2.1 Animals

Adult male Sprague–Dawley rats (weighing 200–240 g, supplied from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China) were used in this study. Rats were randomly grouped and housed in an environment with a temperature (23 ± 2°C), relative humidity (45%–65%), lighting (12-h light/dark cycle). All rats must acclimate to standard conditions for at least 7 days upon arrival, with free access to food and water. Animal procedures were approved by the Ethics Committee of Tongji hospital, Tongji Medical College, Huazhong University of Science and Technology (Number: TJH-202306019) and in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and ARRIVE Guidelines for Reporting Animal Research. All experiments were completed in the Experimental Medicine Research Center of Tongji hospital.

2.2 Chemicals and Reagents

VitD₃ (Calcitriol, cat: HY-10002, purity > 95%) was dissolved in a solution containing 10% DMSO and 90% corn oil. RSL3 (cat: HY-100218A), an activator of ferroptosis through inactivate GPX4, was dissolved in 10% DMSO and 90% corn oil. The PKCα inhibitor bisindolylmaleimide I (BIM-1, cat: HY-13867) was dissolved in 10% DMSO and 40% PEG300 and 5% Tween-80 in sterile saline. The PKCα agonist phorbol 12-myristate 13-acetate (PMA, cat: HY-18739) was dissolved in 10% DMSO and 40% PEG300 and 5% Tween-80 in sterile saline. The vehicles were formulated on the basis of the corresponding solvent medium. All the chemicals employed in the present study were purchased from Med Chem Express (Shanghai, China). Iron Assay Kit (cat: A039-2-1), malondialdehyde (MDA) Assay Kit (cat: A003-1-1), ROS Assay Kit (cat: E004-1-1), Superoxide Dismutase (SOD) Assay Kit (cat: A001-1-1), GSH Assay Kit (cat: A006-2-1), and ATP Assay Kit (cat: A095-1-1) were purchased from Jiancheng Biology (Nanjing, China). Perl's staining Kit was purchased from Servicebio (Wuhan, China). The antibodies used in the molecular studies were showed in Table 1.

2.3 Experimental Design

In the present study, all rats were randomly allocated to different groups using a computer-generated random number table. All the experimental procedures and analyses were blindly performed by researchers unaware of the group assignments. Five main experiments were designed to investigate the analgesic mechanism of VitD₃ in neuropathic pain.

2.4 Neuropathic Pain Model

The SNI rat model of neuropathic pain was induced as previously described [30]. Briefly, the rats were anesthetized with 2.5% isoflurane and were placed in a left decubitus position; an incision of approximately 1 cm was made at the mid-thigh level, the left sciatic nerve and its three branches (the common peroneal, tibial, sural nerves) were carefully exposed and separated. Then, the common peroneal and tibial nerves were ligated together and transected distally, leaving the sural nerve intact. The skin incision was sutured with 3.0 silk and disinfected. The sham-operated animals underwent exactly the same procedure without nerve injury.

2.5 Behavioral Testing

The mechanical allodynia was evaluated by measuring the hind PWT in response to von Frey filament stimuli, as previously described [31]. In brief, rats were individually positioned in test cages with a mesh floor and habituated for 30 min before von Frey testing. The von Frey filaments ranging from 1.4 to 15 g were vertically applied in ascending order on the mid-plantar of the hind paw, bending the filament for 3–5 s. A positive response was defined as quick paw withdrawal, licking, or shaking. The lowest force required to elicit a positive response was recorded as PWTs. All behavioral tests were performed between 9:00 a.m. and 6:00 p.m. by an investigator blinded to the experimental design.

2.6 Intrathecal Catheterization and Drug Administration

For intrathecal drug administration, the intrathecal catheterization was completed at least 5 days prior to sham or SNI surgery as previously reported [32]. Rats were deeply anesthetized with isoflurane (2.5%) and implanted with PE-10 catheters (inner diameter 0.3 mm, outer diameter 0.6 mm) into the intrathecal space at the L4-L6 level. The correct position of the catheter was confirmed by the tail-flick response immediately following the insertion of the catheter. After surgery, 2% lidocaine (10 μL) was delivered through the intrathecal catheter, and both hind limbs of the rats showed temporary paralysis, indicating the success of catheterization. Animals with visible motor dysfunction were excluded from the experiment. In this study, we aimed to investigate a purely spinal mechanism. All drugs were intrathecally injected at 10 μL, followed by 10 μL saline for flushing. Detailed experimental plans for animal assignment and medication are outlined in the respective timeline diagram.

2.7 Western Blot

Under deep anesthetized, the L4-L6 spinal cord was quickly removed and homogenized in ice-cold RIPA lysis-buffer containing a cocktail of protease and phosphatase inhibitors. The lysates were then centrifuged at 12,000 rpm at 4°C for 15 min, and the protein concentration of supernatants was measured using BCA method. Equal amounts of protein samples (40 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gels electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h at RT, the membranes were incubated with the primary antibodies (Table 1) overnight at 4°C. Following washes in TBST three times, the blots were incubated with an HRP-conjugated secondary antibody for 2 h at RT and visualized with Super Lumia ECL Plus HRP Substrate Kit (Abbkine). The bolts were scanned and quantified using the ChemiDoc XRS System (Bio-Rad, CA, USA). The intensity of bands was normalized to the β-Actin and expressed as fold changes over the control group. For successive antibody incubations, the primary and secondary antibodies were removed using a commercial stripping solution (Epizyme Biotech, Shanghai, China), and the membranes were re-immunoblotted for additional molecules.

2.8 Paraffin Section Preparation

After deep anesthesia with 2.5% isoflurane, rats were intracardially perfused with ice-cold 0.1 M PBS followed by 4% paraformaldehyde in PBS. Subsequently, the L4-L6 spinal cord segments were harvested, fixed in 4% PFA at room temperature, dehydrated, and embedded in paraffin. Four-micrometer thick paraffin sections were used for immunofluorescence and Perl's staining.

2.9 Double-Labeling Immunofluorescence

After deparaffinization and hydration, the sections were microwaved in 0.01 M sodium citrate (PH 6.0) for antigen retrieval. The sections were penetrated with 0.3% TritonX-100 for 15 min and blocked with 5% donkey serum for 1 h at RT. Then, the sections were incubated with the primary antibodies (Table 1) overnight at 4°C. After washing with PBS three times, the sections were incubated with the secondary antibodies (Table 1) for 2 h at RT. The nuclei were stained with DAPI for 5 min, and images were captured using a fluorescence microscope (BX51, OLYMPUS, Japan).

2.10 Perl's Staining and Iron Content Detection

Iron deposition was measured in spinal cord by Perl's staining. Perl's staining working solution was added to the deparaffinized and hydrated sections. Then, hemosiderin or ferric iron staining was observed under the microscope. The concentration of Fe²⁺ in the spinal cord expressed as nmol/g protein was measured with a kit assay according to the manufacturer's protocols.

2.11 ROS, MDA, GSH, and SOD Measurements

The contents of ROS, MAD, GSH, and SOD in spinal cord tissues were detected using commercial assay kits according to the manufacturer's protocol. The content of MDA, GSH, and SOD was expressed as nmol/g protein, μmol/g protein, and U/g protein, respectively.

2.12 Measurement of ATP Levels

The ATP levels of the spinal cord were calculated with an assay kit following the manufacturer's operation manual. The concentration of ATP was expressed in the form of μmol/g protein.

2.13 Transmission Electron Microscope

Rats were deeply anesthetized and transcardially perfused with 0.1 M PBS followed by 4% ice-cold PFA, the spinal cord was dissected out and sliced into 1 mm² sections. The sections were fixed in 2% glutaraldehyde, post-fixed in 1% aqueous osmium tetraoxide for 2 h at RT, dehydrated in gradual ethanol, and embedded in epoxy resin. Ultrathin slides were obtained using ultra-micro-tome (Leica, Germany) and double-stained with uranyl acetate and lead citrate. Images were captured by transmission electron microscopy (Thermo, USA).

2.14 Statistical Analysis

All experimental data are presented as mean ± SD and analyzed using GraphPad Prism version 6 (Graph Pad Software, San Diego, CA, USA). All data underwent normality testing using either the Shapiro–Wilk test. Differences in behavioral scores among groups were analyzed by two-way repeated measures analysis of variance (ANOVA), followed by Bonferroni's post hoc test. In other trails, Student's t-tests were used for comparisons between two groups, and one-way ANOVA followed by the Bonferroni test was used for comparisons between multiple groups. For all statistical analyses, p < 0.05 was interpreted as statistically significant.

3.1 SNI Induces Mechanical Allodynia and Ferroptosis in the Spinal Cord

In this study, we adopt SNI rats as an animal model to investigate neuropathic pain, with a focus on mechanical allodynia. The timeline of the surgery and behavior tests is depicted in Figure 1A. There is no significant difference at baseline between the sham and SNI groups regarding the ipsilateral PWT. In contrast to the sham control, SNI rats exhibited a significant reduction in PWT from Day 3 to Day 14 after SNI (Figure 1B), indicating that the SNI surgery successfully evoked significant mechanical hypersensitivity.

To determine the potential involvement of ferroptosis in the development of SNI-induced neuropathic pain, we first examined the time-course expression of the biochemical hallmarks of ferroptosis in the spinal cord on Days 3, 7, and 14 after SNI. As shown in Figure 1C, the spinal levels of Fe²⁺, ROS, and MDA increased on Day 3 after SNI and continued to rise over time. Conversely, the activity of GSH and SOD, the main component of the antioxidant defense system in ferroptosis [33], persistently decreased in the spinal cord of SNI rats compared with the sham control. Perl's staining revealed significant iron deposition that was represented as blue spots in the ipsilateral spinal dorsal horn of SNI rats, when compared to the sham control (Figure 1D). We also observed the dynamic changes of key regulator of ferroptosis, such as SLC7A11 and GPX4, both of which significantly decreased in the spinal cord of SNI rats (Figure 1E,F). These results consistently suggest that ferroptosis is involved in the development of neuropathic pain at the spinal cord level.

3.2 The Analgesic Effect of VitD₃ in SNI Rats

To explore whether VitD₃ could alleviate mechanical allodynia induced by SNI, calcitriol was intrathecally injected into sham and SNI rats. The timeline of this experiment is shown in Figure 2A. Firstly, a different selected dose of calcitriol (0.5, 1, or 2 μg) was given on Day 7 post-surgery. The von Fery test was performed before calcitriol injection and at 1, 2, 4, and 6 h after the first injection. As illustrated in Figure 2B, calcitriol treatment significantly increased the ipsilateral PWT in SNI rats at an effective dose of 1 or 2 μg compared with the SNI + Vehicle group. The effect peaked at 2 h after administration and persisted for at least 4 h. However, a low dose of calcitriol (0.5 μg) was devoid of any activity. Then, to determine whether repeated treatment with calcitriol has cumulative analgesic effects, calcitriol (1 μg) was administrated once daily for 5 consecutive days starting from Day 7 (Figure 2C). The behavioral test was conducted before and 2 h after calcitriol injection. As shown in Figure 2D, repeated calcitriol injection significantly reversed the established mechanical allodynia in SNI rats without tolerance. These results indicate that VitD₃ effectively ameliorates neuropathic pain in a dose-dependent manner.

VitD₃ exerts its biological functions through ligand activation of VDR, which is widely expressed in the peripheral and central neurons involved in pain-sensing and processing [34]. Therefore, we examined the effect of calcitriol on VDR expression in the spinal cord of SNI rats. As shown in Figure 2E,F, western blot results displayed decreased levels of VDR caused by SNI, partially reversed by repeated treatment with calcitriol. Then, to identify the cell type expressing VDR in the spinal dorsal horn, we examined the colocalization of VDR with the markers specific for astrocytes (GFAP), microglial (IBA-1), and neurons (NeuN) by double immunofluorescence staining. The results revealed that VDR predominantly colocalized with NeuN in the spinal dorsal horn, and hardly showed any overlap staining with either GFAP or Iba-1 (Figure 2G). These findings suggest that VDR in spinal dorsal neurons may play a role in the analgesic effects of VitD₃.

3.3 VitD₃ Attenuates Ferroptosis and GABAergic Interneuron Loss in the Spinal Cord of SNI Rats

To investigate whether VitD₃ could inhibit the enhanced ferroptosis induced by SNI, we assessed the changes in ferroptosis-related indicators in the spinal cord. As illustrated in Figure 3A, treatment with calcitriol significantly alleviated the increased level of Fe²⁺, ROS, and MDA in the spinal cord compared with the SNI + Vehicle group. Meanwhile, the suppressed activity of GSH and SOD was reversed by calcitriol treatment. Furthermore, western blot analysis revealed that calcitriol treatment significantly hindered the SNI-induced downregulation of GPX4 and SLC7A11 (Figure 3B,C). These data support the resistant role of VitD₃ in ferroptosis under neuropathic pain conditions.

Considering the neuroprotective effect of VitD₃ in neurodegenerative disease [22], we investigated whether VitD₃ could reduce SNI-induced neuronal injury, particularly the GABAergic interneurons in the spinal cord. Glutamic acid decarboxylase 65 (GAD65) is a GABA-synthesizing enzyme that can be used to indicate the number and functional changes of GABA interneurons [35]. Consequently, we observed a statistically significant decrease in the protein levels of GAD65 in the spinal cord following SNI, and treatment with calcitriol markedly enhanced the expression of GAD65 (Figure 3D,E). To deeply explore whether the protective effect on GABA neurons was attributed to the inhibition of ferroptosis by calcitriol, the ferroptosis inducer RSL3 (5 μg) combined with calcitriol was intrathecally injected into SNI rats and then measured the pain behavior. The timeline of this test is displayed in Figure 3F. As shown in Figure 3G, the behavioral test showed that the RSL3 treatment partially abolished the antinociceptive effect of calcitriol. Moreover, western blot results revealed that administration of RSL3 significantly hindered the upregulation of GAD65 and GPX4 induced by calcitriol treatment (Figure 3H,I). These data hint that VitD₃ protects the spinal GABAergic interneuron against ferroptosis, thereby attenuating neuropathic pain.

3.4 VitD₃ Mitigates SNI-Induced Mitochondrial Dysfunction in the Spinal Cord

Mitochondrial metabolism dysfunction participates in governing ferroptosis and is implicated in the pathogenesis of neuropathic pain [36, 37]. Here, we evaluated the effect of VitD₃ on the activity of mitochondrial oxidative metabolism in the spinal cord. Initially, we measured the ATP content in the spinal cord of sham and SNI rats. We observed that the levels of ATP were time-dependently declined from Day 3 to Day 14 following SNI, a phenomenon that was reversed by calcitriol treatment (Figure 4A,B). In the mitochondrial metabolic pathway, the mitochondrial ETC consists of five protein complexes and plays a pivotal role in ATP production. Subsequently, we analyzed the protein levels of the five mitochondrial ETC complex subunits. As shown in Figure 4C–H, western blot results showed that calcitriol treatment significantly restored the SNI-induced downregulation of ETC Complex III, IV, and V (UQCRC2, MTCO1, and ATP5F1A), as compared to the SNI + Vehicle group. However, the protein levels of Complex I and II (NDUFB8 and SDHB) unexpectedly didn't meet statistical differences. Furthermore, the mitochondrial ultrastructure in spinal neurons by using TEM exhibited swollen mitochondria, cristae rupture, and greater membrane density in response to SNI, which were reversed by calcitriol treatment (Figure 4I). These findings indicate that VitD₃ could regulate the biological activities of mitochondria in the spinal cord.

3.5 PKCα/NOX4 Signaling Pathway Is Involved in the Development of SNI-Induced Neuropathic Pain

It was previously reported that PKCα contributed to pain hypersensitivity in chronic inflammatory pain [38]. Moreover, NOX4 is a downstream effector of PKCα that generates ROS and is also involved in pain processing [39]. Accordingly, we examined the levels of p-PKCα, PKCα, and NOX4 in the spinal cord by western blot. As illustrated in Figure 5A,B, compared with the sham control, the protein expression of p-PKCα, PKCα, and NOX4 consistently increased in the spinal cord from Day 3 to Day 14, peaking on Day 7. Additionally, double immunostaining showed that both p-PKCα and NOX4 were predominately colocalized with neurons in the spinal dorsal horn, but rarely with astrocytes or microglia (Figure 5C,D). These results suggest that the PKCα/NOX4 signaling pathway may function in spinal dorsal neurons in pain processing.

To confirm the role of the PKCα/NOX4 signaling pathway in SNI-induced neuropathic pain, we applied a pharmacological approach using the broad-range PKCα inhibitor BIM-1. The timeline of this test is depicted in Figure 5E. BIM-1(100 μM) was intrathecally injected for 5 consecutive days starting from Day 7 after SNI, and the behavioral test was conducted before and 1.5 h after BIM-1 injection. As illustrated in Figure 5F, the behavioral test showed that the PWT was significantly increased in BIM-1-treated rats compared with the vehicle-treated rats. Importantly, administration of BIM-1 markedly reduced the protein expression of p-PKCα, PKCα, and NOX4 in the spinal cord (Figure 5G,H). These results suggest that the spinal PKCα/NOX4 signaling pathway plays a functional role in neuropathic pain.

3.6 Pharmacological Inhibition of PKCα/NOX4 Signaling Pathway Suppressed Ferroptosis and Mitochondrial Dysfunction

Next, to test whether the PKCα/NOX4 signaling pathway contributed to SNI-induced ferroptosis and mitochondrial dysfunction, we assessed the changes in ferroptosis-related indexes in the spinal cord after intrathecal injection with BIM-1. As shown in Figure 6A, compared with the vehicle-treated group, the level of Fe²⁺, ROS, and MDA were significantly decreased by BIM-1 treatment, while the activity of GSH and SOD were increased. In response to BIM-1 treatment, higher levels of GPX4 and SLC7A11 in the spinal cord were observed in SNI rats (Figure 6B,C). Simultaneously, BIM-1 treatment significantly attenuated SNI-caused decline in ATP contents (Figure 6D). As expected, western blot results showed that BIM-1 administration significantly increased the levels of ETC Complex III, IV, and V in the spinal cord of SNI rats, as compared to the vehicle-treated group (Figure 6E,F). The influence of BIM-1 on mitochondrial morphology was also evaluated, and the mitochondrial morphological injury in spinal neurons caused by SNI was alleviated by BIM-1 treatment (Figure 6G). These results suggest that SNI-triggered ferroptosis and mitochondrial dysfunction in the spinal cord probably in a PKCα/NOX4 signaling pathway-dependent manner.

3.7 VitD₃ Alleviates Pain Hypersensitivity via Inhibiting PKCα/NOX4 Signaling Pathway

The molecular basis for the analgesic effect of VitD₃ remains to be elucidated. Based on our above findings, we further explored the role of the PKCα/NOX4 signaling pathway in VitD₃-mediated antinociception. As a result, western blot results displayed that calcitriol treatment significantly reduced the protein levels of p-PKCα, PKCα, and NOX4 in the spinal cord, as compared to the vehicle-treated SNI group (Figure 7A,B). To further verify the potential relationship between the PKCα/NOX4 signaling pathway and VitD₃ in nociception, we treated naïve rats with the PKCα activator PMA alone or in combination with calcitriol and analyzed the changes in pain behavior. The schedule for the drug delivery and testing is shown in Figure 7C. PMA (2.5 μg) and calcitriol were intrathecally given in a single dose, and the PWT was measured before the injection and 0.5–12 h after the injection. As illustrated in Figure 7D, compared with the vehicle group, rats that received a single dose of PMA exhibited a significantly decreased PWT from 1 h after injection, and lasted at least to 12 h. Moreover, calcitriol treatment attenuated the mechanical allodynia induced by PMA administration. The analgesic effect peaked at 2 h and lasted only for 2 h. Accordingly, the spinal segments were collected at 2 h after drug administration. As expected, western blot results showed that the spinal expression of p-PKCα/PKCα and NOX4 was significantly enhanced in response to PMA injection, which were markedly inhibited by calcitriol treatment (Figure 7E,F). Taken together, these results highlight the involvement of PKCα/NOX4 signaling pathway in the antinociceptive effect of VitD₃.

3.8 VitD₃ Preserves Spinal GABAergic Interneurons via Suppression of PKCα/NOX4 Signaling Pathway-Mediated Ferroptosis and Mitochondrial Dysfunction

To ascertain whether VitD₃ suppressed pain behaviors induced by PKCα/NOX4 pathway activation in naïve rats involves a similar mechanism in SNI rats, we further examined the expression of ferroptosis markers and GAD65 in the spinal cord. Surprisingly, PMA administration induced a dramatic elevation in Fe²⁺, ROS, and MDA, and a decline in GSH and SOD in the spinal cord, all of which were reversed by calcitriol treatment (Figure 8A). Additionally, calcitriol treatment significantly alleviated PMA-induced downregulation of spinal SLC7A11 and GPX4, as well as GAD65 (Figure 8B–E). Furthermore, the effects of VitD₃ on PKCα/NOX4 pathway-mediated mitochondrial function were also explored. As shown in Figure 8F, the ATP content was significantly decreased after PMA administration compared with the vehicle group. Intriguingly, calcitriol treatment significantly restored PMA-induced downregulation of ATP. Simultaneously, the protein levels of ETC Complex III, IV, and V were obviously decreased in the spinal cord of PMA-treated rats, which were dramatically alleviated by calcitriol treatment (Figure 8G,H). Furthermore, TEM results showed swollen mitochondria with reduction in cristae and rupture of the outer membrane in spinal cord neurons after PMA administration (Figure 8I). However, calcitriol treatment attenuated these mitochondrial damages. These findings suggest that the protective effect of VitD₃ on spinal GABAergic interneurons through the suppression of PKCα/NOX4 signaling pathway-mediated ferroptosis and mitochondrial dysfunction.