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Korean Journal of Anesthesiology logoLink to Korean Journal of Anesthesiology
. 2026 Aug 20;79(5):555–564. doi: 10.4097/kja.25771

Analgesic mechanism of Panaphix in spinal nerve-ligated rats: involvement of microglial inflammatory cytokines

Myong-Hwan Karm 1,*, Hyun-Jung Kwon 2,*, Doo-Hwan Kim 3, Jun-Seok Lee 2, Minsu Han 2, Eun Ha Kim 2, Chan-Sik Kim 2, Jin-Woo Shin 2, Seong-Soo Choi 2,✉
PMCID: PMC13624826  PMID: 42685801

Abstract

Background

Neuropathic pain, a chronic and debilitating condition caused by damage to the nervous system, remains challenging to treat due to its complex pathophysiology and resistance to analgesics. This study investigated the analgesic effects of Panaphix (PAX-1), a novel pharmacological agent, in a rat model of neuropathic pain induced by spinal nerve ligation (SNL).

Methods

Male Sprague–Dawley rats underwent SNL to induce neuropathic pain. PAX-1 (0.1, 1, or 10 mg/kg) or vehicle was administered orally once daily for seven consecutive days starting on postoperative day 8. Mechanical allodynia was evaluated using the von Frey filament test, and the dorsal root ganglia were analyzed using an enzyme-linked immunosorbent assay and immunofluorescence to evaluate tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression levels.

Results

PAX-1 significantly alleviated mechanical allodynia induced by SNL in a dose-dependent manner, and 1 mg/kg was selected as the optimal dose for subsequent analyses. PAX-1 markedly reduced IL-1β expression in microglia and partially decreased TNF-α immunoreactivity within the dorsal root ganglion, while showing minimal effects on the expression of astrocytic cytokines.

Conclusions

PAX-1 demonstrated potent anti-allodynic and anti-inflammatory effects in an SNL-induced neuropathic pain model. These findings indicate that PAX-1 may exert analgesic effects primarily through modulation of IL-1β-mediated microglial activation, suggesting its therapeutic potential for neuropathic pain management.

Keywords: Arsenite, Dorsal root ganglion, Interleukin-1β, Ligation, Mechanical allodynia, Microglia, Neuropathic pain, Spinal nerves, Tumor necrosis factor-α

Introduction

Chronic pain significantly affects the quality of life and functional capacity of patients, with neuropathic pain being particularly challenging due to its complex causes and resistance to treatment [1]. This condition, resulting from nervous system damage, is often marked by mechanical allodynia and hyperalgesia, leading to increased sensitivity and substantial morbidity [2,3]. Despite the prevalence of this condition, current treatment options remain limited and are often accompanied by considerable side effects [4]. Therefore, novel therapeutic strategies targeting the underlying mechanisms of neuropathic pain should be explored.

Panaphix (PAX-1, sodium meta-arsenite, or NaAsO2), a newly developed pharmacological agent for cancer treatment, has shown promise in preclinical studies for its anti-inflammatory and antinociceptive properties [5–7]. However, its efficacy in modulating the expression of key inflammatory markers and attenuating mechanical allodynia in models of neuropathic pain induced by spinal nerve ligation (SNL) remains unclear. Understanding these mechanisms could provide critical insights into its therapeutic potential and pave the way for the development of more effective interventions [8]. Given the central role of inflammatory processes in the pathogenesis of neuropathic pain, whether PAX-1 exerts its analgesic effects by regulating pro-inflammatory cytokines should be investigated.

Inflammatory cytokines, particularly tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), are pivotal mediators in the development and maintenance of neuropathic pain [9–15]. Such mediators have been implicated in the sensitization of nociceptors and the promotion of central sensitization [15–17]. Modulation of these pathways is a promising approach to alleviate neuropathic pain [17]. Their upregulation in the dorsal root ganglion (DRG) following nerve injury promotes neuroinflammation, sensitizes nociceptors, and facilitates pain transmission [2]. Despite advances in understanding the pathophysiology of neuropathic pain, current treatments often fail to provide sufficient relief, and novel therapeutic strategies are needed.

This study investigated the therapeutic potential of PAX-1 in alleviating SNL-induced neuropathic pain. The first objective of this study was to determine the analgesic effect of PAX-1 in rats with SNL-induced neuropathic pain. The second objective was to characterize the involvement of TNF-α and IL-1β in the DRG in PAX-induced analgesia.

Materials and Methods

Ethical requirements

The Institutional Animal Care and Use Committee approved the experiments (No. 2020-14-307, No. 2021-14-128, and No. 2023-13-043). This committee follows the guidelines of the Institute of Laboratory Animal Resources. The ethical guidelines of the International Association for the Study of Pain and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health were followed during all procedures. Every effort was made to minimize the number of animals used and their suffering.

Experimental animals

This study utilized male Sprague–Dawley rats (250–300 g, Seongnam, South Korea). The animals were acclimated for 1 week in a controlled environment with a 12-h light/dark cycle, free access to food and water, and a stable temperature of 22 ± 2°C. To familiarize the subjects with the laboratory conditions before surgery, three behavioral tests were conducted, the third of which was adopted as a preoperative measurement. Conventional experimental preparations were performed based on a previous study [18].

SNL for the neuropathic pain model

Neuropathic pain was induced via SNL following the method described by Kim and Chung [19]. Rats were anesthetized with intraperitoneal injections of Zoletil (50 mg/kg) and xylazine (10 mg/kg). A midline incision was made to expose the L5 and L6 spinal nerves. The nerves were ligated using 5-0 black silk sutures distal to the DRG. Wound closure was achieved using 4-0 vicryl for muscle and 3-0 nylon for skin. Sham-operated rats underwent the same surgical procedure without nerve ligation.

Behavioral test

Behavioral tests for mechanical allodynia were conducted from 10:00 AM to 4:00 PM each testing day to reduce experimental variation. The experimental animal was placed in a transparent plastic cage with a wire mesh (8 × 8 mm) at the bottom and remained in the cage for at least 15 min to allow it to adjust to the cage. Once cage investigation and main grooming activities had ended, behavioral accommodation was permitted for 20–30 min. Mechanical allodynia was evaluated using the up–down method with calibrated von Frey filaments (VFF; Stoelting Co.). Eight filaments (0.40–15.10 g) were applied sequentially to the plantar surface of the left hind paw in ascending or descending order. Each filament was applied vertically for 5–6 s until slight bending occurred. A rapid paw withdrawal, flinching, or licking was considered a positive response. According to the up–down paradigm, the next lower force filament was applied after a positive response, whereas a higher force filament was applied after a negative response. The 50% withdrawal threshold was calculated based on at least six responses around the estimated threshold, as previously described [20,21]. All assessments of the antinociceptive properties of PAX-1 were performed by blinded observers. When measuring behavioral responses, the contralateral limb was used as the control for the ipsilateral SNL.

Experiment 1

The study was designed to determine the optimal dose of PAX-1 analgesic effects in the neuropathic pain model using SNL models. After a behavioral test was conducted to confirm the baseline, SNL was performed. Rats in which the SNL model was appropriately established were identified by behavioral testing 1 week after the procedure and subsequently selected. Based on a computer-generated randomization method, an impartial data manager assigned 9 rats to each of 4 groups: 1) Vehicle; 2) PAX-1 0.1 mg/kg; 3) PAX-1 1 mg/kg; and 4) PAX-1 10 mg/kg (Fig. 1A). The person who administered the drug was not blinded, but the evaluator of the behavioral experiment was blinded. PAX-1 or vehicle was administered orally once daily for seven consecutive days starting on day 8 after the SNL surgery. Behavior tests were performed 8, 10, 12, and 14 days after SNL.

Fig. 1.

Fig. 1.

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Flowchart of experiments. (A) The dose-dependent effect of PAX-1 on SNL-induced mechanical allodynia in rats evaluated using the behavioral von Frey filament test. (B) ELISA and immunofluorescence analysis in rat DRG following SNL. DRG: dorsal root ganglion, ELISA: enzyme-linked immunosorbent assay, PAX-1: panaphix, SNL: spinal nerve ligation.

Experiment 2

Eight rats were randomly assigned to each of four groups: 1) the sham (no nerve ligation) group; 2) the PAX (treated with PAX-1) group; 3) the SNL (vehicle-treated after SNL) group; and 4) the SNL + PAX (treated with PAX-1 after SNL) group. The sham group was not administered any drug, the SNL group was administered vehicle, and the PAX and SNL + PAX groups were administered 1 mg/kg of PAX-1 orally once daily for 7 consecutive days starting on the 8th day after SNL (Fig. 1B). Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were performed on samples obtained seven days after drug administration. In addition, upon completion of the experiment, the animal was chemically euthanized using CO2 to terminate the experiment.

ELISA analysis of TNF-α and IL-1β levels

TNF-α and IL-1β levels were analyzed from DRG samples of eight rats from four groups that were not used for behavioral testing using ELISA. The animals were decapitated rapidly under anesthesia on the 7th day after drug administration, and the DRGs of the L5 and L6 segments were removed and flash-frozen in liquid nitrogen. The samples were homogenized in ice-cold RIPA buffer (R0278, Sigma) and protease inhibitor cocktail (P8340, Sigma) and centrifuged (13 000 rpm for 20 min) to obtain protein extracts. Commercially available ELISA kits were used to assess TNF-α (LS Bio. Catalog No. LS-F5193, 40 µg of protein was used per sample) and IL-1β (LS Bio. Catalog No. LS-F5627, 40 µg of protein was used per sample) levels. The Bradford protein assay was used to measure total protein concentration in the tissue supernatant. The microplates were measured using a VICTOR3 V Multilabel Counter (1420, Perkin Elmer), and the data were standardized as picograms of TNF-α and IL-1β to 40 μg of total protein in the supernatant. ELISA measurements were performed in triplicate for each sample to ensure accuracy.

Quadruple labeling immunofluorescence study

On day 7 after drug administration, samples from eight rats from four groups were subjected to histological analysis. The animals were anesthetized with Zoletil 12.5 mg and xylazine 3 mg intraperitoneally and perfused transcranially with 4% buffered paraformaldehyde. The L5 DRG was removed, postfixed in 4% paraformaldehyde, and subsequently embedded in paraffin. Paraffin-embedded DRG was sectioned (3 μm) and placed on slides. Sections were deparaffinized and rehydrated in a graded ethanol series. After blocking with 5% donkey serum, 0.3% Triton X-100, and 1% bovine serum albumin in phosphate-buffered saline with Tween 20 (PBS-T), quadruple label immunofluorescence was performed by incubating the paraffin sections with antibodies to inflammatory cytokines (TNF-α antibody [1:100, Abcam, ab307164, host rabbit] or IL-1β antibody [1:100, Abcam, ab315084, host rabbit]), ionized calcium-binding adaptor molecule 1 (IBa1, microglia marker) antibody (1:100, Abcam, ab283319, donkey anti-mouse IgG), glial fibrillary acidic protein (GFAP, astrocyte marker) antibody (1:100, Thermo, PA1-10004, donkey anti-chicken IgY), or neuronal nuclear protein (NeuN, neuron marker) antibody (1:100, Novus, NBP3-05554, donkey anti-goat IgG), which were applied overnight at 4°C, followed by incubation with Alexa Fluor 405 donkey anti-mouse IgG (blue; 1:500, Thermo, A48257), Alexa Fluor 488 donkey anti-chicken IgY (green; 1:500, Thermo, A78948), Alexa Fluor 555 donkey anti-rabbit IgG (red; 1:500, Thermo, A31572), and Alexa Fluor 633 donkey anti-goat IgG (white; 1:500, Thermo, A21082) for 2 h at room temperature. Sections were rinsed in PBS-T and mounted. The immunoreactions in the DRG were observed using a confocal microscope (Zeiss LSM880). Two slices of the DRG were used for each evaluation to determine immunoreactive cells in the DRG. Co-labeled cells with a clearly definable greenish and reddish cell border were identified as a marker of inflammatory cytokines of astrocytes at 400× magnification for each section. Co-labeled cells with a clearly definable bluish and reddish cell border were identified as a marker of inflammatory cytokines of microglia for each section. Quantitative analysis was performed by measuring the area of co-labeled cells.

Statistical analysis

All statistical analyses were conducted using SPSS (version 21; IBM Corp.) and GraphPad Prism version 8.0.2 (GraphPad Software). Behavioral data are presented as the mean ± standard deviation. The data distribution was tested for normality using the Kolmogorov–Smirnov test. Parametric tests were used for normally distributed data, whereas nonparametric tests were used for non-normally distributed data. For behavioral assessments, two-way repeated-measures analysis of variance (ANOVA) was conducted to evaluate the effects of time (postoperative day, within-subject factor) and treatment group (Vehicle, PAX-1 0.1, 1.0, and 10 mg/kg; between-subject factor) on withdrawal threshold. When a significant time × group interaction was detected, post hoc comparisons between treatment groups were performed at each time point with Holm correction for multiple comparisons. In addition, within-group temporal changes (simple effects of time) were analyzed using repeated-measures ANOVA, followed by baseline-adjusted pairwise comparisons within each group. The results of ELISA and tissue count analyses are expressed as the mean ± standard error of the mean. Statistical significance was set at P value < 0.05. Quantitative imaging analysis of tissue sections was conducted using NIH ImageJ medical imaging software packages (National Institutes of Health).

Results

Experiment 1: Effect of PAX-1 on SNL-induced mechanical allodynia

All the rats exhibited a baseline withdrawal reaction threshold of 15.10 g (maximum mechanical stimulation) to mechanical stimulation before SNL. SNL induced a marked decrease in the mechanical withdrawal threshold, indicating the development of neuropathic pain. Treatment with PAX-1 attenuated the mechanical allodynia induced by SNL in a dose- and time-dependent manner (Fig. 2). To analyze changes over time among treatment groups, a two-way repeated-measures ANOVA was performed with the postoperative day as the within-subject factor and treatment group as the between-subject factor. The analysis revealed a significant interaction between time and treatment group, indicating that time-dependent changes in withdrawal thresholds differed among groups (F [9, 60] = 5.711, P < 0.001). Both main effects of time (F [3, 60] = 59.101, P < 0.001) and treatment group (F [3, 20] = 17.225, P < 0.001) were also significant.

Fig. 2.

Fig. 2.

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Anti-allodynic effect of PAX-1 in spinal nerve-ligated rats. Drugs were administered orally for seven consecutive days in rats with mechanical allodynia 1 week after L5 and L6 spinal nerve ligation. aP < 0.05 compared with the baseline. PAX: panaphix.

Post hoc comparisons at each time point (Holm-adjusted) demonstrated that rats receiving 1.0 mg/kg and 10 mg/kg PAX-1 exhibited significantly higher withdrawal thresholds than the Vehicle group from postoperative day 10 (P < 0.001). In contrast, the 0.1 mg/kg PAX-1 group showed significant differences compared with the Vehicle group from postoperative day 12 (P < 0.001). Within-group analyses further confirmed significant time-dependent increases in withdrawal thresholds in all PAX-1-treated groups. In particular, the 1.0 mg/kg and 10 mg/kg groups showed significant increases from baseline beginning on postoperative day 10, whereas the 0.1 mg/kg group exhibited significant increases from postoperative day 12 (Fig. 2).

Accordingly, both the 1.0 mg/kg and 10 mg/kg doses of PAX-1 significantly inhibited SNL-induced mechanical allodynia from day 3 to day 7 after PAX-1 administration. Based on its consistent analgesic effect and experimental feasibility, 1 mg/kg was selected as the optimal dose for subsequent experiments.

Experiment 2: Involvement of TNF-α and IL-1β in IBa1-, GFAP-, and NeuN-positive cells located at the DRG

The expressions of TNF-α and IL-1β in the DRG of 4 rat groups were analyzed by ELISA (Fig. 3). TNF-α and IL-1β levels in the SNL group were significantly increased compared with those in the sham group (P < 0.001). However, in the SNL + PAX group, TNF-α levels did not change significantly compared with those in the SNL group (Fig. 3A); however, IL-1β levels significantly decreased (P < 0.01, Fig. 3B).

Fig. 3.

Fig. 3.

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Levels of TNF-α and IL-1β in the dorsal root ganglion tissue of spinal nerve-ligated rats. ELISA was performed to assess the expression of TNF-α (A) and IL-1β (B) after oral administration of vehicle or 1 mg/kg of PAX-1 for seven consecutive days in spinal nerve-ligated rats. aP < 0.001 compared with the sham group. bP < 0.01 compared with the SNL group. ELISA: enzyme-linked immunosorbent assay, IL: interleukin, PAX: panaphix, SNL: spinal nerve ligation, TNF: tumor necrosis factor.

The DRG immunofluorescence study showed that SNL markedly increased the expression of TNF-α and IL-1β in IBa1-positive microglia (Figs. 4A and 5A). Quantitative analyses confirmed a significant elevation of TNF-α and IL-1β immunoreactivities (IRs) co-localized with IBa1-positive microglia compared with those of the sham group (Figs. 4B and 5B, P < 0.001 for both). Treatment with PAX-1 attenuated these elevations, resulting in significantly lower TNF-α and IL-1β IRs co-localized with IBa1-positive microglia in the SNL + PAX group than in the SNL group (P < 0.001 for both). However, PAX-1 administration did not completely suppress SNL-induced TNF-α and IL-1β IRs co-localized with IBa1-positive microglia (P < 0.001 compared with sham-treated animals). Moreover, SNL-induced TNF-α and IL-1β IRs were co-localized with GFAP-positive astrocytes (Figs. 4A and 5A). The increase in TNF-α and IL-1β IRs co-localized with GFAP-positive astrocytes in the SNL group was significantly higher than that in the sham group (Figs. 4C and 5C, P < 0.001 for both). PAX-1 treatment did not significantly alter the SNL-induced TNF-α and IL-1β IRs co-localized with GFAP-positive astrocytes unlike those in the microglia. In other words, TNF-α and IL-1β IRs co-localized with GFAP-positive astrocytes of the SNL + PAX group remained comparable to those observed in the SNL group. TNF-α and IL-1β IRs were not co-localized with NeuN-positive cells.

Fig. 4.

Fig. 4.

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Immunofluorescence staining and quantitative analysis of TNF-α in IBa1-, GFAP-, and NeuN-positive cells located at the dorsal root ganglion of spinal nerve-ligated rats. (A) Representative immunofluorescence images from the four experimental groups (sham, PAX-1, SNL, and SNL + PAX-1). Immunofluorescence staining was performed after oral administration of vehicle or 1 mg/kg of PAX-1 for 7 consecutive days in spinal nerve-ligated rats. The scale bar represents 10 μm. (B) Quantified analysis of TNF-α in IBa1-positive cells based on immunofluorescence staining. (C) Quantified analysis of TNF-α in GFAP-positive cells based on immunofluorescence staining. Data represent the mean with standard error. aP < 0.01 and bP < 0.001 compared with the sham group. cP < 0.001 compared with the SNL group. GFAP: glial fibrillary acidic protein, IBa1: ionized calcium-binding adaptor molecule 1, NeuN: neuronal nuclear protein, PAX-1: panaphix, SNL: spinal nerve ligation, TNF: tumor necrosis factor.

Fig. 5.

Fig. 5.

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Immunofluorescence staining and quantitative analysis of IL-1β in IBa1-, GFAP-, and NeuN-positive cells located at the dorsal root ganglion of spinal nerve-ligated rats. (A) Representative immunofluorescence images from the four experimental groups (sham, PAX-1, SNL, and SNL + PAX-1). Immunofluorescence staining was performed after oral administration of vehicle or 1 mg/kg of PAX-1 for 7 consecutive days in spinal nerve-ligated rats. The scale bar represents 10 μm. (B) Quantified analysis of IL-1β in IBa1-positive cells based on immunofluorescence staining. (C) Quantified analysis of IL-1β in GFAP-positive cells based on immunofluorescence staining. Data represent the mean with standard error. aP < 0.05 and bP < 0.001 compared with the sham group. cP < 0.001 compared with the SNL group. GFAP: glial fibrillary acidic protein, IBa1: ionized calcium-binding adaptor molecule 1, IL: interleukin, NeuN: neuronal nuclear protein, PAX-1: panaphix, SNL: spinal nerve ligation.

Discussion

The pro-inflammatory cytokines TNF-α and IL-1β have been strongly implicated in the pathogenesis of neuropathic pain [14]. This study demonstrated that PAX-1 effectively attenuated mechanical allodynia in a rat model of neuropathic pain (Fig. 2) and suggested that PAX-1 may be involved in the expression of TNF-α and IL-1β by microglia in the DRG.

Our behavioral test data revealed a dose-dependent reduction in mechanical allodynia after PAX-1 administration. The 1 and 10 mg/kg doses showed significant improvements in withdrawal thresholds compared with the Vehicle group; 1 mg/kg was selected for subsequent experiments due to its optimal balance of efficacy and feasibility. These findings are consistent with previous research that demonstrated the potential of anti-inflammatory agents to restore nociceptive thresholds in neuropathic pain models [8,14].

The role of pro-inflammatory cytokines such as TNF-α and IL-1β in neuropathic pain is well established. These cytokines promote neuroinflammation and central sensitization, exacerbating nociceptive signaling [1,4]. Our findings align with this understanding, as elevated levels of TNF-α and IL-1β were observed in the SNL group compared with those in the sham group (Fig. 3). The observation that cytokines such as TNF-α and IL-1β did not change significantly when PAX-1 was administered to the general control group confirmed that PAX-1 did not affect the test outcome by decreasing cytokines under normal conditions (i.e., absence of mechanical allodynia). However, PAX-1 administration after SNL significantly reduced IL-1β levels in the DRG but had a limited effect on TNF-α levels (Fig. 3). This selective modulation of cytokines suggests that the analgesic effects of PAX-1 are partially mediated by the suppression of IL-1β-driven pathways, which are crucial in maintaining neuropathic pain [10,12]. The differential effects of PAX-1 on TNF-α and IL-1β warrant further investigation. Previous studies have highlighted the distinct but overlapping roles of these cytokines in neuropathic pain. IL-1β directly sensitized nociceptors and amplified pain signals, whereas the role of TNF-α was more prominent in the initiation phase of neuroinflammation [15,16]. Our results suggest that PAX-1 is more effective in mitigating sustained neuroinflammation than its initial onset.

Immunofluorescence analyses further supported the anti-inflammatory properties of PAX-1. The significant reduction in TNF-α and IL-1β expression in microglia (IBa1-positive cells) in the DRG suggests that PAX-1 modulates neuroinflammation primarily through microglial pathways (Figs. 4 and 5). Microglia play a pivotal role in the propagation of neuroinflammation and the transition to chronic pain states [11,13,22,23], and prior studies have demonstrated that suppression of DRG inflammatory cytokines, including TNF-α and IL-1β, attenuates neuropathic pain behaviors in SNL models [24,25]. SNL induced robust glial upregulation of TNF-α and IL-1β in the DRG (Figs. 4 and 5). PAX-1 preferentially attenuated the expression of TNF-α and IL-1β in microglia (IBa1-positive), but not in astrocytes (GFAP-positive), highlighting a cell-type-specific effect of PAX-1 in modulating neuroinflammation associated with neuropathic pain. By targeting the expression of microglial IL-1β, PAX-1 may effectively disrupt the feedback loop that sustains neuropathic pain. PAX-1 treatment did not reduce the increased expression of TNF-α in overall DRG cells of SNL rats. Moreover, PAX-1 did not significantly alter TNF-α or IL-1β levels in astrocytes (GFAP-positive cells). This observation is consistent with studies indicating that astrocyte-driven upregulation of TNF-α is more resistant to pharmacological intervention than microglial cytokine production [17]. This resistance demonstrates the complexity of glial involvement in neuropathic pain and shows the importance of targeting specific cellular pathways for therapeutic efficacy. Our findings provide convincing evidence for the potential clinical application of PAX-1 in the management of neuropathic pain. By selectively modulating IL-1β levels and alleviating mechanical allodynia, PAX-1 addresses key aspects of the pathophysiology of neuropathic pain. These results also raise important questions about the broader anti-inflammatory and neuroprotective effects of PAX-1, particularly in chronic pain conditions with distinct inflammatory profiles.

The neuropathic pain model induced robust neuroinflammatory responses within the DRG, particularly by upregulating pro-inflammatory cytokines such as TNF-α and IL-1β. Here, we observed significant increases in both cytokine levels in the SNL group, which were predominantly localized to glial cells within the DRG. These findings align with previous reports demonstrating that TNFR1 signaling promotes the production of IL-6 and IL-1β through NF-κB activation following nerve injury, which contributes to hypersensitivity to neuropathic pain [26]. Similarly, systemic administration of granulocyte colony-stimulating factor in the neuropathic pain model suppresses DRG inflammation by reducing the expression of TNF-α and IL-1β and inhibiting microglial activation [27]. Furthermore, receptor for advanced glycation end products (RAGE) signaling is critically involved in the amplification of neuroinflammation in the DRG, with RAGE-neutralizing antibodies reducing the expression of TNF-α, IL-1β, and NF-κB and alleviating mechanical allodynia in SNL rats [28]. Collectively, these findings support the validity of our neuropathic pain model and highlight DRG as a key anatomical site for cytokine-mediated nociceptive sensitization. The observed reduction in IL-1β, but not TNF-α, by PAX-1 administration aligns with the idea that differential modulation of glial-derived cytokines may yield partial but significant analgesic effects in neuropathic pain.

This study provides valuable insights into the analgesic effects of PAX-1 in an SNL-induced neuropathic pain model. Nonetheless, several limitations should be acknowledged. First, the study was conducted exclusively on male Sprague–Dawley rats, which may not fully represent the broader biological diversity in human populations, including potential sex-related differences in drug response. Second, although the results suggest that PAX-1 modulates IL-1β and partially attenuates neuropathic pain, the precise molecular mechanisms underlying its effects remain unclear. For example, the limited impact on TNF-α levels warrants further investigation to determine whether alternative pathways contribute to the analgesic efficacy of PAX-1. Third, the relatively short duration of the experiment (7 days) limits the evaluation of long-term efficacy, neuroplasticity, and safety. Future investigations should include prolonged observation periods and histopathological analyses to determine whether the analgesic and anti-inflammatory effects of PAX-1 are sustained over time. Fourth, considering that PAX-1 (sodium meta-arsenite) was originally developed as an anticancer agent, its potential neurotoxicity requires careful evaluation. In our study, no behavioral or histological signs of neurotoxicity were observed during the 7-day treatment period, and previous studies have reported selective cytotoxicity against tumor cells with minimal effects on normal neural tissues at comparable doses [7]. Nevertheless, future studies should address the long-term safety of PAX-1 and evaluate possible cumulative neurotoxic effects in chronic exposure models. In addition, the use of a SNL model may not fully capture the heterogeneity of clinical neuropathic pain conditions. Moreover, investigating the molecular mechanisms underlying the selective cytokine-induced modulation of PAX-1 could reveal new therapeutic targets for neuropathic pain. Clinical trials are warranted to evaluate the safety, efficacy, and optimal dosing regimens of PAX-1 in human populations. Future studies incorporating diverse animal models and long-term evaluations, as well as clinical trials, are essential to validate and expand on these findings. Understanding the underlying mechanisms and identifying effective treatments could significantly improve the quality of life of individuals experiencing these debilitating pain conditions. By investigating these molecular pathways, this study helps to understand the mechanisms of neuropathic pain and provides a scientific basis for the potential clinical application of PAX-1 in the management of neuropathic pain. If validated, PAX-1 could represent a foundational agent in the development of new analgesics with multi-mechanistic actions, offering relief for patients with complex and refractory pain syndromes. This aligns with the larger clinical goal of improving personalized pain management strategies and ameliorating patient outcomes.

In conclusion, our study demonstrates the anti-allodynic effect of PAX-1 through modulation of cytokines in the DRG of SNL rat models. PAX-1 administration decreased SNL-induced TNF-α and IL-1β levels in microglia, but not in astrocytes, located in the DRG of rats. In other words, the anti-allodynic effect of PAX-1 may be related to inhibition of DRG microglia activation, especially of IL-1β. These findings contribute to the growing body of evidence supporting the therapeutic potential of targeted anti-inflammatory strategies in the management of neuropathic pain. PAX-1 represents a promising candidate for further development and clinical evaluation.

Footnotes

Acknowledgments

The authors would like to thank Seon-Kyung Lee (Department of Anesthesiology and Pain Medicine, Asan Medical Center) for her technical assistance in conducting this research. The authors appreciate the statistical support from Dr. Seong-Sik Cho, MD, MPH, PhD (Department of Occupational and Environmental Medicine, College of Medicine, Dong-A University, Busan, Korea).

Funding

This research was supported by Komipharm International Co., Ltd. However, the funding source did not participate in the study design, implementation, data collection, management, analysis, or interpretation; manuscript preparation, approval, or review; or the decision to submit the manuscript for publication. None of the authors has a personal financial interest in this study.

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Author Contributions

Myong-Hwan Karm (Visualization; Writing – original draft)

Hyun-Jung Kwon (Validation; Writing – original draft)

Doo-Hwan Kim (Data curation; Formal analysis; Investigation; Visualization)

Jun-Seok Lee (Data curation; Validation)

Minsu Han (Validation; Visualization)

Eun Ha Kim (Data curation; Formal analysis)

Chan-Sik Kim (Data curation; Formal analysis)

Jin-Woo Shin (Methodology; Resources; Software)

Seong-Soo Choi (Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Supervision; Writing – review & editing)

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