylation with 2-bromopalmitate significantly attenuated mechanical allodynia after SCI. Resveratrol suppressed SCI-induced δ-catenin palmitoylation and reduced the pathological membrane accumulation of Nav1.6, an effect accompanied by downregulation of the palmitoyl acyltransferase ZDHHC3 (DHHC3). Together, these findings indicate that resveratrol alleviates neuropathic pain after SCI by inhibiting DHHC3-mediated δ-catenin palmitoylation and thereby suppressing the membrane trafficking of Nav1.6 in the spinal dorsal horn.

1. Introduction

Neuropathic pain (NP) is a common and debilitating complication of spinal cord injury (SCI) that affects approximately 40–75% of patients and substantially impairs quality of life.^1,2 Characterized by allodynia and hyperalgesia, ³ SCI-induced NP remains a major clinical challenge, in large part because the cellular and molecular mechanisms that drive neuronal hyperexcitability in the spinal dorsal horn remain incompletely understood.

Voltage-gated sodium (Nav) channels are fundamental determinants of action-potential initiation and neuronal excitability. ⁴ Among the Nav channel subtypes expressed in the adult spinal cord (Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7), Nav1.6 is broadly distributed across all laminae of the spinal cord. ⁵ Increasing evidence indicates that Nav1.6 plays a pivotal role in the development of neuropathic pain by modulating neuronal excitability in the mammalian nervous system. ⁶ Building on our previous findings that membrane upregulation of Nav1.6 in dorsal root ganglion (DRG) neurons drives chemotherapy- or nerve injury-induced pain, ⁷ recent studies indicate that aberrant accumulation of Nav1.6 in the spinal cord also contributes to spasticity after SCI. ⁸ However, the dynamics of membrane Nav1.6 in the spinal dorsal horn during the chronic phase of SCI, and its functional contribution to SCI-induced neuropathic pain, remain poorly understood.

Crucially, the functional output of Nav channels depends not only on their overall expression levels but also critically on membrane surface localization. Emerging evidence implies that the surface localization of Nav channels is governed by scaffold proteins and post-translational modifications. δ-catenin, a neuron-specific adherens junction protein, has been reported to modulate receptor trafficking.^9,10 Our previous studies demonstrated that palmitoylation of δ-catenin promotes the membrane trafficking of Nav1.6 channels in DRG neurons, thereby enhancing neuronal excitability and contributing to neuropathic pain 7. Whether δ-catenin palmitoylation similarly regulates the membrane abundance of Nav1.6 in the spinal dorsal horn after SCI is unknown; if it does, targeting this modification could offer a new therapeutic strategy for SCI-induced neuropathic pain.

Resveratrol, a natural phytoalexin found in many plants and fruits, has anti-inflammatory and neuroprotective effects.^11–14 Accumulating evidence indicates that resveratrol alleviates neuropathic pain induced by nerve injury or chemotherapeutic agents, largely through the activation of silent information regulator 1 (SIRT1).^15–18 Our previous study also showed that resveratrol alleviates chemotherapeutic drug bortezomib-induced neuropathic pain via activation of SIRT1 in the rat spinal dorsal horn. ¹⁹ Notably, SIRT1 is a NAD⁺-dependent deacetylase that regulates multiple post-translational modifications either directly or indirectly by coordinating protein-modifying enzymes and membrane trafficking pathways. ²⁰ Increasing evidence suggests that different post-translational modifications, including palmitoylation and acetylation, exhibit dynamic crosstalk to fine-tune protein stability, localization, and function. ²¹ Because δ-catenin palmitoylation is a key control point for Nav1.6 membrane trafficking and neuronal excitability, SIRT1 activation could plausibly influence the δ-catenin/Nav1.6 axis by modulating palmitoylation-dependent trafficking. Whether resveratrol alleviates SCI-induced neuropathic pain through this axis in the spinal dorsal horn, however, remains unknown.

Therefore, we hypothesized that resveratrol alleviates SCI-induced neuropathic pain by suppressing δ-catenin palmitoylation-mediated Nav1.6 membrane trafficking in the spinal dorsal horn. Accordingly, we examined whether resveratrol attenuates SCI-induced neuropathic pain in association with increased SIRT1 expression and activity, whether it regulates the membrane accumulation of Nav1.6 through δ-catenin palmitoylation, and whether it acts by inhibiting DHHC3-dependent δ-catenin palmitoylation and Nav1.6 membrane trafficking in the spinal dorsal horn.

2. Materials and methods

2.1. Animals

Adult male Sprague-Dawley rats (220-250g) were obtained from the Institute of Experimental Animals of Sun Yat-sen University. The animals were housed in standard cages under controlled conditions (23 ± 1°C, 55 ± 3% humidity) on a 12 h light/dark cycle with food and water available ad libitum. All rats were randomly assigned to experimental groups. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Approval No. SYSU-IACUC-2026-B2081) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study was designed and reported in accordance with the ARRIVE guidelines. Efforts were made to minimize animal suffering and to reduce the number of animals used.

2.2. Experimental models and drug treatment

The SCI model was established as previously described. ²² Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), with adequate anesthetic depth confirmed by loss of the pedal withdrawal reflex, and body temperature was maintained at approximately 37 °C throughout surgery. A moderate compression injury was then produced at the T9 level after a laminectomy that exposed the cord with the dura left intact: a metal rod fitted with a plastic plate was advanced onto the dura with a stereotaxic device at 0.5 mm/min to the predefined depth, held in place briefly, and withdrawn within 1 min. Sham-operated rats underwent the same procedure without cord compression. Perioperative analgesia was standardized across all surgical groups as buprenorphine (0.05 mg/kg, subcutaneously) given 30–60 min before incision and every 12 h for 48 h after surgery.^23,24 Successful establishment of SCI was confirmed by the presence of local spinal cord hematoma, spasmodic tail flicking, and hindlimb twitching immediately after surgery. On postoperative day 3, locomotor function was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Animals with BBB scores < 7 were excluded from subsequent experiments. During and after the operation, all efforts were made to relieve the suffering of the rats used in the study.

For pharmacological interventions, shRNAs targeting rat Nav1.6 and DHHC3 mRNA, as well as scrambled control shRNA, were designed and synthesized by Obio Biotechnology (China) as our previous study reported. ⁷ shRNAs (10 μl) were administered intrathecally once daily for 7 consecutive days, starting 30 min prior to SCI surgery. The broad-spectrum palmitoylation inhibitor 2-bromopalmitate (2-BP; 15 μg in 10 μl; Tocris Bioscience, USA) was first dissolved in DMSO and then diluted in saline to a final concentration of 5% DMSO, yielding a fine suspension, and injected intrathecally, followed by a saline flush; the matched vehicle control received the same volume of 5% DMSO in saline on the same schedule. ²⁵

Resveratrol (Sigma-Aldrich, USA) was administered as a fine suspension in 2% DMSO/98% saline and injected intraperitoneally in a fixed volume of 1 mL per rat, ²⁶ with the concentration of the suspension adjusted to body weight to deliver 50, 100, and 200 mg/kg; the suspension was re-vortexed immediately before each injection, and vehicle-control rats received 1 mL of the same vehicle without resveratrol. Resveratrol and vehicle were given 30 min before SCI surgery and once daily for 7 consecutive days. Drug dosages were chosen on the basis of previously published studies. ²⁷

2.3. Basso, beattie and bresnahan (BBB) score

The hindlimb motor function of rats was evaluated using the BBB locomotor rating scale. ²⁸ Briefly, the rats were acclimated to the open-field environment for 5 min per day for 3 consecutive days prior to testing. On the test day, rats were placed individually in the open field for 5 min, and hindlimb locomotor activity was scored on a scale from 0 to 21 according to the previously described criteria ²⁸ by trained evaluators who were blind to the experimental group. Animals that died before the scheduled endpoint were excluded from analysis.

2.4. Intrathecal injection

Lumbar subarachnoid catheterization for intrathecal drug administration was performed according to a previously described method. ²⁹ In brief, rats were anesthetized, and a laminectomy was performed at the L5 vertebral level. After gentle puncture of the dura mater using a 22-gauge needle, a PE-10 catheter was inserted through the L5/L6 intervertebral space into the subarachnoid space, with the catheter tip positioned at the L5 spinal segment. Rats were allowed to recover for at least 5 days before drug administration. Animals exhibiting motor deficits following catheter implantation were excluded from the study.

2.5. Behavioral assessment

Mechanical allodynia was assessed with the von Frey test. ³⁰ Briefly, rats were placed in the testing environment for 15 min daily for three consecutive days. On the test day, the rats were tested using von Frey filaments with a specific bending force to the midplantar surface of the hind paw. A brisk paw withdrawal or paw flinching after filament application was defined as a positive nociceptive response. Each test was performed three times at 2 min intervals. The 50% paw-withdrawal threshold was determined using the validated up-down method. ³¹ All behavioral testing was performed by an experimenter blinded to treatment.

2.6. Assay of SIRT1 activity

The activity of SIRT1 was determined by using the SIRT1 fluorometric kit (Abcam, UK). Briefly, after the protein was extracted from L4 and L5 spinal dorsal horn tissues, nuclear extract was purified by immunoprecipitation using a rabbit anti-SIRT1 antibody (Elabscience Biotechnology, China) and Protein A Agarose Beads (Cell Signaling Technology Inc, USA). The reaction mixture was added, and the NAD-dependent deacetylase activity was assessed according to the manufacturer’s instructions. Fluorescence intensity was recorded at excitation/emission wavelengths of 355/460 nm every 2 min for 60 min using a microplate reader (Thermo Scientific, USA).

2.7. RNA extraction and real-time quantitative PCR

Total RNA was harvested from L4 and L5 spinal dorsal horn tissues of rats using Trizol reagent (Invitrogen, USA). Evo M-MLV reverse transcriptase Premix (AG, China) was used to perform the reverse transcription reactions. The synthesized cDNA from total RNA was evaluated by specific primers using SYBR Green Premix qPCR Kit (AG, China). The calculation method of relative expression ratio of mRNA was the 2^^−△△CT method. ³² Sequences of the primers used for PCR were shown in Table 1.

Table 1.

Primer sequences used for quantitative real-time PCR.

Gene	Primer	Sequence
β-actin (rat)	Forward	5′-AGG GAA ATC GTG CGT GAC AT-3′
​	Reverse	5′-GAA CCG CTC ATT GCC GAT AG-3′
SIRT1 (rat)	Forward	5′-TTG GCA CCG ATC CTC GAA-3′
​	Reverse	5′-ACA GAA ACC CCA GCT CCA-3′

Abbreviation: qPCR, quantitative real-time PCR.

2.8. Western blotting

Western blotting was performed as described previously. ³³ Briefly, L4 and L5 spinal dorsal horn tissue was homogenized in lysis buffer, and protein concentrations were determined. For membrane Nav1.6, a plasma-membrane–enriched fraction was prepared with a Plasma Membrane Protein Extraction Kit (Invent Biotechnologies, USA). Equal amounts of protein were separated by gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. Membranes were pre-incubated with blocking buffer for 1 h at room temperature and then probed with primary antibodies against SIRT1 (1:2000, #ab110304, Abcam, UK), Nav1.6 (1:200, #ASC-009, Alomone Labs, Israel), GAPDH (1:1000, #SC-32233, Santa Cruz, USA), and transferrin receptor (TfR; 1:1000, #ab84036, Abcam, UK) at 4 °C overnight. Next, membranes were incubated in HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized in the membranes by chemiluminescent HRP substrate. Bands were quantified in ImageJ (NIH); membrane Nav1.6 was normalized to transferrin receptor (TfR) and total-protein targets to GAPDH.

2.9. Statistical analysis

All data are presented as mean ± SEM and were analyzed using SPSS version 25.0. Data were analyzed using an unpaired two-tailed Student’s t-test (two independent samples), two-way repeated-measures ANOVA, or one-way ANOVA followed by Tukey’s post hoc test when variances were equal or Dunnett’s T3 test when variances were unequal. Data that did not meet normality were analyzed by the permutation test. A P value of < 0.05 was considered statistically significant.

3. Results

3.1. SCI impairs functional motor recovery, reduces paw withdrawal threshold and downregulates SIRT1 expression and activity in the spinal dorsal horn

Previous evidence shows that SCI caused a decrease in SIRT1 expression in the spinal cord, and that silencing SIRT1 in the spinal cord aggravated SCI.^34,35 Similarly, studies also indicate that upregulating SIRT1 expression in the spinal cord by resveratrol exerted neuroprotective effects on SCI.^15,16 To confirm whether SIRT1 participated in SCI-induced neuropathic pain, the rat model of spinal cord compression injury was established, and functional motor recovery, pain behavior, and SIRT1 expression were assessed. The BBB scores utilized to determine locomotor recovery were significantly decreased on days 3, 7, 14 and 21 post-SCI (Figure 1(a)). Similarly, mechanical paw withdrawal thresholds (PWT) utilized to determine mechanical allodynia were markedly reduced on days 7, 14 and 21 post-SCI (Figure 1(b)). In addition, the expression and activity of SIRT1 were significantly downregulated in the spinal dorsal horn of rats. Notably, the time course of this SIRT1 decline paralleled the development of mechanical allodynia (Figure 1(c)–(e)). These results suggest a potential association between SIRT1 function and SCI-induced neuropathic pain.

Figure 1.

SCI impairs motor functional recovery, induces mechanical allodynia, and decreases the activity of SIRT1 in the spinal dorsal horn. (a) Hindlimb motor function assessed by the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale shows a significant decrease in rats after SCI (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group). (b) Mechanical allodynia was evaluated by measuring the 50% paw withdrawal thresholds. SCI rats exhibited a significant reduction in paw withdrawal thresholds compared with sham-operated rats (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group). (c) SIRT1 activity, measured by a fluorometric assay, was significantly decreased in the spinal dorsal horn on days 7, 14 and 21 after SCI (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group). (d) and (e) SIRT1 mRNA and protein expression, assessed by RT-qPCR and western blotting, respectively, were significantly reduced in the spinal dorsal horn on days 7, 14, and 21 after SCI (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group).

3.2. Resveratrol promotes functional motor recovery and alleviates neuropathic pain after SCI in association with increased SIRT1 expression and activity

Resveratrol is known to activate SIRT1 and attenuate chronic constriction injury (CCI)-induced neuropathic pain. ³⁶ To explore the effect of resveratrol on SCI-induced neuropathic pain, functional motor recovery and mechanical allodynia at different time points were assessed in SCI rats following resveratrol intervention. The results showed that resveratrol significantly promoted functional motor recovery and attenuated mechanical allodynia after SCI (Figure 2(a) and (b)). To investigate the mechanism, the expression and activity of SIRT1 were further examined in the spinal dorsal horn of rats with resveratrol administration on day 14 post-SCI. The results showed that resveratrol reversed the decrease in SIRT1 expression and activity in the spinal dorsal horn of rats after SCI (Figure 2(c)–(e)). In summary, resveratrol intervention significantly improved locomotion function and alleviated mechanical allodynia after SCI, in association with increased SIRT1 expression and activity in the spinal dorsal horn.

Figure 2.

Resveratrol improves motor function recovery, attenuates mechanical allodynia, and restores SIRT1 activity in the spinal dorsal horn after SCI. (a) Resveratrol administered intraperitoneally (i.p.) at the dose of 100 mg/kg and 200 mg/kg, but not 50 mg/kg, significantly increased BBB score after SCI compared with the vehicle-treated SCI rats (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group); # p < 0.05, ## p < 0.01 and && p < 0.01 (vs. vehicle-treated SCI group). (b) Resveratrol (100 mg/kg, i.p.) significantly attenuated the mechanical allodynia after SCI compared with the vehicle-treated SCI rats (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group). (c) Resveratrol (100 mg/kg, i.p.) significantly increased SIRT1 activity in the spinal dorsal horn on day 14 after SCI compared with the vehicle-treated SCI rats (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group). (d) and (e) Resveratrol (100 mg/kg, i.p.) significantly upregulated SIRT1 mRNA and protein expression in the spinal dorsal horn on day 14 after SCI compared with vehicle-treated SCI rats (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle- treated SCI group).

3.3. Resveratrol suppresses the pathological Nav1.6 membrane localization and alleviates SCI-induced neuropathic pain

Studies demonstrate that resveratrol suppresses Nav1.7 expression in the DRG and alleviates neuropathic pain in CCI rats, ³⁶ and elevated Nav1.6 in spinal lumbar motoneurons of adult rats with SCI promotes spasticity. ⁸ Meanwhile, our previous study showed that increased membrane trafficking of Nav1.6 in sensory neurons promotes neuropathic pain. Here, this study focused on whether resveratrol exerted an analgesic effect on SCI-induced neuropathic pain by regulating the membrane localization of Nav1.6 in the spinal dorsal horn. First, the expression of membrane Nav1.6 in the spinal dorsal horn of SCI rats was determined after resveratrol administration. The results showed that resveratrol significantly suppressed the upregulation of membrane Nav1.6 expression in the spinal dorsal horn after SCI (Figure 3(a)). In addition, intrathecal injection of Nav1.6 shRNA inhibited the upregulation of membrane Nav1.6 in the spinal dorsal horn after SCI and attenuated SCI-induced neuropathic pain (Figure 3(b) and (c)). These results suggest that resveratrol intervention significantly suppressed the increase in membrane Nav1.6 in the spinal dorsal horn, thereby attenuating SCI-induced mechanical allodynia.

Figure 3.

Resveratrol alleviates mechanical allodynia by suppressing SCI-induced upregulation of membrane Nav1.6 in the spinal dorsal horn. (a) Resveratrol (100 mg/kg, i.p.) significantly reduced SCI-induced increases in membrane Nav1.6 in the dorsal horn (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group). (b) Nav1.6 shRNA (i.t.) significantly inhibited SCI-induced upregulation of membrane Nav1.6 in the spinal dorsal horn (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham + scramble shRNA group), && p < 0.01 (vs. scramble shRNA-treated SCI group). (c) Nav1.6 shRNA (i.t.) significantly attenuated SCI-induced mechanical allodynia (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. scramble shRNA-treated SCI group).

3.4. Resveratrol suppresses δ-catenin palmitoylation-mediated membrane accumulation of Nav1.6 after SCI

Our previous study revealed that palmitoylation of δ-catenin increases membrane trafficking of Nav1.6 in sensory neurons to promote oxaliplatin- or nerve injury-induced neuropathic pain 7. Here, palmitoylation of δ-catenin in the spinal dorsal horn of SCI rats was examined after resveratrol injection. The results showed that resveratrol significantly suppressed SCI-induced δ-catenin palmitoylation in the spinal dorsal horn (Figure 4(a)). In addition, intrathecal administration of the broad-spectrum palmitoylation inhibitor 2-bromopalmitate (2-BP) markedly attenuated SCI-induced mechanical allodynia and the increase of membrane Nav1.6 expression in the spinal dorsal horn induced by SCI (Figure 4(b) and (c)). Overall, resveratrol suppressed δ-catenin palmitoylation-mediated membrane accumulation of Nav1.6 after SCI, thereby attenuating mechanical allodynia.

Figure 4.

Resveratrol suppresses δ-catenin palmitoylation-mediated upregulation of membrane Nav1.6 after SCI. (a) Resveratrol (100 mg/kg, i.p.) significantly reduced SCI-induced increase in δ-catenin palmitoylation in the spinal dorsal horn (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group). Membrane Nav1.6 was normalized to transferrin receptor (TfR); full-length uncropped blots are provided as Source Data. (b) 2-BP (i.t.) relieved SCI-induced mechanical allodynia (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group). (c) 2-BP (i.t.) significantly inhibited SCI-induced upregulation of membrane Nav1.6 in the spinal dorsal horn (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group).

3.5. Resveratrol inhibits δ-catenin palmitoylation in the spinal dorsal horn after SCI by decreasing DHHC3 expression

Based on our previous results that DHHC3-mediated palmitoylation of δ-catenin in the DRG plays an important role in oxaliplatin- or nerve injury-induced neuropathic pain, ⁷ we examined the expression of DHHC3 in the spinal dorsal horn of SCI rats following resveratrol treatment. The results showed that resveratrol significantly suppressed the SCI-induced increase of DHHC3 in the spinal dorsal horn (Figure 5(a)). In addition, intrathecal administration of DHHC3 shRNA markedly inhibited δ-catenin palmitoylation in the spinal dorsal horn and attenuated mechanical allodynia after SCI (Figure 5(b) and 5(c)). In summary, resveratrol intervention significantly inhibited δ-catenin palmitoylation in the spinal dorsal horn after SCI by decreasing DHHC3 expression, thereby attenuating SCI-induced mechanical allodynia.

Figure 5.

Resveratrol inhibits δ-catenin palmitoylation by suppressing DHHC3 upregulation after SCI. (a) Resveratrol (100 mg/kg, i.p.) significantly reduced SCI-induced upregulation of DHHC3 in the spinal dorsal horn (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. vehicle-treated SCI group). (b) DHHC3 shRNA (i.t.) significantly reduced SCI-induced increase in δ-catenin palmitoylation in the spinal dorsal horn (n = 3). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. scramble shRNA-treated SCI group). (c) DHHC3 shRNA (i.t.) significantly attenuated SCI-induced mechanical allodynia (n = 6). Data are presented as mean ± SEM. **p < 0.01 (vs. sham group), && p < 0.01 (vs. scramble shRNA-treated SCI group).

4. Discussion

Spinal cord injury-induced neuropathic pain is a complex process that involves altered gene expression, neuroinflammation, and neuronal hyperexcitability throughout the nervous system.^37–39 Resveratrol, a natural phytoalexin and a known activator of SIRT1, has been reported to exert analgesic and anti-inflammatory effects in various neuropathic pain models.^{18,36,40–42} In the current study, we provide novel mechanistic insights into the therapeutic effects of resveratrol on SCI-induced neuropathic pain. We demonstrated that systemic administration of resveratrol not only improved functional motor recovery but also significantly attenuated mechanical hypersensitivity. The core finding of our work is that resveratrol exerts its analgesic effects by enhancing SIRT1 expression and activity in the spinal dorsal horn and suppressing the membrane localization of Nav1.6. This is consistent with our previous findings showing that resveratrol alleviates chemotherapy-induced neuropathic pain through SIRT1 activation, ¹⁹ as well as other reports indicating its neuroprotective actions via SIRT1/NF-κB and SIRT1/AMPK signaling pathways following SCI,^41,42 consistent with an important role for SIRT1 signaling in SCI. Because the behavioral readout was hind-paw mechanical sensitivity, the molecular assays were performed on the L4 and L5 dorsal horn rather than at the T9 lesion site, because the lumbar enlargement receives hind-paw primary afferent input^43,44 and shows below-level neuroinflammatory and excitability changes after thoracic SCI. ⁴⁵

A key advance of our study is the identification of Nav1.6 as a downstream target of resveratrol. Whereas earlier work on resveratrol and sodium channels focused on Nav1.7, ³⁶ we found that SCI leads to a marked upregulation of Nav1.6 on the neuronal membrane, which contributes to pain maintenance. Consistent with this, intrathecal knockdown of Nav1.6 attenuated SCI-induced hypersensitivity, and resveratrol reduced the pathological membrane abundance of Nav1.6.

Mechanistically, our data revealed that resveratrol produced analgesia by suppressing δ-catenin palmitoylation, a process that occurred in association with increased SIRT1 expression and activity, although we did not test a direct causal role for SIRT1. SCI-induced Nav1.6 membrane upregulation was mediated by increased palmitoylation of δ-catenin, a neuron-specific adherens junction protein highly expressed in the central nervous system.^7,46–48 Inhibition of δ-catenin palmitoylation by 2-BP, the broad-spectrum palmitoylation inhibitor, suppressed Nav1.6 membrane trafficking and alleviated mechanical hypersensitivity, indicating that palmitoylation-dependent regulation of Nav1.6 represents an important pathological process after SCI. Resveratrol likewise reduced δ-catenin palmitoylation and limited the membrane accumulation of Nav1.6 in the spinal dorsal horn after SCI. Consistent with a palmitoylation-based mechanism, S-palmitoylation of Nav1.6 itself modulates channel activity and neuronal excitability. ⁴⁹

To further elucidate the upstream mechanism regulating δ-catenin palmitoylation, we focused on DHHC3, a palmitoyl acyltransferase. Our previous work indicated that DHHC3 plays a pivotal role in regulating δ-catenin palmitoylation in the dorsal root ganglion (DRG), and its suppression relieves oxaliplatin- or nerve injury-induced neuropathic pain 7. This study further shows that DHHC3 was markedly upregulated in the dorsal horn following SCI and local knockdown of DHHC3 by intrathecal administration of DHHC3 shRNA significantly prevented δ-catenin palmitoylation in the spinal dorsal horn and mechanical allodynia after SCI. Moreover, to our knowledge, our study provides evidence that resveratrol alleviates SCI-induced neuropathic pain by targeting the DHHC3/δ-catenin palmitoylation pathway, thereby inhibiting Nav1.6 membrane trafficking in the dorsal horn. This newly identified mechanism highlights palmitoylation modulation as a potential therapeutic strategy for neuropathic pain management.

Several limitations should be acknowledged. First, resveratrol is a multi-target compound, and although we identified the DHHC3/δ-catenin/Nav1.6 axis as an important target, it may also act through other pathways. Future studies using more specific genetic or pharmacological approaches are needed to further determine the relative contribution of this pathway compared with other potential mechanisms. Second, resveratrol increased SIRT1 expression and activity, but we did not knock down SIRT1 during treatment, so the SIRT1-DHHC3/δ-catenin/Nav1.6 sequence remains a proposed model rather than a causally established pathway. Third, our interventions began before injury, so the study addresses prevention of SCI-induced changes rather than reversal of established chronic pain, and post-onset dosing studies will be needed before efficacy against established neuropathic pain can be claimed. Fourth, we did not perform a trafficking-selective rescue experiment for Nav1.6, and 2-bromopalmitate inhibits palmitoylation broadly with recognized off-target effects, ⁵⁰ so these interventions implicate but do not isolate Nav1.6 trafficking. Finally, although the von Frey response is primarily a spinal reflex, severe motor impairment after SCI can affect paw placement, ⁵¹ so a residual contribution of improved motor function to the sensory outcomes cannot be fully excluded. The translational potential and optimal dosing of resveratrol in patients also require further study.

In conclusion, our findings suggest that resveratrol, as a phytotherapeutic compound, may help prevent the development of neuropathic pain after SCI by inhibiting DHHC3-mediated δ-catenin palmitoylation and reducing the membrane accumulation of Nav1.6, although studies using post-injury dosing will be needed to establish efficacy against established neuropathic pain. These results are consistent with the role of SIRT1 in pain relief reported in chemotherapy-induced pain models, ¹⁹ and they point to palmitoylation-dependent regulation of Nav1.6 as a potential therapeutic avenue for SCI-associated neuropathic pain.

ORCID iDs

Ling-Jun Xu https://orcid.org/0000-0003-0959-8363

Cui-Cui Liu https://orcid.org/0000-0002-5960-5851

Ethical considerations

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Approval No. SYSU-IACUC-2026-B2081) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Data availability statement

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