Activation of PPARγ Attenuates Neuropathic Pain by Modulating Spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation

Activation of PPARγ Attenuates Neuropathic Pain by Modulating Spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Activation of PPARγ Attenuates Neuropathic Pain by Modulating Spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation Qingling Xu, Guoxu Ling, Yuanzhi Lv, Tingting Su, Hengyi Ning, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8756915/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 18 You are reading this latest preprint version Abstract Background Cuprotosis is a recently identified form of programmed cell death driven by copper accumulation. Increasing evidence suggests that copper dyshomeostasis contributes to neuroinflammatory processes. Peroxisome proliferator-activated receptor gamma (PPARγ) activation has been implicated in the regulation of cuprotosis; however, its role in cuprotosis-associated neuropathic pain remains poorly understood. This study aimed to investigate whether spinal PPARγ modulates cuprotosis and thereby influences neuropathic pain in a rat model of chronic constriction injury (CCI). Methods Neuropathic pain behaviors were assessed using mechanical and thermal sensitivity tests. Intrathecal catheterization was performed for spinal drug administration. Mitochondrial ultrastructure was examined by transmission electron microscopy. Copper concentration, protein expression, oxidative stress, mitochondrial function and inflammatory signaling were evaluated using western blotting, immunofluorescence staining, dihydroethidium staining and the enzyme activity assay kit. Results CCI induced a time-dependent increase in spinal copper levels, accompanied by upregulation of the copper transport-related proteins SLC31A1, ATP7a, and SCO1, and downregulation of the cuprotosis-related protein FDX1. These changes were coincided with excessive mitochondrial structural damage, ROS accumulation, reduced mitochondrial respiratory chain complex IV activity and progressive activation of the NF-κB signaling pathway. Activation of spinal PPARγ significantly alleviated CCI-induced mechanical allodynia and thermal hyperalgesia. This effect was associated with restoration of copper homeostasis, suppression of SLC31A1, ATP7a, and SCO1 expression, enhancement of FDX1 expression, improvement of mitochondrial function and inhibition of NF-κB p65 phosphorylation in the spinal cord. Conclusions These findings suggest that PPARγ activation alleviates neuropathic pain by modulating copper dyshomeostasis, mitochondrial dysfunction, and neuroinflammatory signaling associated with cuprotosis. Targeting the PPARγ regulated spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation may represent a promising therapeutic strategy for neuropathic pain. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience Pioglitazone Cuprotosis Mitochondria ROS Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Neuropathic pain is a debilitating clinical condition resulting from injury or dysfunction of the peripheral or central nervous system [ 1 ] . Epidemiological studies estimate that approximately 7–10% of the adult population experiences chronic neuropathic pain, imposing a substantial socioeconomic burden worldwide [ 2 ] . In the United States alone, direct healthcare costs associated with neuropathic pain exceed $ 10 billion annually, underscoring its significant public health impact [ 3 ] . Despite its high prevalence and economic burden, the pathophysiological mechanisms of neuropathic pain remain incompletely understood, and current therapeutic options provide limited efficacy. Consequently, there is an urgent need to elucidate novel mechanisms and develop more effective treatment strategies. Innate immune activation and the subsequent neuroinflammation are pivotal to the pathogenesis of neuropathic pain. Accumulating evidence indicates that mitochondrial dysfunction, aberrant inflammatory signaling, and disruption of ionic homeostasis are key pathological mechanisms underlying neuropathic pain progression [ 4 ] . Copper (Cu) ions are essential trace elements for life, have redox activity, and play a vital role in various physiological processes in the human body [ 5 ] .Under normal physiological conditions, copper ions enter cells mainly through the copper transporter 1 receptor (High affinity copper uptake protein 1, SLC31A1/CTR1).SLC31A1 is a transmembrane transporter on the cell membrane, with the C-terminus inside the cell and the N-terminus outside the cell [ 6 ] . It is arranged on the cell membrane in the form of a homotrimer, transporting extracellular copper ions into the cell, and cooperating with ATPase copper transporter a (ATP7a) and ATPase copper transporter b (ATP7b) to regulate the transmembrane dynamic balance of copper from the trans-Golgi network to post-Golgi vesicles. These copper-loaded vesicles can fuse with the plasma membrane and release copper into the extracellular environment [ 7 ] .Ferredoxin 1 (FDX1) is an iron-sulfur cluster protein present in mitochondria. It is a key regulatory gene for cuprotosis.It is mainly involved in key metabolic processes such as electron transfer, steroidogenesis, heme synthesis and lipoic acid modification [ 8 ] . The mitochondrial copper partner protein (Synthesis of Cytochrome C Oxidase 1, SCO1) can selectively transfer copper ions according to the ring recognition, and plays a key role in the assembly of cytochrome C oxidase and the steady state regulation of copper ions [ 9 ] .Abnormal copper metabolism and transport are associated with a variety of pathological physiology in the body, especially when genes regulating copper homeostasis mutate, it can lead to the occurrence of a variety of diseases such as Wilson's disease, Huntington's disease and Alzheimer's disease [ 10 – 12 ] .Cuproptosis is a new cell death mode first proposed in 2022, different from known cell death modes including apoptosis, necrosis, pyroptosis or ferroptosis, characterized by copper dependence and regulation of mitochondrial respiration [ 13 ] . However, there is currently very little research on the relationship between cuprotosis and neuropathic pain, and the specific molecular mechanism is still unclear, which deserves further in depth study. Mitochondria are organelles with a double membrane structure in cells, serving as the primary source of cellular energy and are essential for maintaining cellular homeostasis. Beyond their role in ATP production, mitochondria critically regulate apoptosis, redox balance, and reactive oxygen species (ROS) generation [ 14 ] . Mitochondrial dysfunction has been increasingly recognized as a potent trigger of innate immune activation and chronic inflammatory responses [ 15 ] . Excessive copper exposure profoundly disrupts mitochondrial homeostasis through multiple mechanisms. Copper can directly catalyze redox reactions resembling Fenton chemistry, leading to excessive ROS production, while simultaneously impairing antioxidant defenses, including superoxide dismutase and glutathione systems. As a consequence, oxidative stress is markedly enhanced, resulting in damage to mitochondrial proteins, lipids, and mitochondrial DNA, and ultimately compromising mitochondrial membrane integrity [ 16 ] . In parallel, copper toxicity induces loss of mitochondrial membrane potential, disrupts ionic gradients across the mitochondrial membranes, and impairs oxidative phosphorylation and ATP synthesis. These alterations promote the opening of the mitochondrial permeability transition pore (MPTP), which further exacerbates mitochondrial dysfunction and cell injury. MPTP opening also amplifies ROS generation and activates inflammatory signaling pathways, particularly NF-κB, thereby establishing a self-perpetuating cycle of oxidative stress, mitochondrial damage, and inflammation [ 17 ] . Peroxisome proliferator-activated receptor γ (PPARγ) belongs to the nuclear hormone receptor superfamily and has been reported to be expressed in the spinal cord. [ 18 ] .Emerging evidence indicates that activation of PPARγ on sensory afferent and dorsal horn(DH) neurons play an important role in inflammation-induced pain,cancer-induced pain and neuropathic pain [ 19 , 20 ] .In recent years, it has been found that PPARγ plays an important role in the mechanism of cell death [ 21 ] . Cuprotosis, as a newly discovered cell death mechanism, is closely related to mitochondrial respiration, and the activation of PPARγ may play a regulatory role in this process [ 22 ] , influencing the occurrence and development of cuprotosis through multiple pathways.In atherosclerosis models, the activation of PPARγ can upregulate the expression of antioxidant enzymes, reducing ROS levels and thereby mitigating copper ion-induced cellular damage [ 23 ] . Additionally, the activation of PPARγ may influence the metabolism and excretion of copper ions in cells [ 24 ] . PPARγ agonists can affect the sensitivity of cells to copper ions by modulating lipid metabolism and oxidative stress responses, thus regulating the occurrence of cuprotosis [ 25 ] .Nevertheless, whether spinal PPARγ regulates cuprotosis and its downstream mitochondrial and inflammatory consequences in neuropathic pain remains largely unknown. Given the relationship of PPARγ and cuprotosis, mitochondrial damage and inflammatory responses.Here, this study aimed to test the hypothesis that activation of spinal PPARγ alleviates neuropathic pain by regulating cuprotosis-associated mitochondrial dysfunction and neuroinflammation. 2. Materials and methods 2.1.Animals and Ethical Statement Adult male specific pathogen-free (SPF) Sprague–Dawley rats (220–250 g) were obtained from the Experimental Animal Center of Guangxi Medical University (license NO. SCXK (Gui) 2020–0003). Animals were housed under controlled conditions (12-h light/dark cycle, 23 ± 1°C, 50% humidity) with ad libitum access to food and water. Behavioral testing was conducted between 09:00 am and 6:00 pm.All experimental procedures were approved by the Animal Care and Use Committee of Guangxi Medical University (approval NO. 2022-KT-031) and complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) [ 26 ] . The study was reported in accordance with ARRIVE guidelines. Rats were acclimated for at least 1 week prior to experimentation. All efforts were made to minimize animal suffering and to reduce the number of animals used. 2.2.Experimental designs and animal groups In the present study, all rats were randomly assigned to three experimental groups, which were subjected to the following three experiments. The experimental groups and number of animals used in experiments are listed in Fig. 1 . Experimental Design 1 :To investigate the temporal alterations in copper ion levels, mitochondrial ultrastructure of the spinal dorsal horn, reactive oxygen species (ROS) generation, cuproptosis-related proteins (SLC31A1, FDX1, ATP7A, and SCO1), phosphorylated nuclear factor-κB p65 (p-NF-κB p65), and mitochondrial respiratory chain complex IV activity following chronic constriction injury (CCI), rats were allocated to different experimental cohorts at multiple post-injury time points.A total of 30 rats were randomly divided into five groups for behavioral assessments ( n = 6 per group): sham group rats evaluated at 21 days (Sham 21d) and CCI rats examined at 3, 7, 14, and 21 days post-injury (CCI 3d, CCI 7d, CCI 14d, and CCI 21d). Upon completion of behavioral testing, rats were euthanized by an overdose of pentobarbital sodium (200 mg/kg, i.p.). After deep anesthesia was achieved, cervical dislocation was performed as a secondary method in accordance with AVMA guidelines. Death was confirmed by the absence of breathing, heartbeat, and corneal reflex. L 4 -L 6 segments of the spinal cord were harvested for copper concentration measurement, mitochondrial enzyme activity assays, and western blot (WB) analyses.16 rats were assigned to four groups ( n = 4 per group) for immunofluorescence (IF) staining and dihydroethidium (DHE) detection: Sham 21d, CCI 7d, CCI 14d, and CCI 21d. The L 4 -L 6 spinal cord sections from these animals were used for the respective analyses.To assess mitochondrial ultrastructural changes, 8 rats were divided into two groups ( n = 4 per group): Sham 14d and CCI 14d. Mitochondrial morphology in the dorsal horn of the spinal cord was examined using transmission electron microscopy. Experimental Design 2 :To evaluate the potential analgesic effects of pioglitazone in the CCI model, pioglitazone was administered once daily for eight consecutive days, from day 7 to day 14 post-CCI surgery. A total of 24 rats were randomly allocated into four experimental groups ( n = 6 per group): Sham + DMSO, CCI + DMSO, Sham + Pioglitazone, and CCI + Pioglitazone. Pain-related behavioral tests were performed at baseline (0 d) and on days 3, 7, 14, and 21 after surgery. Following the completion of behavioral assessments, spinal cord segments L 4 –L 6 were collected at 0, 7, 14, and 21 days for subsequent analyses. Western blotting(each with n = 4 rats per group per time point) and immunofluorescence staining (each with n = 4 rats per group per time point) were conducted to examine the expression and spatial localization of PPARγ. Experimental Design 3 :To investigate whether repeated administration of a PPARγ activator could modulate cuproptosis during the development of neuropathic pain, 80 rats were randomly divided into four experimental groups ( n = 4 per group): Sham + DMSO, CCI + DMSO, Sham + Pioglitazone, and CCI + Pioglitazone. Two hours after the final injection, L 4 –L 6 spinal cord segments were collected for subsequent analyses. Tissue samples were assessed via transmission electron microscopy (TEM), a copper content detection kit, enzyme activity assay kits, western blotting (WB), immunofluorescence (IF), and dihydroethidium (DHE) staining. These methods were used to quantify copper ion levels, mitochondrial ultrastructure, reactive oxygen species (ROS) production, expression of cuproptosis-related proteins (SLC31A1, FDX1, ATP7A, and SCO1), phosphorylated NF-κB p65 (p-NF-κB p65), and the activity of mitochondrial respiratory chain complex IV. 2.3.CCI model preparation and Drug Treatments The chronic constriction injury (CCI) model was established in rats according to the method described by Bennett and Xie [ 27 ] . Briefly, A longitudinal skin incision (~ 1 cm) was made on the lateral surface of the left thigh at the mid-femoral level. The biceps femoris muscle was bluntly separated to expose the sciatic nerve using a glass hook.Four loose ligatures (4 − 0 chromic gut) were tied around the sciatic nerve approximately 2 mm proximal to the trifurcation, with ~ 1 mm spacing between ligatures. The ligatures were tightened until a brief twitch of the ipsilateral hindlimb was observed. The muscle and skin were then sutured in layers.Sham-operated rats underwent the same surgical exposure without nerve ligation. After surgery, animals were placed on a heating pad until fully recovered from anesthesia and then returned to their home cages. To evaluate the role of PPARγ in neuropathic pain, rats received daily intrathecal injections of the PPARγ agonist pioglitazone (100 µg in 10 µl, dissolved in 5% DMSO) from postoperative day 7 to day 14. Control rats received an equal volume of 5% DMSO via intrathecal injection. 2.4.Placement and verification of intrathecal catheter Intrathecal catheterization was performed in rats as previously described by Pogatzki et al. [ 28 ] . After adequate anesthesia was achieved, the rats were placed in a prone position and the surgical area was aseptically prepared.The L 5 –L 6 intervertebral space was identified by palpation. A midline skin incision (~ 1 cm) was made to expose the L5–L6 intervertebral region. A 22-G needle was carefully inserted into the subarachnoid space. Entry into the intrathecal space was confirmed by a tail-flick response and the appearance of clear cerebrospinal fluid.A PE-10 catheter was then advanced approximately 2 cm cranially into the subarachnoid space. Catheter patency was verified by gentle injection of sterile saline. The external end of the catheter was heat-sealed and secured to the paraspinal muscles near the iliac crest. The catheter was tunneled subcutaneously and exteriorized at the back of the neck, leaving approximately 3 cm exposed and fixed in place.To prevent catheter damage, rats were housed individually in transparent cages with visual contact with other animals. Exclusion criteria included postoperative motor deficits (e.g., hindlimb paralysis or hemiplegia), catheter loss, or insufficient catheter length after surgery.On postoperative day 3, correct catheter placement was verified by intrathecal injection of 10 µl of 2% lidocaine followed by 10 µl of saline. Transient bilateral hindlimb paralysis occurring within ~ 10 s and resolving within 30 min was considered confirmation of correct intrathecal catheter placement. 2.5.Pain behavior quantification All behavioral assessments were performed and analyzed by an experimenter blinded to group allocation. Rats were allowed to acclimatize to the testing environment for at least 30 min before each session. Behavioral tests were conducted at baseline (before CCI) and on days 3, 7, 14, and 21 after CCI. In the pioglitazone-treated groups, nociceptive behaviors were assessed at the same time points.Testing was conducted in a quiet room under controlled temperature and lighting conditions. Paw withdrawal mechanical threshold (PWMT) was assessed as previously described [ 29 ] . Rats were individually placed in elevated Plexiglas chambers (20 × 20 × 25 cm) on a metal mesh floor and allowed to acclimate for 30–60 min. Mechanical stimuli were applied to the mid-plantar surface of the left hind paw using calibrated von Frey filaments (Stoelting, USA).Testing began with a 2.0 g filament, followed by an ascending series of filaments. Each filament was applied perpendicularly with sufficient force to produce slight bending for approximately 4 s. Paw lifting or licking was considered a positive response. The PWMT was defined as the lowest force that evoked a withdrawal response. Values above 15 g or below 1 g were recorded as 15 g and 1 g, respectively. Paw withdrawal thermal latency (PWTL) was measured using a thermal testing apparatus (BME410A, China) as previously described [ 30 ] . After acclimation, a radiant heat source was focused on the mid-plantar surface of the left hind paw. The latency to paw withdrawal was automatically recorded when the rat withdrew its paw.A cut-off time of 25 s was set to prevent tissue damage. Each rat was tested five times at 5-min intervals, and the mean of the last three measurements was used as the PWTL value. 2.6.Measurement of Copper ion content According to the method of the copper content detection kit instructions, copper was extracted from the lumbar enlargement of the spinal cord and used a cryogenic grinder to homogenize on ice.Precool at 4℃ and centrifuge at 10,000 g for 10 min.Aspirate the supernatant and determined protein concentration.Detection of tissue copper content using fully automatic microplate reader. Calculation of tissue copper content: Tissue copper content (µmol/g prot)=Δ determination/(Δ standard/C standard)*V extraction/(Cpr*V extraction) = 80*Δ determination/Δ standard/Cpr. C standard: standard concentration, 80 µmol/L; V extraction: pre-treatment distilled water volume,0.001L; Cpr: sample protein concentration, mg/mL. 2.7.Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure. L 4 -L 6 spinal cord were fixed in 2.5% glutaraldehyde at 4°C for 4 h, followed by postfixation in 1% osmium tetroxide for 2 h at 4°C.Samples were dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, and 100%), followed by acetone dehydration. The tissues were then infiltrated with epoxy resin and embedded. Polymerization was performed at 35°C and 45°C for 12 h each, and finally at 60°C for 24 h.Ultrathin sections were prepared and examined using a transmission electron microscope (HT7800, Hitachi, Japan). Mitochondrial morphology in the spinal dorsal horn was evaluated, including mitochondrial size, cristae integrity, swelling, and vacuolization. 2.8.Immunofluorescence staining The immunofluorescence staining was performed as described in our previous study [ 31 ] .The L 4 -L 6 spinal cord segments were dissected and post-fixed in 4% PFA for 8 h, then cryoprotected in 30% sucrose at 4°C until the tissue sank.Spinal cords were embedded and sectioned at 20 µm thickness using a cryostat. Sections were collected in PBS.For immunofluorescence staining, sections were blocked with 5% normal goat serum in PBS for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: p-PPARγ (1:1000, rabbit, Servicebio), SLC31A1 (1:100, rabbit, Zhengneng), FDX1 (1:100, rabbit, ABclonal), ATP7A (1:70, rabbit, Affinity), SCO1 (1:100, rabbit, Zhengneng), Iba1 (1:200, mouse, GeneTex), GFAP (1:400, mouse, Cell Signaling Technology), and NeuN (1:400, mouse, Novus).After washing with PBS, sections were incubated for 2 h at room temperature with CoraLite594-conjugated goat anti-mouse IgG (H + L) or CoraLite488-conjugated goat anti-rabbit IgG (H + L) secondary antibodies (1:500, Proteintech).Sections were rinsed and mounted with glycerol-based mounting medium and stored at 4°C in the dark until imaging. Fluorescence images were acquired using an Olympus BX53 fluorescence microscope. 2.9.Western blot analysis L 4 -L 6 spinal cord segments were collected and homogenized to prepare protein lysates. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein (20µg) were separated on 10% SDS–PAGE gels and subsequently transferred onto PVDF membranes using a wet transfer system.Membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with the following primary antibodies: p-PPARγ (1:1000, rabbit, Servicebio), PPARγ (1:1000, rabbit, Affinity), ATP7A (1:500, rabbit, Affinity), SLC31A1 (1:1000, rabbit, Zhengneng), FDX1 (1:1000, rabbit, ABclonal), SCO1 (1:700, rabbit, Zhengneng), p-NF-κB p65 (1:700, rabbit, Zhengneng), NF-κB p65 (1:700, rabbit, Zhengneng), and β-actin (1:1000, mouse, Abcam).After washing with TBST (3 × 10 min), membranes were incubated with goat anti-rabbit IgG (H + L) DyLight™ 800 4X PEG secondary antibody (1:10,000, Thermo Fisher Scientific) or goat anti-mouse IgG (H + L) DyLight™ 800 4X PEG secondary antibody (1:10,000, Thermo Fisher Scientific).Protein bands were visualized using an Odyssey infrared imaging system. Band intensities were quantified using ImageJ software. Phosphorylated protein levels were normalized to their corresponding total protein levels, whereas total protein levels were normalized to β-actin. The mean value of the sham group was defined as 100%. 2.10.Measurement of ROS production Dihydroethidium (DHE) staining was used to detect ROS production in the spinal cord. Rats were perfused and spinal cord tissues were prepared as described above. L 4 -L6 spinal cord segments were post-fixed in 4% paraformaldehyde for 8 h and cryoprotected in 30% sucrose at 4°C. Tissues were sectioned at 20 µm using a cryostat.Sections were incubated with DHE solution (1:200 dilution, Beyotime Biotechnology, Nantong, China) at 37°C for 30 min in a humidified, light-protected chamber. After incubation, sections were washed with PBS and coverslipped.Fluorescence images were acquired using an Olympus BX53 fluorescence microscope. ROS levels were quantified as mean fluorescence intensity using Image-Pro Plus 6.0 software. 2.11.Respiratory chain complex IV enzymatic activity Mitochondrial respiratory chain complex IV (cytochrome c oxidase) activity was measured using a commercial assay kit (Solarbio, Beijing, China) according to the manufacturer’s instructions [ 32 ] .Briefly, the enzymatic reaction was monitored by measuring absorbance at 550 nm using a Thermo Multiskan microplate reader. For each sample and blank control, absorbance values were recorded at 10 s (A1) and 1 min (A2). The change in absorbance for each reaction was calculated as ΔA = A1 − A2. Net absorbance change was obtained by subtracting the blank value from the sample value.Complex IV activity (U/mg protein) was calculated using the formula provided by the manufacturer:Activity (U/mg protein) = 1832 × ΔA / C,where ΔA represents the corrected absorbance change and C denotes the protein concentration (mg/mL).Enzyme activities were normalized to the control group and expressed as fold changes. 2.12.Statistical analysis All data are expressed as mean ± standard error of the mean (SEM) unless otherwise indicated. Normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated by Levene’s or Brown–Forsythe test as appropriate.For behavioral assessments (paw withdrawal mechanical threshold [PWMT] and paw withdrawal thermal latency [PWTL]) measured over multiple time points, repeated-measures two-way ANOVA was applied, with factors of group and time, followed by Bonferroni’s post hoc test for pairwise comparisons.For endpoint measurements including immunofluorescence intensity, western blot densitometry, spinal copper content, ROS levels (DHE staining), and mitochondrial respiratory chain complex IV activity, two-way ANOVA with post hoc Bonferroni correction was used to assess differences between groups and time points. If only single-factor comparisons were performed, one-way ANOVA followed by Tukey’s or Bonferroni post hoc test was used. In cases where data did not meet assumptions of normality or homogeneity, non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test was applied.All statistical analyses were conducted using IBM SPSS Statistics (version 23.0, SPSS Inc., Chicago, IL, USA). Graphs were generated with GraphPad Prism (version 6, GraphPad Software, San Diego, CA, USA). A P value < 0.05 was considered statistically significant. 3. Results 3.1. CCI induces progressive copper accumulation in the spinal dorsal horn Copper is an essential trace element involved in numerous cellular functions, and increasing evidence implicates dysregulated copper homeostasis in neurological dysfunction and degeneration [ 33 , 34 ] . To assess whether spinal dorsal horn (DH) copper levels are altered following chronic constriction injury (CCI), copper content in spinal cord tissue was evaluated at multiple post-injury intervals using the tissue copper ion content detection kit. As shown in Fig. 2 A, CCI induced a marked elevation in copper ion accumulation within the spinal cord, displaying a clear temporal progression. A significant increase was first detected on postoperative day 3 and persisted through day 21 ( * P < 0.05, ** P < 0.01 compared with the sham group; # P < 0.05, ## P < 0.01 for comparisons between CCI time points; n = 4 per group). In contrast, sham group animals exhibited no appreciable changes in spinal copper levels throughout the experimental period. 3.2. CCI induces dysregulated expression of cuprotosis-related proteins in the spinal dorsal horn SLC31A1,FDX1,ATP7a and SCO1 are the critical proteins involved in cuprotosis regulation. To examine whether CCI triggers alterations in the expression of cuproptosis-associated proteins, their spatial distribution and temporal expression profiles in the spinal dorsal horn (DH) were assessed at multiple post-injury time points using immunofluorescence staining and western blot analysis. As illustrated in Fig. 2 B-I, all four proteins exhibited prominent immunoreactivity within the superficial laminae of the DH. Following CCI, the fluorescence intensities of SLC31A1, ATP7A, and SCO1 were markedly elevated beginning on day 7 and remained at maximal levels through day 21 (Fig. 2 B-C, 2 F-G, and 2 H–I; * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 for comparisons among post-CCI time points; n = 4 per group). In contrast, FDX1 immunoreactivity displayed a significant reduction from day 7 onward and persisted at reduced levels until day 21 after injury (Fig. 2 D–E; * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 among post-CCI groups; n = 4 per group).Consistent with the immunofluorescence findings, western blot analysis of total spinal cord protein collected at 0, 3, 7, 14, and 21 days after CCI or sham surgery revealed pronounced alterations in cuproptosis-related protein expression. Quantitative analysis demonstrated that SLC31A1, ATP7A, and SCO1 protein levels increased progressively after CCI, with significant upregulation first detected at day 7 and sustained through day 21 (Fig. 3 A-B and 3 D-E; * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 among post-CCI time points; n = 4 per group), while their expression remained minimal in sham-operated controls. Conversely, FDX1 protein abundance exhibited a robust and sustained decline from day 7 to day 21 following CCI (Fig. 3 A and 3 C; * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 among post-CCI groups; n = 4 per group). No significant temporal changes in these proteins were observed in sham group animals. To determine what types of cells in the spinal cord express cuproptosis-associated proteins following CCI, double immunofluorescence staining was performed at 14 days post-injury using cell-type–specific markers, including NeuN for neurons, GFAP for astrocytes, and IBA1 for microglia. As illustrated in Fig. 3 F and Fig. 4 A-C, SLC31A1, FDX1, ATP7A, and SCO1 signals were predominantly detected in NeuN-positive neurons in the spinal cord of CCI-treated rats,partly colocalization with IBA1-labeled microglia was also observed, whereas overlap with GFAP-positive astrocytes was minimal. 3.3. Cuprotosis-associated mitochondrial dysfunction and NF-κB activation contribute to CCI-induced neuropathic pain Cuproptosis is closely associated with mitochondrial function [ 5 ] .To further substantiate the association between neuronal mitochondrial impairment and cuproptosis in neurons following CCI, ultrastructural alterations of mitochondria were examined. Owing to its superior spatial resolution, transmission electron microscopy (TEM) was employed to visualize mitochondrial morphology at the subcellular level. TEM analysis revealed pronounced mitochondrial abnormalities in neurons of CCI-treated rats, including extensive swelling, disruption and disappearance of cristae, and formation of vacuole-like structures within the spinal cord. In contrast, mitochondria in sham group retained intact membranes and well-organized cristae, with no evident ultrastructural damage (Fig. 5 A).Given that mitochondria represent a principal intracellular source of reactive oxygen species (ROS), ROS generation within the spinal cord was subsequently assessed. As shown in Fig. 5 B–C, ROS-associated fluorescence intensity increased progressively beginning at day 7 after CCI and remained elevated through day 21 ( * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 for comparisons among post-CCI time points; n = 4 per group). Copper is an important cofactor for several enzymes including the mitochondrial cytochrome C oxidase, complex IV of the respiratory chain [ 35 ] .To determine alterations in the activity of mitochondrial respiratory chain complex IV after CCI, enzymatic activity of mitochondrial respiratory chain complex IV in spinal cord tissue was quantified at successive time points using a enzyme activity kit. As presented in Fig. 5 F, complex IV activity in the spinal cord exhibited a pronounced decline after CCI, with significant reductions evident from day 7 through day 21 ( * P < 0.05, ** P < 0.01 vs. Sham group; # P < 0.05, ## P < 0.01 for comparisons among post-CCI time points; n = 4 per group). In contrast, sham group rats maintained stable and relatively high complex IV activity throughout the observation period. To further evaluate neuroinflammatory activation induced by CCI, the nuclear factor κB (NF-κB) signaling cascade in the spinal cord were analyzed at multiple post-injury time points using western blotting. As shown in Fig. 5 D-E, phosphorylation of the NF-κB p65 subunit was markedly enhanced following CCI, exhibiting a progressive increase beginning on day 7 and persisting through day 21 ( * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 for comparisons among post-CCI time points; n = 4 per group). In contrast, phosphorylated NF-κB p65 levels remained minimal in sham-operated animals. In contrast, total NF-κB p65 protein abundance did not differ significantly between groups across all examined time points. 3.4.Activation of spinal PPARγ attenuates CCI-induced neuropathic pain To investigate the involvement of PPARγ in CCI-induced pathological changes, spinal cord samples were subjected to western blot analysis. Quantitative assessment demonstrated a marked reduction in PPARγ activity beginning at day 3 after CCI, with a progressive decline observed through day 21 (Fig. 6 A–B; * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 for comparisons among post-CCI time points; n = 4 per group). In contrast, PPARγ activity in sham-operated animals remained consistently elevated throughout the experimental period.Consistent with these western blot findings, immunofluorescence analysis revealed prominent PPARγ immunoreactivity localized predominantly to the ipsilateral superficial dorsal horn, which exhibited a significant temporal decrease following CCI (Fig. 6 C–D; * P < 0.05, ** P < 0.01 vs. sham group; # P < 0.05, ## P < 0.01 among post-CCI groups; n = 4 per group). Together, these complementary results suggest that altered PPARγ signaling is closely associated with the development of neuropathic pain after CCI. To evaluate whether sustained activation of PPARγ alleviates established nociceptive hypersensitivity in CCI rats, the PPARγ agonist pioglitazone (100 µg/10 µl) was administered intrathecally once daily from days 7 to 14 after injury. This repeated treatment produced a robust attenuation of both paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), with significant improvement observed from days 7 to 21 following CCI and no evidence of analgesic tolerance over the treatment period. In contrast, identical pioglitazone administration in sham group rats failed to elicit any measurable changes in PWMT or PWTL. These behavioral outcomes are summarized in Fig. 6 E–F ( * P < 0.05, ** P < 0.01 vs. sham + DMSO group; # P < 0.05, ## P < 0.01 vs. CCI + DMSO group; n = 6 per group). 3.5.Activation of spinal PPARγ suppresses copper accumulation and reprograms cuprotosis-related protein expression To determine whether PPARγ activation following CCI is linked to alterations in SLC31A1 and FDX1 expression, double immunofluorescence staining was performed on spinal cord sections at postoperative day 14. The results showed that phosphorylated PPARγ exhibited clear spatial overlap with both SLC31A1 and FDX1 within the superficial laminae of the dorsal horn in CCI rats (Fig. 6 G). To further investigate the role of PPARγ activation to CCI-associated cuproptosis, the PPARγ agonist pioglitazone (100 µg/10 µl) was delivered intrathecally once per day from postoperative days 7 to 14. Subsequently, spinal cord tissues were harvested 2 h after the final administration.As shown in Fig. 7 A, pioglitazone treatment markedly reduced the elevation of copper ion levels induced by CCI ( * P < 0.05, ** P < 0.01 vs. sham + DMSO group ; # P < 0.05, ## P < 0.01 vs. CCI + DMSO group; n = 4 per group).The expression of PPARγ and cuproptosis-related proteins was evaluated by western blotting. As illustrated in Fig. 6 H–I and Fig. 7 B–F, activation of PPARγ with pioglitazone significantly suppressed the expression of SLC31A1, ATP7A, and SCO1, while concurrently enhancing PPARγ and FDX1 protein levels in CCI rats ( * P < 0.05, ** P < 0.01 vs. sham + DMSO group; # P < 0.05, ## P < 0.01 vs. CCI + DMSO group; n = 4 per group). Collectively, these findings indicate that PPARγ activation is linked to the modulation of cuproptosis. 3.6.Activation of spinal PPARγ ameliorates cuprotosis-associated mitochondrial dysfunction and NF-κB signaling To determine whether spinal activation of PPARγ directly ameliorates mitochondrial impairment and modulates NF-κB signaling following CCI, the PPARγ agonist pioglitazone (100 µg/10 µl) was administered intrathecally once daily for eight consecutive days (postoperative days 7–14). Ultrastructural assessment revealed that pioglitazone treatment markedly alleviated CCI-induced mitochondrial abnormalities, including cristae loss, fragmentation, vacuole formation, and swelling, thereby preserving overall mitochondrial integrity (Fig. 7 G). In parallel, mitochondrial respiratory chain complex IV activity in the spinal cord was significantly restored by pioglitazone administration (Fig. 7 H; * P < 0.05, ** P < 0.01 vs. sham + DMSO group; # P < 0.05, ## P < 0.01 vs. CCI + DMSO; n = 4 per group). Given the close association between mitochondrial dysfunction and oxidative stress, reactive oxygen species (ROS) production was subsequently evaluated. As shown in Fig. 7 I–J, dihydroethidium (DHE) fluorescence intensity in the spinal cord was significantly reduced after pioglitazone treatment in CCI rats ( * P < 0.05, ** P < 0.01 vs. sham + DMSO group; # P < 0.05, ## P < 0.01 vs. CCI + DMSO group; n = 4 per group). Furthermore, pioglitazone markedly suppressed the nuclear translocation of phosphorylated NF-κB p65 without altering total NF-κB p65 protein levels (Fig. 7 K–L; * P < 0.05, ** P < 0.01 vs. sham + DMSO group; # P < 0.05, ## P < 0.01 vs. CCI + DMSO group; n = 4 per group). Collectively, these findings demonstrate that PPARγ activation exerts a protective effect against cuprotosis-associated mitochondrial dysfunction and inflammatory signaling in the spinal cord. 4.Discussion The present study demonstrates that chronic constriction injury (CCI) induces a distinct cuproptosis-related pathological profile in the spinal cord that parallels the development of mechanical allodynia and thermal hyperalgesia. During neuropathic pain progression, cuproptosis was associated with profound mitochondrial abnormalities, including disrupted ultrastructure and suppressed activity of mitochondrial respiratory chain complex IV, accompanied by excessive ROS accumulation. These changes occurred alongside a progressive increase in the expression of cuproptosis-associated proteins, including SLC31A1, ATP7A, and SCO1, as well as activation of the NF-κB p65 signaling pathway, while FDX1 and PPARγ expression levels declined in a time-dependent manner. Importantly, selective activation of spinal PPARγ markedly alleviated CCI-induced nociceptive hypersensitivity. PPARγ activation reduced spinal copper accumulation, preserved mitochondrial structural integrity, restored complex IV activity, and attenuated oxidative stress. Concomitantly, PPARγ stimulation reversed the dysregulation of cuproptosis-related proteins by suppressing SLC31A1, ATP7A, SCO1, and phosphorylated NF-κB p65, while enhancing FDX1 expression in the spinal cord. Collectively, these findings indicate that PPARγ functions as a critical modulator linking copper dyshomeostasis to mitochondrial dysfunction and neuroinflammatory signaling in neuropathic pain,and suggest that therapeutic targeting of PPARγ-regulated cuprotosis-associated mitochondrial dysfunction and neuroinflammation may represent a promising strategy for the treatment of neuropathic pain. A variety of experimental models have been developed to facilitate investigation of the mechanisms underlying neuropathic pain. Among these, the chronic constriction injury (CCI) of the sciatic nerve is widely recognized as a classical and reliable model. The CCI model induces robust pain hypersensitivity and aberrant nociceptive behaviors while largely preserving peripheral nerve afferent and efferent conduction, thereby closely resembling clinical conditions such as low back and leg pain or sciatica resulting from intervertebral disc herniation or foraminal stenosis [ 36 , 37 ] . Beyond triggering acute injury-related neural discharge, CCI provokes sustained pathophysiological alterations in sensory axon cell bodies, leading to persistent ectopic firing and long-term hypersensitivity [ 27 ] .In the present study, both paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) began to decline as early as day 3 following CCI, with pronounced reductions observed between days 7 and 14, after which these behavioral abnormalities remained relatively stable through day 21 post-surgery. In contrast, sham-operated animals exhibited no significant changes in PWMT or PWTL throughout the experimental period. These behavioral outcomes are consistent with previous reports, confirming that CCI elicits a rapid-onset and sustained neuropathic pain phenotype. Copper functions as an essential trace element involved in multiple biological processes, including cellular energy metabolism, mitochondrial respiration, and antioxidant defense, primarily through its role as a catalytic cofactor. Intracellular copper homeostasis is tightly regulated by an integrated transport system comprising copper-dependent enzymes, molecular chaperones, and membrane transporters, which collectively control copper uptake, distribution, and efflux to maintain physiological balance. Disruption of this finely tuned regulatory network can profoundly affect neuronal function, and growing evidence links copper dyshomeostasis to the pathogenesis of neurological disorders [ 38 ] .In the present study, spinal cord copper levels were significantly elevated as early as day 3 following CCI induction and remained persistently increased through day 21 compared with sham-operated controls. Notably, this temporal pattern of copper accumulation closely paralleled the development and maintenance of pain-related behaviors in CCI rats. These findings suggest that progressive copper imbalance within the spinal dorsal horn may contribute to the initiation and persistence of neuropathic pain, highlighting dysregulated copper homeostasis as a potential pathogenic factor. Previous studies have shown that elevated extracellular copper concentrations are known to enhance SLC31A1-mediated copper uptake, while ATP7a functions as a key compensatory regulator by facilitating copper efflux or intracellular compartmentalization to prevent toxic accumulation. Upregulation of ATP7a is therefore widely regarded as an adaptive response to copper overload [ 39 ] . In parallel, FDX1, a mitochondrial matrix enzyme essential for copper-dependent metabolic processes, has emerged as a central regulator of cuprotosis, whose dysregulation mechanistically differentiates this pathway from ferroptosis, apoptosis, and other forms of regulated cell death. In this study,immunofluorescence results showed that, compared with the sham group, the expression of ATP7a, SLC31A1, and SCO1 was upregulated in the CCI group, while the expression of FDX1 was downregulated, suggests a profound disturbance of neuronal copper trafficking in neuropathic pain. Furthermore, SLC31A1, FDX1, ATP7a, and SCO1 were all expressed in the lamina I-IV of the dorsal horn of the spinal cord, consistently, the double immunofluorescence results showed that the SLC31A1, FDX1, ATP7a, and SCO1 was colocalized mostly with neurons in the spinal cord dorsal horn. Regions and cell types that are central to nociceptive transmission and central sensitization.Therefore, it is speculated that cuprotosis-related proteins SLC31A1, FDX1, ATP7a, and SCO1 may be involved in the development and progression of neuropathic pain. Furthermore, western blot analysis confirmed the upregulation of the copper transporters SLC31A1 and ATP7a, as well as the mitochondrial copper chaperone SCO1, accompanied by a significant decrease in FDX1 protein expression. Together with the observed behavioral hypersensitivity and spinal copper accumulation, these molecular alterations provide further evidence that cuproptosis-related mechanisms are closely linked to the initiation and progression of neuropathic pain. Inflammation represents a core component of the innate immune response within the central nervous system [ 40 ] . Mitochondria, ubiquitous organelles in eukaryotic cells, serve as critical regulators of innate immunity by releasing danger-associated molecular patterns (DAMPs) and reactive oxygen species (ROS), thereby initiating and amplifying inflammatory signaling cascades [ 41 ] . Notably, cuprotosis has emerged as a copper-dependent form of cell death that directly targets mitochondrial integrity in neurons, leading to structural disruption, excessive ROS generation, and inflammatory activation [ 16 ] .Copper homeostasis plays a pivotal role in maintaining mitochondrial respiratory chain function. SCO1 is a key regulator of mitochondrial copper transport and is essential for the assembly and activity of cytochrome c oxidase (complex IV) [ 42 , 43 ] . Dysregulation of SCO1 can disrupt early complex IV subassembly [ 44 ] , impair copper-dependent enzymatic activity, and promote mitochondrial dysfunction [ 45 ] . Studies suggest excessive mitochondrial copper accumulation has been shown to inhibit complex IV activity and enhance ROS production, thereby creating a pro-oxidative intracellular environment. Valnot found that mutations in the SCO1 gene lead to mitochondrial cytochrome oxidase C deficiency [ 46 ] . Hosseini et al. showed that excessive copper leads to decreased activity of mitochondrial respiratory chain complex IV enzymes and increased ROS production [ 47 ] . In the present study, transmission electron microscopy revealed pronounced mitochondrial abnormalities in the spinal dorsal horn following CCI, including severe swelling, cristae disruption, and vacuolar degeneration, indicating substantial mitochondrial structural damage during neuropathic pain development. Consistently, mitochondrial ROS levels in the spinal dorsal horn increased progressively after CCI, suggesting sustained oxidative stress. Concomitantly, we observed a significant reduction in mitochondrial respiratory chain complex IV activity alongside increased SCO1 protein expression in the spinal cord of CCI rats, indicating impaired mitochondrial respiratory function under conditions of copper dyshomeostasis.Excessive mitochondrial ROS not only reflect functional impairment but also serve as potent secondary messengers that activate inflammatory signaling pathways. Accordingly, our results demonstrated a marked increase in phosphorylated NF-κB p65 without changes in total p65 expression by western blot analysis, indicating activation of the NF-κB pathway. NF-κB activation subsequently drives the transcription of pro-inflammatory cytokines, thereby initiating and amplifying neuroinflammatory responses that facilitate pain signal transmission [ 48 ] .Taken together, these findings demonstrate that CCI-induced copper dyshomeostasis promotes mitochondrial structural and functional impairment via SCO1-associated complex IV dysfunction, leading to excessive ROS generation and activation of NF-κB-dependent inflammatory signaling. This feed-forward axis linking copper imbalance, mitochondrial dysfunction, and neuroinflammation may play a critical role in the persistence and amplification of neuropathic pain. Accumulating evidence indicates that activation of peroxisome proliferator-activated receptor γ (PPARγ) plays a critical role in the modulation of pathological pain conditions. In the present study, pioglitazone was administered intrathecally to deliver the drug directly into the spinal cord, thereby circumventing the blood–brain barrier, enhancing local drug availability, reducing the required dosage, and minimizing systemic and off-target effects compared with intravenous administration [ 49 ] . Continuous intrathecal treatment was initiated from day 7 to day 14 after surgery, a time window that more closely reflects the clinical use of pharmacological interventions during the chronic phase of neuropathic pain.Behavioral assessments demonstrated that intrathecal administration of the PPARγ agonist pioglitazone attenuated pain-related behaviors in a dose-dependent manner. Among the tested doses, 100 µg of pioglitazone produced a significantly greater improvement in mechanical allodynia and thermal hyperalgesia than the 10 µg and 30 µg doses, while not producing additional adverse effects [ 50 ] . Based on these observations, a dose of 100 µg was selected for continuous intrathecal administration in CCI rats.To further elucidate the role of PPARγ in the maintenance of neuropathic pain, pioglitazone was administered intrathecally during the established pain phase. This intervention effectively alleviated both mechanical and thermal hypersensitivity in CCI rats, indicating that spinal PPARγ activation remains therapeutically effective during the maintenance phase of neuropathic pain. Collectively, these findings suggest that enhanced PPARγ activity plays an important role in sustaining analgesic modulation during chronic neuropathic pain. Western blotting and immunofluorescence analyses demonstrated that PPARγ expression was progressively downregulated following CCI. Immunofluorescence further revealed that PPARγ was predominantly localized within laminae I-IV of the spinal dorsal horn, a key region for the integration and transmission of peripheral nociceptive signals [ 51 ] . These findings suggest that CCI-induced suppression of PPARγ signaling occurs at a critical spinal site involved in pain processing.Given the central role of copper dyshomeostasis and mitochondrial dysfunction in CCI-induced neuroinflammation, we next examined whether PPARγ is functionally linked to cuprotosis-related pathways. Double-label immunofluorescence demonstrated that PPARγ colocalized with the copper transporter SLC31A1 and the cuprotosis regulator FDX1 in the spinal cord, this spatial association suggests that a potential regulatory between PPARγ signaling and cuprotosis under neuropathic pain conditions. Consistent with this association, Qi et al. reported that activation of the PPARγ-FDX1 signaling axis suppresses cuprotosis in mice [ 52 ] .Extending these findings, we observed that intrathecal administration of the PPARγ agonist pioglitazone significantly reduced copper accumulation in the spinal dorsal horn. This reduction was accompanied by coordinated modulation of cuprotosis-related proteins, including downregulation of SLC31A1, ATP7a, and SCO1, together with upregulation of FDX1, suggesting partial restoration of copper homeostasis.Mechanistically, the decrease in copper concentration mediated by PPARγ may exert its effect through a dual mechanism. On the one hand, by downregulating SLC31A1 to reduce cellular copper uptake, and on the other hand, by upregulating ATP7a to promote copper efflux [ 53 ] .This bidirectional regulatory effect is significantly different from the hepatic copper metabolism regulation mechanism, suggesting that the nervous system may have a unique copper homeostasis or compensatory mechanism.On the other hand, PPARγ may enhance the biological activity of FDX1 through direct transcriptional regulation or protein-protein interaction, effectively inhibiting copper death and thus exerting a protective effect [ 52 ] . Several reports have suggest that cuprotosis promotes mitochondrial dysfunction by inducing oxidative stress, increasing expression of the mitochondrial copper chaperone SCO1, and impairing respiratory chain activity [ 54 – 56 ] . Excessive mitochondria-derived reactive oxygen species (mtROS) generated under these conditions can activate the IKK/IκB/NF-κB signaling cascade, leading to inflammatory gene expression and sustained neuroinflammation [ 57 ] . This study has several limitations that warrant consideration. First, although pharmacological activation of PPARγ using pioglitazone revealed a close association between PPARγ activity and cuprotosis-related regulation in the spinal dorsal horn, genetic approaches such as PPARγ knockout or knockdown models were not employed to systematically assess the temporal progression or intensity of pain behaviors following CCI, which restricts a more definitive evaluation of PPARγ involvement in CCI-induced nociception. Second, a range of pharmacological doses was not explored. Drug concentrations were selected based on prior literature and preliminary experimental observations, yielding a demonstrably effective dose; however, this single-dose strategy may not reflect the optimal therapeutic window. Consequently, future investigations incorporating dose–response analyses will be necessary to identify the maximally efficacious dosing regimens. Third, the findings were obtained from a single neuropathic pain model, and further validation in additional pain models and experimental conditions will be necessary to fully assess the generalizability of these results. 5. Conclusion In summary, the present study demonstrates that CCI leads to pronounced spinal copper accumulation, accompanied by upregulation of the cuprotosis-associated proteins SLC31A1, ATP7a, and SCO1, downregulation of FDX1, mitochondrial dysfunction, and activation of the NF-κB signaling pathway. Importantly, activation of spinal PPARγ markedly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. This analgesic effect was associated with restoration of copper homeostasis, suppression of SLC31A1, ATP7a, and SCO1 expression, enhancement of FDX1 expression, improvement of mitochondrial function, and inhibition of NF-κB pathway activation. Collectively, targeting a PPARγ-regulated cuprotosis-associated mitochondrial dysfunction and neuroinflammation may represent a promising therapeutic strategy for the treatment of neuropathic pain. Declarations Declaration of Competing Interest The authors declare that there are no conflicts of interest. Funding Author Contribution Qingling Xu: Software, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing-original draft. Guoxu Ling:Data curation,Formal analysis, Methodology, Software. Yuanzhi Lv:Data curation, Methodology, Validation, Software. Tingting Su: Methodology, Project administration. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8756915","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":591313747,"identity":"922a4861-792f-4274-a8fd-7effea555dd6","order_by":0,"name":"Qingling Xu","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingling","middleName":"","lastName":"Xu","suffix":""},{"id":591313748,"identity":"8fa661e9-185b-4b8d-baa9-9aa592b29a63","order_by":1,"name":"Guoxu Ling","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guoxu","middleName":"","lastName":"Ling","suffix":""},{"id":591313752,"identity":"a3a1818a-4235-41b5-89d2-ef2794713802","order_by":2,"name":"Yuanzhi Lv","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanzhi","middleName":"","lastName":"Lv","suffix":""},{"id":591313753,"identity":"fd650fe3-b36d-470a-bc9f-969963df55a6","order_by":3,"name":"Tingting Su","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Su","suffix":""},{"id":591313754,"identity":"a2612949-9e31-48e5-8925-ffc97f9e3a9e","order_by":4,"name":"Hengyi Ning","email":"","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hengyi","middleName":"","lastName":"Ning","suffix":""},{"id":591313755,"identity":"b88f1dee-cd45-4387-964c-ebefd1171d26","order_by":5,"name":"Yu Zhong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIie3RsQrCMBCA4RwHnU6yNrTQV7hNxKKvUhCcHBwdI4WsXfVFnBO66yy+RAsOHW19gusmmH/Jch/hOKVisZ8MrVLHkrS2YgLjKO9zc/HzSFuyrYSgaIIzb34QKw9dfxD9EVyW84uWaNFcbwKCcHZZOpKV9QkuJCRBmMid2FdCQgk407GfQVKCeq14R+YSatkuRdOG53DabLWuQ9dLyBTS95kOJA0G+WwsFov9Yx+FfjBabB7kQgAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Guangxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhong","suffix":""}],"badges":[],"createdAt":"2026-02-01 14:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8756915/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8756915/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102798995,"identity":"4472798c-b045-47ca-940a-01f9ecb0fc18","added_by":"auto","created_at":"2026-02-16 20:05:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart showing the timeline of the experimental procedures used in the study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were allocated to sham or CCI groups, protein expression levels and cellular localization of SLC31A1, FDX1, ATP7a, SCO1, and NF-κB p65 were assessed by western blotting and immunofluorescence staining. Subsequently, rats received either vehicle (5% DMSO) or the PPARγ agonist pioglitazone. Behavioral pain assessments, western blotting, immunofluorescence staining, transmission electron microscopy, and enzymatic activity assays were conducted at the indicated time points. The number of animals used in each group is shown in parentheses.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/35f913a83f790ab87423b2b5.png"},{"id":102798996,"identity":"e283ddf1-775f-4b89-9f29-3ec1b6acc0b5","added_by":"auto","created_at":"2026-02-16 20:05:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":280012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of copper ion levels and the expression and distribution of cuprotosis-related proteins in the dorsal horn (DH) of the spinal cord following CCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Copper ion levels in sham rats and at 3, 7, 14, and 21 days post-CCI.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for intergroup comparisons. \u003cem\u003en\u003c/em\u003e=4 rats per group.(B-C), (D-E), (F-G), and (H-I) Quantification of immunofluorescence staining for SLC31A1, FDX1, ATP7A, and SCO1 in the DH at indicated time points.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/e771113cbc7140dd6c2a1008.png"},{"id":102798989,"identity":"17f49ebc-8497-4dd0-8f78-1277b87171d1","added_by":"auto","created_at":"2026-02-16 20:05:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":637549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in cuprotosis-related protein expression and cellular localization of SLC31A1\u003c/strong\u003e \u003cstrong\u003ein the spinal cord DH after CCI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative western blots and quantitative analysis of SLC31A1, FDX1, ATP7A, and SCO1 at indicated time points. (A-E) Protein levels normalized to β-actin.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group. (F) Double immunofluorescence showing SLC31A1 localization primarily in neurons, partially in microglia, and rarely in astrocytes in the ipsilateral DH at day 14 post-CCI. Original magnification: 200× (scale bar=100 μm) and 400× (scale bar=50 μm).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/631fa55a41375210ec2b6c68.png"},{"id":102798994,"identity":"609d704b-c59f-421a-8935-41c0433905d9","added_by":"auto","created_at":"2026-02-16 20:05:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1289244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular distribution of cuprotosis-related proteins in the DH of the spinal cord of CCI rat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Double immunofluorescence showing cuprotosis-associated proteins FDX1(green), ATP7A(green), and SCO1 (green) were predominantly colocalized with NeuN (neuronal marker, red), partially with IBA1 (microglial marker, red), and showed minimal colocalization with GFAP (astrocytic marker, red) at day 14 post-CCI. Original magnification: 200× (scale bar=100 μm) and 400× (scale bar=50 μm).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/5dbb9a6b7aab2f29f9f70f93.png"},{"id":103049323,"identity":"b054e30c-ff06-49c2-baef-81144a33cd18","added_by":"auto","created_at":"2026-02-20 07:39:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":514495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial dysfunction and NF-κB pathway are involved in CCI-induced cuprotosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A)Representative transmission electron microscopy (TEM) images of neuronal mitochondria in the spinal cord DH at day 14 post-CCI. Mitochondria are indicated by red arrows. Scale bar = 500 nm. (B-C) Dihydroethidium (DHE) staining showing ROS production at indicated time points.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group.(D-E) Western blot analysis of p-NF-κB p65 normalized to total NF-κB p65 at indicated time points. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group. (F)Changes in mitochondrial respiratory chain complex IV activity following CCI at indicated time points. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/1c116044159f4876ed0f4af8.png"},{"id":102798993,"identity":"fb62c220-7ef9-48b2-9ef1-df870ed71a85","added_by":"auto","created_at":"2026-02-16 20:05:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":444257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular localization of PPARγ in the DH of the spinal cord and activation of PPARγ in the spinal cord attenuates CCI-induced pain behaviors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Western blot analysis of pPPARγ in the spinal cord DH at indicated time points.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group.(C-D)Quantification of pPPARγ immunofluorescence in the spinal cord DH at indicated time points.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 for comparisons among post-CCI time points. \u003cem\u003en\u003c/em\u003e=4 rats per group. (E-F)Pioglitazone treatment (100μg/10μl, days 7-14 post-CCI) alleviated mechanical and thermal hypersensitivity. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham + DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the CCI + DMSO group. \u003cem\u003en\u003c/em\u003e = 6 rats per group. (G) Double immunofluorescence showing colocalization of pPPARγ (green) and SLC31A1 (red), FDX1 (red) in the spinal cord DH at day 14 post-CCI. Original magnification: 200× (scale bar=100 μm) and 400× (scale bar=50 μm). (H-I) Effects of pioglitazone on pPPARγ expression across experimental groups. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the sham + DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the CCI + DMSO group. \u003cem\u003en\u003c/em\u003e = 4 rats per group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/3a57ae8e8b3100ccf4f04c01.png"},{"id":102962833,"identity":"9ffedd04-61bd-482c-8070-102f516cc496","added_by":"auto","created_at":"2026-02-19 04:11:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":521759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of PPAR-γ receptor on the copper ion concentration,the expression of cuprotosis related proteins mitochondrial dysfunction and NF-κB pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntrathecal pioglitazone treatment (100 μg/10 μl, once daily from days 7 to 14 post-CCI) modulated copper ion concentration, cuprotosis related proteins, mitochondrial function, oxidative stress, and inflammatory signaling in the spinal cord DH.(A) Copper ion concentration in the spinal cord following pioglitazone treatment.\u003csup\u003e⁎\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e⁎⁎\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 versus the sham+DMSO control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the CCI+DMSO model group.\u003cem\u003e n\u003c/em\u003e=4 rats in each group.(B–F) Western blot analysis of the cuprotosis-related proteins SLC31A1, FDX1, ATP7A, and SCO1 collected 2 h after the final intrathecal injection.\u003csup\u003e⁎\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e⁎⁎\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 versus the sham+DMSO control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the CCI+DMSO model group.\u003cem\u003e n\u003c/em\u003e=4 rats in each group.(G) Representative transmission electron microscopy images showing mitochondrial ultrastructure in neurons of the spinal cord DH.Scale bar=500 nm. (H) Activity of mitochondrial respiratory chain complex IV in the spinal cord DH.\u003csup\u003e⁎\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e⁎⁎\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 versus the sham+DMSO control group.\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus the CCI+DMSO model group.\u003cem\u003e n\u003c/em\u003e=4 rats in each group.(I–J) Reactive oxygen species (ROS) production detected by dihydroethidium (DHE) staining.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the sham+DMSO control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 versus the CCI+DMSO model group. \u003cem\u003en\u003c/em\u003e=4 rats in each group.(K-L) Western blot analysis of p-NF-κB p65 and total NF-κB p65 in L\u003csub\u003e4\u003c/sub\u003e–L\u003csub\u003e6\u003c/sub\u003e spinal cord tissues following intrathecal pioglitazone treatment. Protein levels were normalized to NF-κB p65.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the sham+DMSO group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.01 versus the CCI+DMSO group. \u003cem\u003en\u003c/em\u003e=4 rats in each group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/5fc733117c02a50de5b7446e.png"},{"id":104397536,"identity":"27d29ed0-f7ce-49f5-81db-b128e75d2d62","added_by":"auto","created_at":"2026-03-11 11:50:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5499658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/298edaf6-c027-4d35-b58b-33b24848e069.pdf"},{"id":102798991,"identity":"4b065094-815a-4768-86ae-9c2098ef34b7","added_by":"auto","created_at":"2026-02-16 20:05:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":293827,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8756915/v1/10c18c15c6b0eddaba6bfe63.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Activation of PPARγ Attenuates Neuropathic Pain by Modulating Spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNeuropathic pain is a debilitating clinical condition resulting from injury or dysfunction of the peripheral or central nervous system\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Epidemiological studies estimate that approximately 7\u0026ndash;10% of the adult population experiences chronic neuropathic pain, imposing a substantial socioeconomic burden worldwide\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. In the United States alone, direct healthcare costs associated with neuropathic pain exceed \u003cspan\u003e$\u003c/span\u003e10\u0026nbsp;billion annually, underscoring its significant public health impact\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Despite its high prevalence and economic burden, the pathophysiological mechanisms of neuropathic pain remain incompletely understood, and current therapeutic options provide limited efficacy. Consequently, there is an urgent need to elucidate novel mechanisms and develop more effective treatment strategies.\u003c/p\u003e \u003cp\u003eInnate immune activation and the subsequent neuroinflammation are pivotal to the pathogenesis of neuropathic pain. Accumulating evidence indicates that mitochondrial dysfunction, aberrant inflammatory signaling, and disruption of ionic homeostasis are key pathological mechanisms underlying neuropathic pain progression\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Copper (Cu) ions are essential trace elements for life, have redox activity, and play a vital role in various physiological processes in the human body\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.Under normal physiological conditions, copper ions enter cells mainly through the copper transporter 1 receptor (High affinity copper uptake protein 1, SLC31A1/CTR1).SLC31A1 is a transmembrane transporter on the cell membrane, with the C-terminus inside the cell and the N-terminus outside the cell\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. It is arranged on the cell membrane in the form of a homotrimer, transporting extracellular copper ions into the cell, and cooperating with ATPase copper transporter a (ATP7a) and ATPase copper transporter b (ATP7b) to regulate the transmembrane dynamic balance of copper from the trans-Golgi network to post-Golgi vesicles. These copper-loaded vesicles can fuse with the plasma membrane and release copper into the extracellular environment\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.Ferredoxin 1 (FDX1) is an iron-sulfur cluster protein present in mitochondria. It is a key regulatory gene for cuprotosis.It is mainly involved in key metabolic processes such as electron transfer, steroidogenesis, heme synthesis and lipoic acid modification\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. The mitochondrial copper partner protein (Synthesis of Cytochrome C Oxidase 1, SCO1) can selectively transfer copper ions according to the ring recognition, and plays a key role in the assembly of cytochrome C oxidase and the steady state regulation of copper ions\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.Abnormal copper metabolism and transport are associated with a variety of pathological physiology in the body, especially when genes regulating copper homeostasis mutate, it can lead to the occurrence of a variety of diseases such as Wilson's disease, Huntington's disease and Alzheimer's disease\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.Cuproptosis is a new cell death mode first proposed in 2022, different from known cell death modes including apoptosis, necrosis, pyroptosis or ferroptosis, characterized by copper dependence and regulation of mitochondrial respiration\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, there is currently very little research on the relationship between cuprotosis and neuropathic pain, and the specific molecular mechanism is still unclear, which deserves further in depth study.\u003c/p\u003e \u003cp\u003eMitochondria are organelles with a double membrane structure in cells, serving as the primary source of cellular energy and are essential for maintaining cellular homeostasis. Beyond their role in ATP production, mitochondria critically regulate apoptosis, redox balance, and reactive oxygen species (ROS) generation\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Mitochondrial dysfunction has been increasingly recognized as a potent trigger of innate immune activation and chronic inflammatory responses\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Excessive copper exposure profoundly disrupts mitochondrial homeostasis through multiple mechanisms. Copper can directly catalyze redox reactions resembling Fenton chemistry, leading to excessive ROS production, while simultaneously impairing antioxidant defenses, including superoxide dismutase and glutathione systems. As a consequence, oxidative stress is markedly enhanced, resulting in damage to mitochondrial proteins, lipids, and mitochondrial DNA, and ultimately compromising mitochondrial membrane integrity\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. In parallel, copper toxicity induces loss of mitochondrial membrane potential, disrupts ionic gradients across the mitochondrial membranes, and impairs oxidative phosphorylation and ATP synthesis. These alterations promote the opening of the mitochondrial permeability transition pore (MPTP), which further exacerbates mitochondrial dysfunction and cell injury. MPTP opening also amplifies ROS generation and activates inflammatory signaling pathways, particularly NF-κB, thereby establishing a self-perpetuating cycle of oxidative stress, mitochondrial damage, and inflammation\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeroxisome proliferator-activated receptor γ (PPARγ) belongs to the nuclear hormone receptor superfamily and has been reported to be expressed in the spinal cord.\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.Emerging evidence indicates that activation of PPARγ on sensory afferent and dorsal horn(DH) neurons play an important role in inflammation-induced pain,cancer-induced pain and neuropathic pain\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.In recent years, it has been found that PPARγ plays an important role in the mechanism of cell death\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Cuprotosis, as a newly discovered cell death mechanism, is closely related to mitochondrial respiration, and the activation of PPARγ may play a regulatory role in this process\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, influencing the occurrence and development of cuprotosis through multiple pathways.In atherosclerosis models, the activation of PPARγ can upregulate the expression of antioxidant enzymes, reducing ROS levels and thereby mitigating copper ion-induced cellular damage\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Additionally, the activation of PPARγ may influence the metabolism and excretion of copper ions in cells\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. PPARγ agonists can affect the sensitivity of cells to copper ions by modulating lipid metabolism and oxidative stress responses, thus regulating the occurrence of cuprotosis\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e.Nevertheless, whether spinal PPARγ regulates cuprotosis and its downstream mitochondrial and inflammatory consequences in neuropathic pain remains largely unknown.\u003c/p\u003e \u003cp\u003eGiven the relationship of PPARγ and cuprotosis, mitochondrial damage and inflammatory responses.Here, this study aimed to test the hypothesis that activation of spinal PPARγ alleviates neuropathic pain by regulating cuprotosis-associated mitochondrial dysfunction and neuroinflammation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Animals and Ethical Statement\u003c/h2\u003e \u003cp\u003eAdult male specific pathogen-free (SPF) Sprague\u0026ndash;Dawley rats (220\u0026ndash;250 g) were obtained from the Experimental Animal Center of Guangxi Medical University (license NO. SCXK (Gui) 2020\u0026ndash;0003). Animals were housed under controlled conditions (12-h light/dark cycle, 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 50% humidity) with ad libitum access to food and water. Behavioral testing was conducted between 09:00 am and 6:00 pm.All experimental procedures were approved by the Animal Care and Use Committee of Guangxi Medical University (approval NO. 2022-KT-031) and complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health)\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The study was reported in accordance with ARRIVE guidelines. Rats were acclimated for at least 1 week prior to experimentation. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2.Experimental designs and animal groups\u003c/h2\u003e \u003cp\u003eIn the present study, all rats were randomly assigned to three experimental groups, which were subjected to the following three experiments. The experimental groups and number of animals used in experiments are listed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental Design 1\u003c/b\u003e:To investigate the temporal alterations in copper ion levels, mitochondrial ultrastructure of the spinal dorsal horn, reactive oxygen species (ROS) generation, cuproptosis-related proteins (SLC31A1, FDX1, ATP7A, and SCO1), phosphorylated nuclear factor-κB p65 (p-NF-κB p65), and mitochondrial respiratory chain complex IV activity following chronic constriction injury (CCI), rats were allocated to different experimental cohorts at multiple post-injury time points.A total of 30 rats were randomly divided into five groups for behavioral assessments (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 per group): sham group rats evaluated at 21 days (Sham 21d) and CCI rats examined at 3, 7, 14, and 21 days post-injury (CCI 3d, CCI 7d, CCI 14d, and CCI 21d). Upon completion of behavioral testing, rats were euthanized by an overdose of pentobarbital sodium (200 mg/kg, i.p.). After deep anesthesia was achieved, cervical dislocation was performed as a secondary method in accordance with AVMA guidelines. Death was confirmed by the absence of breathing, heartbeat, and corneal reflex. L\u003csub\u003e4\u003c/sub\u003e-L\u003csub\u003e6\u003c/sub\u003e segments of the spinal cord were harvested for copper concentration measurement, mitochondrial enzyme activity assays, and western blot (WB) analyses.16 rats were assigned to four groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group) for immunofluorescence (IF) staining and dihydroethidium (DHE) detection: Sham 21d, CCI 7d, CCI 14d, and CCI 21d. The L\u003csub\u003e4\u003c/sub\u003e-L\u003csub\u003e6\u003c/sub\u003e spinal cord sections from these animals were used for the respective analyses.To assess mitochondrial ultrastructural changes, 8 rats were divided into two groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group): Sham 14d and CCI 14d. Mitochondrial morphology in the dorsal horn of the spinal cord was examined using transmission electron microscopy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental Design 2\u003c/b\u003e:To evaluate the potential analgesic effects of pioglitazone in the CCI model, pioglitazone was administered once daily for eight consecutive days, from day 7 to day 14 post-CCI surgery. A total of 24 rats were randomly allocated into four experimental groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 per group): Sham\u0026thinsp;+\u0026thinsp;DMSO, CCI\u0026thinsp;+\u0026thinsp;DMSO, Sham\u0026thinsp;+\u0026thinsp;Pioglitazone, and CCI\u0026thinsp;+\u0026thinsp;Pioglitazone. Pain-related behavioral tests were performed at baseline (0 d) and on days 3, 7, 14, and 21 after surgery. Following the completion of behavioral assessments, spinal cord segments L\u003csub\u003e4\u003c/sub\u003e\u0026ndash;L\u003csub\u003e6\u003c/sub\u003e were collected at 0, 7, 14, and 21 days for subsequent analyses. Western blotting(each with \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 rats per group per time point) and immunofluorescence staining (each with \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 rats per group per time point) were conducted to examine the expression and spatial localization of PPARγ.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperimental Design 3\u003c/b\u003e:To investigate whether repeated administration of a PPARγ activator could modulate cuproptosis during the development of neuropathic pain, 80 rats were randomly divided into four experimental groups (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group): Sham\u0026thinsp;+\u0026thinsp;DMSO, CCI\u0026thinsp;+\u0026thinsp;DMSO, Sham\u0026thinsp;+\u0026thinsp;Pioglitazone, and CCI\u0026thinsp;+\u0026thinsp;Pioglitazone. Two hours after the final injection, L\u003csub\u003e4\u003c/sub\u003e\u0026ndash;L\u003csub\u003e6\u003c/sub\u003e spinal cord segments were collected for subsequent analyses. Tissue samples were assessed via transmission electron microscopy (TEM), a copper content detection kit, enzyme activity assay kits, western blotting (WB), immunofluorescence (IF), and dihydroethidium (DHE) staining. These methods were used to quantify copper ion levels, mitochondrial ultrastructure, reactive oxygen species (ROS) production, expression of cuproptosis-related proteins (SLC31A1, FDX1, ATP7A, and SCO1), phosphorylated NF-κB p65 (p-NF-κB p65), and the activity of mitochondrial respiratory chain complex IV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3.CCI model preparation and Drug Treatments\u003c/h2\u003e \u003cp\u003eThe chronic constriction injury (CCI) model was established in rats according to the method described by Bennett and Xie\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Briefly, A longitudinal skin incision (~\u0026thinsp;1 cm) was made on the lateral surface of the left thigh at the mid-femoral level. The biceps femoris muscle was bluntly separated to expose the sciatic nerve using a glass hook.Four loose ligatures (4\u0026thinsp;\u0026minus;\u0026thinsp;0 chromic gut) were tied around the sciatic nerve approximately 2 mm proximal to the trifurcation, with ~\u0026thinsp;1 mm spacing between ligatures. The ligatures were tightened until a brief twitch of the ipsilateral hindlimb was observed. The muscle and skin were then sutured in layers.Sham-operated rats underwent the same surgical exposure without nerve ligation. After surgery, animals were placed on a heating pad until fully recovered from anesthesia and then returned to their home cages.\u003c/p\u003e \u003cp\u003eTo evaluate the role of PPARγ in neuropathic pain, rats received daily intrathecal injections of the PPARγ agonist pioglitazone (100 \u0026micro;g in 10 \u0026micro;l, dissolved in 5% DMSO) from postoperative day 7 to day 14. Control rats received an equal volume of 5% DMSO via intrathecal injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4.Placement and verification of intrathecal catheter\u003c/h2\u003e \u003cp\u003eIntrathecal catheterization was performed in rats as previously described by Pogatzki et al.\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. After adequate anesthesia was achieved, the rats were placed in a prone position and the surgical area was aseptically prepared.The L\u003csub\u003e5\u003c/sub\u003e\u0026ndash;L\u003csub\u003e6\u003c/sub\u003e intervertebral space was identified by palpation. A midline skin incision (~\u0026thinsp;1 cm) was made to expose the L5\u0026ndash;L6 intervertebral region. A 22-G needle was carefully inserted into the subarachnoid space. Entry into the intrathecal space was confirmed by a tail-flick response and the appearance of clear cerebrospinal fluid.A PE-10 catheter was then advanced approximately 2 cm cranially into the subarachnoid space. Catheter patency was verified by gentle injection of sterile saline. The external end of the catheter was heat-sealed and secured to the paraspinal muscles near the iliac crest. The catheter was tunneled subcutaneously and exteriorized at the back of the neck, leaving approximately 3 cm exposed and fixed in place.To prevent catheter damage, rats were housed individually in transparent cages with visual contact with other animals. Exclusion criteria included postoperative motor deficits (e.g., hindlimb paralysis or hemiplegia), catheter loss, or insufficient catheter length after surgery.On postoperative day 3, correct catheter placement was verified by intrathecal injection of 10 \u0026micro;l of 2% lidocaine followed by 10 \u0026micro;l of saline. Transient bilateral hindlimb paralysis occurring within ~\u0026thinsp;10 s and resolving within 30 min was considered confirmation of correct intrathecal catheter placement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5.Pain behavior quantification\u003c/h2\u003e \u003cp\u003eAll behavioral assessments were performed and analyzed by an experimenter blinded to group allocation. Rats were allowed to acclimatize to the testing environment for at least 30 min before each session. Behavioral tests were conducted at baseline (before CCI) and on days 3, 7, 14, and 21 after CCI. In the pioglitazone-treated groups, nociceptive behaviors were assessed at the same time points.Testing was conducted in a quiet room under controlled temperature and lighting conditions.\u003c/p\u003e \u003cp\u003ePaw withdrawal mechanical threshold (PWMT) was assessed as previously described\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Rats were individually placed in elevated Plexiglas chambers (20 \u0026times; 20 \u0026times; 25 cm) on a metal mesh floor and allowed to acclimate for 30\u0026ndash;60 min. Mechanical stimuli were applied to the mid-plantar surface of the left hind paw using calibrated von Frey filaments (Stoelting, USA).Testing began with a 2.0 g filament, followed by an ascending series of filaments. Each filament was applied perpendicularly with sufficient force to produce slight bending for approximately 4 s. Paw lifting or licking was considered a positive response. The PWMT was defined as the lowest force that evoked a withdrawal response. Values above 15 g or below 1 g were recorded as 15 g and 1 g, respectively.\u003c/p\u003e \u003cp\u003ePaw withdrawal thermal latency (PWTL) was measured using a thermal testing apparatus (BME410A, China) as previously described\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. After acclimation, a radiant heat source was focused on the mid-plantar surface of the left hind paw. The latency to paw withdrawal was automatically recorded when the rat withdrew its paw.A cut-off time of 25 s was set to prevent tissue damage. Each rat was tested five times at 5-min intervals, and the mean of the last three measurements was used as the PWTL value.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6.Measurement of Copper ion content\u003c/h2\u003e \u003cp\u003eAccording to the method of the copper content detection kit instructions, copper was extracted from the lumbar enlargement of the spinal cord and used a cryogenic grinder to homogenize on ice.Precool at 4℃ and centrifuge at 10,000 g for 10 min.Aspirate the supernatant and determined protein concentration.Detection of tissue copper content using fully automatic microplate reader.\u003c/p\u003e \u003cp\u003eCalculation of tissue copper content:\u003c/p\u003e \u003cp\u003eTissue copper content (\u0026micro;mol/g prot)=Δ determination/(Δ standard/C standard)*V extraction/(Cpr*V extraction)\u0026thinsp;=\u0026thinsp;80*Δ determination/Δ standard/Cpr.\u003c/p\u003e \u003cp\u003eC standard: standard concentration, 80 \u0026micro;mol/L; V extraction: pre-treatment distilled water volume,0.001L; Cpr: sample protein concentration, mg/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7.Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy (TEM) was used to examine mitochondrial ultrastructure. L\u003csub\u003e4\u003c/sub\u003e-L\u003csub\u003e6\u003c/sub\u003e spinal cord were fixed in 2.5% glutaraldehyde at 4\u0026deg;C for 4 h, followed by postfixation in 1% osmium tetroxide for 2 h at 4\u0026deg;C.Samples were dehydrated in a graded ethanol series (50%, 70%, 80%, 90%, and 100%), followed by acetone dehydration. The tissues were then infiltrated with epoxy resin and embedded. Polymerization was performed at 35\u0026deg;C and 45\u0026deg;C for 12 h each, and finally at 60\u0026deg;C for 24 h.Ultrathin sections were prepared and examined using a transmission electron microscope (HT7800, Hitachi, Japan). Mitochondrial morphology in the spinal dorsal horn was evaluated, including mitochondrial size, cristae integrity, swelling, and vacuolization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8.Immunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe immunofluorescence staining was performed as described in our previous study\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.The L\u003csub\u003e4\u003c/sub\u003e-L\u003csub\u003e6\u003c/sub\u003e spinal cord segments were dissected and post-fixed in 4% PFA for 8 h, then cryoprotected in 30% sucrose at 4\u0026deg;C until the tissue sank.Spinal cords were embedded and sectioned at 20 \u0026micro;m thickness using a cryostat. Sections were collected in PBS.For immunofluorescence staining, sections were blocked with 5% normal goat serum in PBS for 1 h at room temperature and then incubated overnight at 4\u0026deg;C with the following primary antibodies: p-PPARγ (1:1000, rabbit, Servicebio), SLC31A1 (1:100, rabbit, Zhengneng), FDX1 (1:100, rabbit, ABclonal), ATP7A (1:70, rabbit, Affinity), SCO1 (1:100, rabbit, Zhengneng), Iba1 (1:200, mouse, GeneTex), GFAP (1:400, mouse, Cell Signaling Technology), and NeuN (1:400, mouse, Novus).After washing with PBS, sections were incubated for 2 h at room temperature with CoraLite594-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) or CoraLite488-conjugated goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibodies (1:500, Proteintech).Sections were rinsed and mounted with glycerol-based mounting medium and stored at 4\u0026deg;C in the dark until imaging. Fluorescence images were acquired using an Olympus BX53 fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9.Western blot analysis\u003c/h2\u003e \u003cp\u003eL\u003csub\u003e4\u003c/sub\u003e-L\u003csub\u003e6\u003c/sub\u003e spinal cord segments were collected and homogenized to prepare protein lysates. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of protein (20\u0026micro;g) were separated on 10% SDS\u0026ndash;PAGE gels and subsequently transferred onto PVDF membranes using a wet transfer system.Membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4\u0026deg;C with the following primary antibodies: p-PPARγ (1:1000, rabbit, Servicebio), PPARγ (1:1000, rabbit, Affinity), ATP7A (1:500, rabbit, Affinity), SLC31A1 (1:1000, rabbit, Zhengneng), FDX1 (1:1000, rabbit, ABclonal), SCO1 (1:700, rabbit, Zhengneng), p-NF-κB p65 (1:700, rabbit, Zhengneng), NF-κB p65 (1:700, rabbit, Zhengneng), and β-actin (1:1000, mouse, Abcam).After washing with TBST (3 \u0026times; 10 min), membranes were incubated with goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) DyLight\u0026trade; 800 4X PEG secondary antibody (1:10,000, Thermo Fisher Scientific) or goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) DyLight\u0026trade; 800 4X PEG secondary antibody (1:10,000, Thermo Fisher Scientific).Protein bands were visualized using an Odyssey infrared imaging system. Band intensities were quantified using ImageJ software. Phosphorylated protein levels were normalized to their corresponding total protein levels, whereas total protein levels were normalized to β-actin. The mean value of the sham group was defined as 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10.Measurement of ROS production\u003c/h2\u003e \u003cp\u003eDihydroethidium (DHE) staining was used to detect ROS production in the spinal cord. Rats were perfused and spinal cord tissues were prepared as described above. L\u003csub\u003e4\u003c/sub\u003e-L6 spinal cord segments were post-fixed in 4% paraformaldehyde for 8 h and cryoprotected in 30% sucrose at 4\u0026deg;C. Tissues were sectioned at 20 \u0026micro;m using a cryostat.Sections were incubated with DHE solution (1:200 dilution, Beyotime Biotechnology, Nantong, China) at 37\u0026deg;C for 30 min in a humidified, light-protected chamber. After incubation, sections were washed with PBS and coverslipped.Fluorescence images were acquired using an Olympus BX53 fluorescence microscope. ROS levels were quantified as mean fluorescence intensity using Image-Pro Plus 6.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11.Respiratory chain complex IV enzymatic activity\u003c/h2\u003e \u003cp\u003eMitochondrial respiratory chain complex IV (cytochrome c oxidase) activity was measured using a commercial assay kit (Solarbio, Beijing, China) according to the manufacturer\u0026rsquo;s instructions\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.Briefly, the enzymatic reaction was monitored by measuring absorbance at 550 nm using a Thermo Multiskan microplate reader. For each sample and blank control, absorbance values were recorded at 10 s (A1) and 1 min (A2). The change in absorbance for each reaction was calculated as ΔA\u0026thinsp;=\u0026thinsp;A1\u0026thinsp;\u0026minus;\u0026thinsp;A2. Net absorbance change was obtained by subtracting the blank value from the sample value.Complex IV activity (U/mg protein) was calculated using the formula provided by the manufacturer:Activity (U/mg protein)\u0026thinsp;=\u0026thinsp;1832\u0026thinsp;\u0026times;\u0026thinsp;ΔA / C,where ΔA represents the corrected absorbance change and C denotes the protein concentration (mg/mL).Enzyme activities were normalized to the control group and expressed as fold changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12.Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) unless otherwise indicated. Normality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test, and homogeneity of variances was evaluated by Levene\u0026rsquo;s or Brown\u0026ndash;Forsythe test as appropriate.For behavioral assessments (paw withdrawal mechanical threshold [PWMT] and paw withdrawal thermal latency [PWTL]) measured over multiple time points, repeated-measures two-way ANOVA was applied, with factors of group and time, followed by Bonferroni\u0026rsquo;s post hoc test for pairwise comparisons.For endpoint measurements including immunofluorescence intensity, western blot densitometry, spinal copper content, ROS levels (DHE staining), and mitochondrial respiratory chain complex IV activity, two-way ANOVA with post hoc Bonferroni correction was used to assess differences between groups and time points. If only single-factor comparisons were performed, one-way ANOVA followed by Tukey\u0026rsquo;s or Bonferroni post hoc test was used. In cases where data did not meet assumptions of normality or homogeneity, non-parametric Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s multiple comparisons test was applied.All statistical analyses were conducted using IBM SPSS Statistics (version 23.0, SPSS Inc., Chicago, IL, USA). Graphs were generated with GraphPad Prism (version 6, GraphPad Software, San Diego, CA, USA). A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. CCI induces progressive copper accumulation in the spinal dorsal horn\u003c/h2\u003e\n \u003cp\u003eCopper is an essential trace element involved in numerous cellular functions, and increasing evidence implicates dysregulated copper homeostasis in neurological dysfunction and degeneration \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. To assess whether spinal dorsal horn (DH) copper levels are altered following chronic constriction injury (CCI), copper content in spinal cord tissue was evaluated at multiple post-injury intervals using the tissue copper ion content detection kit. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, CCI induced a marked elevation in copper ion accumulation within the spinal cord, displaying a clear temporal progression. A significant increase was first detected on postoperative day 3 and persisted through day 21 (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 compared with the sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for comparisons between CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). In contrast, sham group animals exhibited no appreciable changes in spinal copper levels throughout the experimental period.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. CCI induces dysregulated expression of cuprotosis-related proteins in the spinal dorsal horn\u003c/h2\u003e\n \u003cp\u003eSLC31A1,FDX1,ATP7a and SCO1 are the critical proteins involved in cuprotosis regulation. To examine whether CCI triggers alterations in the expression of cuproptosis-associated proteins, their spatial distribution and temporal expression profiles in the spinal dorsal horn (DH) were assessed at multiple post-injury time points using immunofluorescence staining and western blot analysis. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB-I, all four proteins exhibited prominent immunoreactivity within the superficial laminae of the DH. Following CCI, the fluorescence intensities of SLC31A1, ATP7A, and SCO1 were markedly elevated beginning on day 7 and remained at maximal levels through day 21 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB-C, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF-G, and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH\u0026ndash;I; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for comparisons among post-CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). In contrast, FDX1 immunoreactivity displayed a significant reduction from day 7 onward and persisted at reduced levels until day 21 after injury (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 among post-CCI groups; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group).Consistent with the immunofluorescence findings, western blot analysis of total spinal cord protein collected at 0, 3, 7, 14, and 21 days after CCI or sham surgery revealed pronounced alterations in cuproptosis-related protein expression. Quantitative analysis demonstrated that SLC31A1, ATP7A, and SCO1 protein levels increased progressively after CCI, with significant upregulation first detected at day 7 and sustained through day 21 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-B and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD-E; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 among post-CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group), while their expression remained minimal in sham-operated controls. Conversely, FDX1 protein abundance exhibited a robust and sustained decline from day 7 to day 21 following CCI (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 among post-CCI groups; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). No significant temporal changes in these proteins were observed in sham group animals.\u003c/p\u003e\n \u003cp\u003eTo determine what types of cells in the spinal cord express cuproptosis-associated proteins following CCI, double immunofluorescence staining was performed at 14 days post-injury using cell-type\u0026ndash;specific markers, including NeuN for neurons, GFAP for astrocytes, and IBA1 for microglia. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF and Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, SLC31A1, FDX1, ATP7A, and SCO1 signals were predominantly detected in NeuN-positive neurons in the spinal cord of CCI-treated rats,partly colocalization with IBA1-labeled microglia was also observed, whereas overlap with GFAP-positive astrocytes was minimal.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Cuprotosis-associated mitochondrial dysfunction and NF-\u0026kappa;B activation contribute to CCI-induced neuropathic pain\u003c/h2\u003e\n \u003cp\u003eCuproptosis is closely associated with mitochondrial function\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.To further substantiate the association between neuronal mitochondrial impairment and cuproptosis in neurons following CCI, ultrastructural alterations of mitochondria were examined. Owing to its superior spatial resolution, transmission electron microscopy (TEM) was employed to visualize mitochondrial morphology at the subcellular level. TEM analysis revealed pronounced mitochondrial abnormalities in neurons of CCI-treated rats, including extensive swelling, disruption and disappearance of cristae, and formation of vacuole-like structures within the spinal cord. In contrast, mitochondria in sham group retained intact membranes and well-organized cristae, with no evident ultrastructural damage (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA).Given that mitochondria represent a principal intracellular source of reactive oxygen species (ROS), ROS generation within the spinal cord was subsequently assessed. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;C, ROS-associated fluorescence intensity increased progressively beginning at day 7 after CCI and remained elevated through day 21 (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for comparisons among post-CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\n \u003cp\u003eCopper is an important cofactor for several enzymes including the mitochondrial cytochrome C oxidase, complex IV of the respiratory chain\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.To determine alterations in the activity of mitochondrial respiratory chain complex IV after CCI, enzymatic activity of mitochondrial respiratory chain complex IV in spinal cord tissue was quantified at successive time points using a enzyme activity kit. As presented in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF, complex IV activity in the spinal cord exhibited a pronounced decline after CCI, with significant reductions evident from day 7 through day 21 (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. Sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for comparisons among post-CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). In contrast, sham group rats maintained stable and relatively high complex IV activity throughout the observation period.\u003c/p\u003e\n \u003cp\u003eTo further evaluate neuroinflammatory activation induced by CCI, the nuclear factor \u0026kappa;B (NF-\u0026kappa;B) signaling cascade in the spinal cord were analyzed at multiple post-injury time points using western blotting. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD-E, phosphorylation of the NF-\u0026kappa;B p65 subunit was markedly enhanced following CCI, exhibiting a progressive increase beginning on day 7 and persisting through day 21 (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for comparisons among post-CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). In contrast, phosphorylated NF-\u0026kappa;B p65 levels remained minimal in sham-operated animals. In contrast, total NF-\u0026kappa;B p65 protein abundance did not differ significantly between groups across all examined time points.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4.Activation of spinal PPAR\u0026gamma; attenuates CCI-induced neuropathic pain\u003c/h2\u003e\n \u003cp\u003eTo investigate the involvement of PPAR\u0026gamma; in CCI-induced pathological changes, spinal cord samples were subjected to western blot analysis. Quantitative assessment demonstrated a marked reduction in PPAR\u0026gamma; activity beginning at day 3 after CCI, with a progressive decline observed through day 21 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;B; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 for comparisons among post-CCI time points; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). In contrast, PPAR\u0026gamma; activity in sham-operated animals remained consistently elevated throughout the experimental period.Consistent with these western blot findings, immunofluorescence analysis revealed prominent PPAR\u0026gamma; immunoreactivity localized predominantly to the ipsilateral superficial dorsal horn, which exhibited a significant temporal decrease following CCI (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026ndash;D; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 among post-CCI groups; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). Together, these complementary results suggest that altered PPAR\u0026gamma; signaling is closely associated with the development of neuropathic pain after CCI.\u003c/p\u003e\n \u003cp\u003eTo evaluate whether sustained activation of PPAR\u0026gamma; alleviates established nociceptive hypersensitivity in CCI rats, the PPAR\u0026gamma; agonist pioglitazone (100 \u0026micro;g/10 \u0026micro;l) was administered intrathecally once daily from days 7 to 14 after injury. This repeated treatment produced a robust attenuation of both paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL), with significant improvement observed from days 7 to 21 following CCI and no evidence of analgesic tolerance over the treatment period. In contrast, identical pioglitazone administration in sham group rats failed to elicit any measurable changes in PWMT or PWTL. These behavioral outcomes are summarized in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE\u0026ndash;F (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham\u0026thinsp;+\u0026thinsp;DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CCI\u0026thinsp;+\u0026thinsp;DMSO group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5.Activation of spinal PPAR\u0026gamma; suppresses copper accumulation and reprograms cuprotosis-related protein expression\u003c/h2\u003e\n \u003cp\u003eTo determine whether PPAR\u0026gamma; activation following CCI is linked to alterations in SLC31A1 and FDX1 expression, double immunofluorescence staining was performed on spinal cord sections at postoperative day 14. The results showed that phosphorylated PPAR\u0026gamma; exhibited clear spatial overlap with both SLC31A1 and FDX1 within the superficial laminae of the dorsal horn in CCI rats (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e\n \u003cp\u003eTo further investigate the role of PPAR\u0026gamma; activation to CCI-associated cuproptosis, the PPAR\u0026gamma; agonist pioglitazone (100 \u0026micro;g/10 \u0026micro;l) was delivered intrathecally once per day from postoperative days 7 to 14. Subsequently, spinal cord tissues were harvested 2 h after the final administration.As shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA, pioglitazone treatment markedly reduced the elevation of copper ion levels induced by CCI (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham\u0026thinsp;+\u0026thinsp;DMSO group ; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CCI\u0026thinsp;+\u0026thinsp;DMSO group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group).The expression of PPAR\u0026gamma; and cuproptosis-related proteins was evaluated by western blotting. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH\u0026ndash;I and Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB\u0026ndash;F, activation of PPAR\u0026gamma; with pioglitazone significantly suppressed the expression of SLC31A1, ATP7A, and SCO1, while concurrently enhancing PPAR\u0026gamma; and FDX1 protein levels in CCI rats ( \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham\u0026thinsp;+\u0026thinsp;DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CCI\u0026thinsp;+\u0026thinsp;DMSO group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). Collectively, these findings indicate that PPAR\u0026gamma; activation is linked to the modulation of cuproptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6.Activation of spinal PPAR\u0026gamma; ameliorates cuprotosis-associated mitochondrial dysfunction and NF-\u0026kappa;B signaling\u003c/h2\u003e\n \u003cp\u003eTo determine whether spinal activation of PPAR\u0026gamma; directly ameliorates mitochondrial impairment and modulates NF-\u0026kappa;B signaling following CCI, the PPAR\u0026gamma; agonist pioglitazone (100 \u0026micro;g/10 \u0026micro;l) was administered intrathecally once daily for eight consecutive days (postoperative days 7\u0026ndash;14). Ultrastructural assessment revealed that pioglitazone treatment markedly alleviated CCI-induced mitochondrial abnormalities, including cristae loss, fragmentation, vacuole formation, and swelling, thereby preserving overall mitochondrial integrity (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG). In parallel, mitochondrial respiratory chain complex IV activity in the spinal cord was significantly restored by pioglitazone administration (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham\u0026thinsp;+\u0026thinsp;DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CCI\u0026thinsp;+\u0026thinsp;DMSO; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\n \u003cp\u003eGiven the close association between mitochondrial dysfunction and oxidative stress, reactive oxygen species (ROS) production was subsequently evaluated. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eI\u0026ndash;J, dihydroethidium (DHE) fluorescence intensity in the spinal cord was significantly reduced after pioglitazone treatment in CCI rats (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham\u0026thinsp;+\u0026thinsp;DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CCI\u0026thinsp;+\u0026thinsp;DMSO group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). Furthermore, pioglitazone markedly suppressed the nuclear translocation of phosphorylated NF-\u0026kappa;B p65 without altering total NF-\u0026kappa;B p65 protein levels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eK\u0026ndash;L; \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. sham\u0026thinsp;+\u0026thinsp;DMSO group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. CCI\u0026thinsp;+\u0026thinsp;DMSO group; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 per group). Collectively, these findings demonstrate that PPAR\u0026gamma; activation exerts a protective effect against cuprotosis-associated mitochondrial dysfunction and inflammatory signaling in the spinal cord.\u003c/p\u003e\u003cbr\u003e\n\u003c/div\u003e"},{"header":"4.Discussion","content":"\u003cp\u003eThe present study demonstrates that chronic constriction injury (CCI) induces a distinct cuproptosis-related pathological profile in the spinal cord that parallels the development of mechanical allodynia and thermal hyperalgesia. During neuropathic pain progression, cuproptosis was associated with profound mitochondrial abnormalities, including disrupted ultrastructure and suppressed activity of mitochondrial respiratory chain complex IV, accompanied by excessive ROS accumulation. These changes occurred alongside a progressive increase in the expression of cuproptosis-associated proteins, including SLC31A1, ATP7A, and SCO1, as well as activation of the NF-\u0026kappa;B p65 signaling pathway, while FDX1 and PPAR\u0026gamma; expression levels declined in a time-dependent manner. Importantly, selective activation of spinal PPAR\u0026gamma; markedly alleviated CCI-induced nociceptive hypersensitivity. PPAR\u0026gamma; activation reduced spinal copper accumulation, preserved mitochondrial structural integrity, restored complex IV activity, and attenuated oxidative stress. Concomitantly, PPAR\u0026gamma; stimulation reversed the dysregulation of cuproptosis-related proteins by suppressing SLC31A1, ATP7A, SCO1, and phosphorylated NF-\u0026kappa;B p65, while enhancing FDX1 expression in the spinal cord. Collectively, these findings indicate that PPAR\u0026gamma; functions as a critical modulator linking copper dyshomeostasis to mitochondrial dysfunction and neuroinflammatory signaling in neuropathic pain,and suggest that therapeutic targeting of PPAR\u0026gamma;-regulated cuprotosis-associated mitochondrial dysfunction and neuroinflammation may represent a promising strategy for the treatment of neuropathic pain.\u003c/p\u003e\n\u003cp\u003eA variety of experimental models have been developed to facilitate investigation of the mechanisms underlying neuropathic pain. Among these, the chronic constriction injury (CCI) of the sciatic nerve is widely recognized as a classical and reliable model. The CCI model induces robust pain hypersensitivity and aberrant nociceptive behaviors while largely preserving peripheral nerve afferent and efferent conduction, thereby closely resembling clinical conditions such as low back and leg pain or sciatica resulting from intervertebral disc herniation or foraminal stenosis\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Beyond triggering acute injury-related neural discharge, CCI provokes sustained pathophysiological alterations in sensory axon cell bodies, leading to persistent ectopic firing and long-term hypersensitivity\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.In the present study, both paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) began to decline as early as day 3 following CCI, with pronounced reductions observed between days 7 and 14, after which these behavioral abnormalities remained relatively stable through day 21 post-surgery. In contrast, sham-operated animals exhibited no significant changes in PWMT or PWTL throughout the experimental period. These behavioral outcomes are consistent with previous reports, confirming that CCI elicits a rapid-onset and sustained neuropathic pain phenotype.\u003c/p\u003e\n\u003cp\u003eCopper functions as an essential trace element involved in multiple biological processes, including cellular energy metabolism, mitochondrial respiration, and antioxidant defense, primarily through its role as a catalytic cofactor. Intracellular copper homeostasis is tightly regulated by an integrated transport system comprising copper-dependent enzymes, molecular chaperones, and membrane transporters, which collectively control copper uptake, distribution, and efflux to maintain physiological balance. Disruption of this finely tuned regulatory network can profoundly affect neuronal function, and growing evidence links copper dyshomeostasis to the pathogenesis of neurological disorders\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.In the present study, spinal cord copper levels were significantly elevated as early as day 3 following CCI induction and remained persistently increased through day 21 compared with sham-operated controls. Notably, this temporal pattern of copper accumulation closely paralleled the development and maintenance of pain-related behaviors in CCI rats. These findings suggest that progressive copper imbalance within the spinal dorsal horn may contribute to the initiation and persistence of neuropathic pain, highlighting dysregulated copper homeostasis as a potential pathogenic factor.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that elevated extracellular copper concentrations are known to enhance SLC31A1-mediated copper uptake, while ATP7a functions as a key compensatory regulator by facilitating copper efflux or intracellular compartmentalization to prevent toxic accumulation. Upregulation of ATP7a is therefore widely regarded as an adaptive response to copper overload\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. In parallel, FDX1, a mitochondrial matrix enzyme essential for copper-dependent metabolic processes, has emerged as a central regulator of cuprotosis, whose dysregulation mechanistically differentiates this pathway from ferroptosis, apoptosis, and other forms of regulated cell death. In this study,immunofluorescence results showed that, compared with the sham group, the expression of ATP7a, SLC31A1, and SCO1 was upregulated in the CCI group, while the expression of FDX1 was downregulated, suggests a profound disturbance of neuronal copper trafficking in neuropathic pain. Furthermore, SLC31A1, FDX1, ATP7a, and SCO1 were all expressed in the lamina I-IV of the dorsal horn of the spinal cord, consistently, the double immunofluorescence results showed that the SLC31A1, FDX1, ATP7a, and SCO1 was colocalized mostly with neurons in the spinal cord dorsal horn. Regions and cell types that are central to nociceptive transmission and central sensitization.Therefore, it is speculated that cuprotosis-related proteins SLC31A1, FDX1, ATP7a, and SCO1 may be involved in the development and progression of neuropathic pain. Furthermore, western blot analysis confirmed the upregulation of the copper transporters SLC31A1 and ATP7a, as well as the mitochondrial copper chaperone SCO1, accompanied by a significant decrease in FDX1 protein expression. Together with the observed behavioral hypersensitivity and spinal copper accumulation, these molecular alterations provide further evidence that cuproptosis-related mechanisms are closely linked to the initiation and progression of neuropathic pain.\u003c/p\u003e\n\u003cp\u003eInflammation represents a core component of the innate immune response within the central nervous system\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Mitochondria, ubiquitous organelles in eukaryotic cells, serve as critical regulators of innate immunity by releasing danger-associated molecular patterns (DAMPs) and reactive oxygen species (ROS), thereby initiating and amplifying inflammatory signaling cascades\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Notably, cuprotosis has emerged as a copper-dependent form of cell death that directly targets mitochondrial integrity in neurons, leading to structural disruption, excessive ROS generation, and inflammatory activation\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.Copper homeostasis plays a pivotal role in maintaining mitochondrial respiratory chain function. SCO1 is a key regulator of mitochondrial copper transport and is essential for the assembly and activity of cytochrome c oxidase (complex IV)\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Dysregulation of SCO1 can disrupt early complex IV subassembly\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, impair copper-dependent enzymatic activity, and promote mitochondrial dysfunction\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Studies suggest excessive mitochondrial copper accumulation has been shown to inhibit complex IV activity and enhance ROS production, thereby creating a pro-oxidative intracellular environment. Valnot found that mutations in the SCO1 gene lead to mitochondrial cytochrome oxidase C deficiency\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Hosseini et al. showed that excessive copper leads to decreased activity of mitochondrial respiratory chain complex IV enzymes and increased ROS production\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. In the present study, transmission electron microscopy revealed pronounced mitochondrial abnormalities in the spinal dorsal horn following CCI, including severe swelling, cristae disruption, and vacuolar degeneration, indicating substantial mitochondrial structural damage during neuropathic pain development. Consistently, mitochondrial ROS levels in the spinal dorsal horn increased progressively after CCI, suggesting sustained oxidative stress. Concomitantly, we observed a significant reduction in mitochondrial respiratory chain complex IV activity alongside increased SCO1 protein expression in the spinal cord of CCI rats, indicating impaired mitochondrial respiratory function under conditions of copper dyshomeostasis.Excessive mitochondrial ROS not only reflect functional impairment but also serve as potent secondary messengers that activate inflammatory signaling pathways. Accordingly, our results demonstrated a marked increase in phosphorylated NF-\u0026kappa;B p65 without changes in total p65 expression by western blot analysis, indicating activation of the NF-\u0026kappa;B pathway. NF-\u0026kappa;B activation subsequently drives the transcription of pro-inflammatory cytokines, thereby initiating and amplifying neuroinflammatory responses that facilitate pain signal transmission\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e.Taken together, these findings demonstrate that CCI-induced copper dyshomeostasis promotes mitochondrial structural and functional impairment via SCO1-associated complex IV dysfunction, leading to excessive ROS generation and activation of NF-\u0026kappa;B-dependent inflammatory signaling. This feed-forward axis linking copper imbalance, mitochondrial dysfunction, and neuroinflammation may play a critical role in the persistence and amplification of neuropathic pain.\u003c/p\u003e\n\u003cp\u003eAccumulating evidence indicates that activation of peroxisome proliferator-activated receptor \u0026gamma; (PPAR\u0026gamma;) plays a critical role in the modulation of pathological pain conditions. In the present study, pioglitazone was administered intrathecally to deliver the drug directly into the spinal cord, thereby circumventing the blood\u0026ndash;brain barrier, enhancing local drug availability, reducing the required dosage, and minimizing systemic and off-target effects compared with intravenous administration\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Continuous intrathecal treatment was initiated from day 7 to day 14 after surgery, a time window that more closely reflects the clinical use of pharmacological interventions during the chronic phase of neuropathic pain.Behavioral assessments demonstrated that intrathecal administration of the PPAR\u0026gamma; agonist pioglitazone attenuated pain-related behaviors in a dose-dependent manner. Among the tested doses, 100 \u0026micro;g of pioglitazone produced a significantly greater improvement in mechanical allodynia and thermal hyperalgesia than the 10 \u0026micro;g and 30 \u0026micro;g doses, while not producing additional adverse effects\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Based on these observations, a dose of 100 \u0026micro;g was selected for continuous intrathecal administration in CCI rats.To further elucidate the role of PPAR\u0026gamma; in the maintenance of neuropathic pain, pioglitazone was administered intrathecally during the established pain phase. This intervention effectively alleviated both mechanical and thermal hypersensitivity in CCI rats, indicating that spinal PPAR\u0026gamma; activation remains therapeutically effective during the maintenance phase of neuropathic pain. Collectively, these findings suggest that enhanced PPAR\u0026gamma; activity plays an important role in sustaining analgesic modulation during chronic neuropathic pain.\u003c/p\u003e\n\u003cp\u003eWestern blotting and immunofluorescence analyses demonstrated that PPAR\u0026gamma; expression was progressively downregulated following CCI. Immunofluorescence further revealed that PPAR\u0026gamma; was predominantly localized within laminae I-IV of the spinal dorsal horn, a key region for the integration and transmission of peripheral nociceptive signals\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that CCI-induced suppression of PPAR\u0026gamma; signaling occurs at a critical spinal site involved in pain processing.Given the central role of copper dyshomeostasis and mitochondrial dysfunction in CCI-induced neuroinflammation, we next examined whether PPAR\u0026gamma; is functionally linked to cuprotosis-related pathways. Double-label immunofluorescence demonstrated that PPAR\u0026gamma; colocalized with the copper transporter SLC31A1 and the cuprotosis regulator FDX1 in the spinal cord, this spatial association suggests that a potential regulatory between PPAR\u0026gamma; signaling and cuprotosis under neuropathic pain conditions. Consistent with this association, Qi et al. reported that activation of the PPAR\u0026gamma;-FDX1 signaling axis suppresses cuprotosis in mice\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.Extending these findings, we observed that intrathecal administration of the PPAR\u0026gamma; agonist pioglitazone significantly reduced copper accumulation in the spinal dorsal horn. This reduction was accompanied by coordinated modulation of cuprotosis-related proteins, including downregulation of SLC31A1, ATP7a, and SCO1, together with upregulation of FDX1, suggesting partial restoration of copper homeostasis.Mechanistically, the decrease in copper concentration mediated by PPAR\u0026gamma; may exert its effect through a dual mechanism. On the one hand, by downregulating SLC31A1 to reduce cellular copper uptake, and on the other hand, by upregulating ATP7a to promote copper efflux\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e.This bidirectional regulatory effect is significantly different from the hepatic copper metabolism regulation mechanism, suggesting that the nervous system may have a unique copper homeostasis or compensatory mechanism.On the other hand, PPAR\u0026gamma; may enhance the biological activity of FDX1 through direct transcriptional regulation or protein-protein interaction, effectively inhibiting copper death and thus exerting a protective effect\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSeveral reports have suggest that cuprotosis promotes mitochondrial dysfunction by inducing oxidative stress, increasing expression of the mitochondrial copper chaperone SCO1, and impairing respiratory chain activity\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Excessive mitochondria-derived reactive oxygen species (mtROS) generated under these conditions can activate the IKK/I\u0026kappa;B/NF-\u0026kappa;B signaling cascade, leading to inflammatory gene expression and sustained neuroinflammation\u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study has several limitations that warrant consideration. First, although pharmacological activation of PPAR\u0026gamma; using pioglitazone revealed a close association between PPAR\u0026gamma; activity and cuprotosis-related regulation in the spinal dorsal horn, genetic approaches such as PPAR\u0026gamma; knockout or knockdown models were not employed to systematically assess the temporal progression or intensity of pain behaviors following CCI, which restricts a more definitive evaluation of PPAR\u0026gamma; involvement in CCI-induced nociception. Second, a range of pharmacological doses was not explored. Drug concentrations were selected based on prior literature and preliminary experimental observations, yielding a demonstrably effective dose; however, this single-dose strategy may not reflect the optimal therapeutic window. Consequently, future investigations incorporating dose\u0026ndash;response analyses will be necessary to identify the maximally efficacious dosing regimens. Third, the findings were obtained from a single neuropathic pain model, and further validation in additional pain models and experimental conditions will be necessary to fully assess the generalizability of these results.\u003c/p\u003e"},{"header":"5. Conclusion","content":" \u003cp\u003eIn summary, the present study demonstrates that CCI leads to pronounced spinal copper accumulation, accompanied by upregulation of the cuprotosis-associated proteins SLC31A1, ATP7a, and SCO1, downregulation of FDX1, mitochondrial dysfunction, and activation of the NF-κB signaling pathway. Importantly, activation of spinal PPARγ markedly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. This analgesic effect was associated with restoration of copper homeostasis, suppression of SLC31A1, ATP7a, and SCO1 expression, enhancement of FDX1 expression, improvement of mitochondrial function, and inhibition of NF-κB pathway activation. Collectively, targeting a PPARγ-regulated cuprotosis-associated mitochondrial dysfunction and neuroinflammation may represent a promising therapeutic strategy for the treatment of neuropathic pain.\u003c/p\u003e "},{"header":"Declarations","content":" \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eQingling Xu: Software, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing-original draft. Guoxu Ling:Data curation,Formal analysis, Methodology, Software. Yuanzhi Lv:Data curation, Methodology, Validation, Software. Tingting Su: Methodology, Project administration. Hengyi Ning: Investigation, Formal analysis. Yu Zhong: Writing\u0026ndash;review \u0026amp; editing, Supervision, Funding acquisition, Conceptualization.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data generated during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBARON, R. \u0026amp; BINDER, A. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment [J]. \u003cem\u003eLancet Neurol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (8), 807\u0026ndash;819 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMOISSET, X. \u0026amp; PAG\u0026eacute;, M. G. 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Increased mitochondrial fission promotes autophagy and hepatocellular carcinoma cell survival through the ROS-modulated coordinated regulation of the NFKB and TP53 pathways [J]. \u003cem\u003eAutophagy\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (6), 999\u0026ndash;1014 (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pioglitazone, Cuprotosis, Mitochondria, ROS, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-8756915/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8756915/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCuprotosis is a recently identified form of programmed cell death driven by copper accumulation. Increasing evidence suggests that copper dyshomeostasis contributes to neuroinflammatory processes. Peroxisome proliferator-activated receptor gamma (PPARγ) activation has been implicated in the regulation of cuprotosis; however, its role in cuprotosis-associated neuropathic pain remains poorly understood. This study aimed to investigate whether spinal PPARγ modulates cuprotosis and thereby influences neuropathic pain in a rat model of chronic constriction injury (CCI).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNeuropathic pain behaviors were assessed using mechanical and thermal sensitivity tests. Intrathecal catheterization was performed for spinal drug administration. Mitochondrial ultrastructure was examined by transmission electron microscopy. Copper concentration, protein expression, oxidative stress, mitochondrial function and inflammatory signaling were evaluated using western blotting, immunofluorescence staining, dihydroethidium staining and the enzyme activity assay kit.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCCI induced a time-dependent increase in spinal copper levels, accompanied by upregulation of the copper transport-related proteins SLC31A1, ATP7a, and SCO1, and downregulation of the cuprotosis-related protein FDX1. These changes were coincided with excessive mitochondrial structural damage, ROS accumulation, reduced mitochondrial respiratory chain complex IV activity and progressive activation of the NF-κB signaling pathway. Activation of spinal PPARγ significantly alleviated CCI-induced mechanical allodynia and thermal hyperalgesia. This effect was associated with restoration of copper homeostasis, suppression of SLC31A1, ATP7a, and SCO1 expression, enhancement of FDX1 expression, improvement of mitochondrial function and inhibition of NF-κB p65 phosphorylation in the spinal cord.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that PPARγ activation alleviates neuropathic pain by modulating copper dyshomeostasis, mitochondrial dysfunction, and neuroinflammatory signaling associated with cuprotosis. Targeting the PPARγ regulated spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation may represent a promising therapeutic strategy for neuropathic pain.\u003c/p\u003e","manuscriptTitle":"Activation of PPARγ Attenuates Neuropathic Pain by Modulating Spinal cuprotosis-associated mitochondrial dysfunction and neuroinflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 20:05:40","doi":"10.21203/rs.3.rs-8756915/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-07T04:23:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T21:10:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T20:32:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T00:31:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T11:05:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73411509645029055069765507634719009352","date":"2026-02-16T10:06:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T07:56:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234716172197703001389950595236810140089","date":"2026-02-13T18:07:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86825607942546732747563587059021389","date":"2026-02-11T21:13:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11994108512634520417603452158885824492","date":"2026-02-11T20:46:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127605145877212690476209941940085892786","date":"2026-02-11T16:57:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265912115187854058828830117485760510390","date":"2026-02-11T15:15:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332330178213013146250011322222883136628","date":"2026-02-11T15:14:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-11T15:00:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-11T14:58:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-09T12:16:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T21:38:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-06T21:30:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"282c1901-aa1c-419b-bf9c-dbe1622e0cd9","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62927613,"name":"Biological sciences/Cell biology"},{"id":62927614,"name":"Health sciences/Diseases"},{"id":62927615,"name":"Health sciences/Neurology"},{"id":62927616,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-04-07T04:39:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 20:05:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8756915","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8756915","identity":"rs-8756915","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}