CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis‒Inflammation Axis via the ERK/Nrf2 Pathway

CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis‒Inflammation Axis via the ERK/Nrf2 Pathway | 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 Research Article CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis‒Inflammation Axis via the ERK/Nrf2 Pathway Ye Yuan, Yu Jiang, Saisai Du, Guohong Yuan, Zhenjun Yang, Pei Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7846278/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Mar, 2026 Read the published version in Inflammation → Version 1 posted 9 You are reading this latest preprint version Abstract Peripheral nerve injury (PNI) is often limited by the activation of the ferroptosis‒inflammation axis. Although CXCL12 is a crucial neuroregenerative factor, the precise mechanism by which it promotes nerve repair through regulating this axis remains unclear. This study systematically investigated the role and underlying mechanism of CXCL12 via clinical samples, cell models, and animal experiments. Clinical data revealed a significant increase in serum CXCL12 levels in PNI patients, suggesting its potential involvement in nerve injury regulation. In an LPS-induced Schwann cell (SC) injury model, CXCL12 attenuated ferroptosis and oxidative damage by activating the ERK/Nrf2 signaling pathway to upregulate GPX4 and FSP1 while suppressing ACSL4 expression. Concurrently, CXCL12 inhibited the activation of the NF-κB signaling pathway, thereby reducing the secretion of TNF-α and IL-1β and alleviating the inflammatory response. The antiferroptotic effect of CXCL12 was reversed by the ERK inhibitor U0126. Furthermore, ferrous ammonium citrate (FAC)-induced iron overload experiments confirmed that ferroptosis is a critical mechanism bridging CXCL12 regulation of inflammation and that NF-κB overexpression weakens its anti-inflammatory effects. In animal experiments further demonstrated that CXCL12 improved the mitochondrial structure, reduced the accumulation of Fe2+ and lipid peroxidation in injured nerve tissue, and promoted axon and myelin regeneration after PNI. Overall, CXCL12 promotes PNI repair by activating the ERK/Nrf2 pathway to inhibit SC ferroptosis, which subsequently downregulates the NF-κB-mediated inflammatory response. This study is the first to elucidate the bridging role of ferroptosis in CXCL12-mediated inflammation regulation, suggesting a new theoretical basis for targeting CXCL12 as a potential therapeutic strategy for PNI. Nerve regeneration Ferroptosis Inflammatory response CXCL12 ERK/Nrf2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Introduction Peripheral nerve injury (PNI), often caused by trauma, ischemia, or metabolic disorders, leads to impaired motor and sensory function [1,2] . Despite continuous advancements in treatments such as surgical suturing and nerve transplantation [3] , the process of nerve regeneration remains limited by challenges, including slow axon regrowth, persistent inflammatory responses, and inadequate reconstruction of myelin sheaths [4,5] . Recent studies have shown that ferroptosis, a form of iron-dependent programmed cell death, plays a critical role in various neurological disorders, such as Alzheimer's disease and spinal cord injury. Furthermore, ferroptosis is closely linked to neuroinflammation, suggesting that it may be a significant mechanism hindering nerve regeneration. In a clinical context, PNI is often accompanied by dysregulated iron metabolism and increased oxidative stress. This leads to the abnormal accumulation of iron ions within Schwann cells (SCs), which then catalyze the production of large amounts of highly reactive hydroxyl radicals (·OH) via the Fenton reaction. This process can induce lipid peroxidation, thereby triggering ferroptosis in SCs [6-8] . However, ferroptosis is not just a passive result of ROS; it is also a significant source. Continuous ROS production during ferroptosis can create a positive feedback loop, further aggravating SC damage [9,10] . Additionally, ROS can oxidatively modify and enhance the activity of NF-κB subunits, promoting the nuclear translocation of the p65/p50 dimer. This, in turn, upregulates the expression of IL-1β and TNF-α, exacerbating local inflammation and hindering nerve regeneration [11-13] . Therefore, the mechanism by which ferroptosis regulates SC inflammation through iron-dependent ROS production requires further investigation. CXCL12 (C-X-C motif chemokine ligand 12), also known as stromal cell-derived factor-1 (SDF-1), belongs to the CXC chemokine family and was first cloned and identified in the 1990s [14,15] . By binding to its two main receptors, CXCR4 and CXCR7, CXCL12 participates in a variety of biological processes, including cell migration, survival, proliferation, and tissue homeostasis [16-18] . This molecule has several splice variants, with CXCL12α being the most common and widely studied isoform [19,20] . The protein structure of CXCL12 includes a typical chemokine core fold (a three-stranded β-sheet and a C-terminal α-helix). Its N-terminal region is responsible for receptor activation (entering the binding pocket of CXCR4 and triggering the signal), whereas the C-terminus binds to glycosaminoglycans/heparan sulfate proteoglycans, which helps with its localization and the formation of concentration gradients within the tissue microenvironment [21-23] . Upon binding to CXCL12, CXCR4, a G protein-coupled receptor, can activate multiple downstream signaling pathways, including the PI3K/Akt, ERK/MAPK, and JAK/STAT pathways, which then regulate gene expression and cellular responses [24-26] . In contrast, CXCR7 mainly regulates the availability and distribution of CXCL12 through β-arrestin-mediated signal transduction and endocytosis [27-29] . In the central nervous system, the CXCL12/CXCR4 axis is involved in the migration of neuroblasts and the formation of brain regions during embryonic development [30,31] . In adulthood, it plays crucial roles in hippocampal neurogenesis, neuronal survival, and maintenance of the blood‒brain barrier [32,33] . Studies have shown that CXCL12 plays dual roles in diseases such as ischemic stroke, Alzheimer's disease, and multiple sclerosis. For example, in a model of cerebral ischemia, CXCL12 can promote vascular regeneration and the recovery of behavioral function [34] . However, during the acute phase of stroke, its signaling activity may also exacerbate the inflammatory response [35] , and some reports link its elevated expression to inflammatory cell infiltration and neuronal apoptosis in these diseases [36,37] . In recent years, the function of CXCL12 in the process of peripheral nerve injury and repair has garnered increasing attention. Research indicates that the CXCL12/CXCR4 axis can promote both axon growth and angiogenesis and is also involved in the migration and myelin repair of SCs [38,39] . Furthermore, stimuli such as oxidative stress and inflammatory factors released after tissue injury can also induce CXCL12 expression [40] . However, it remains unclear whether CXCL12 is involved in the regulation of ferroptosis following PNI, particularly its role in the ferroptosis‒inflammation axis. Therefore, our study utilized clinical, in vitro, and in vivo experiments to investigate the mechanism by which CXCL12 promotes nerve regeneration after PNI by regulating SC ferroptosis and mitigating the inflammatory response. This research aims to provide new insights and potential therapeutic strategies for the clinical treatment of PNI. 2. Materials and Methods 2.1 Ethical approval 2.1.1 Clinical Sample Collection and Ethics A total of 20 patients with peripheral nerve injury (PNI) who presented to the Affiliated Hospital of Chengde Medical University between November 2024 and May 2025 were enrolled in this study. Concurrently, 20 healthy volunteers matched for sex and age were recruited. All participants provided informed consent for their participation. The collection and use of human samples were approved by the Ethics Committee of The Affiliated Hospital of Chengde Medical University. The submission and approval of ethical standards adhered to the Declaration of Helsinki. The ethical approval number is CYFYLL2024077. 2.1.2 Animal Subjects and Ethics Healthy adult male Sprague–Dawley rats weighing approximately 200 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental rats were housed under standard barrier conditions (temperature 23±1°C, 12-h light/dark cycle). All animal procedures were approved by the Animal Care and Use Committee of The Affiliated Hospital of Chengde Medical University. The ethical approval number is CYFYLL2025006. 2.2 Clinical Study 2.2.1 Inclusion and exclusion criteria for peripheral nerve injury patients To observe the changes in the expression of CXCL12 in the serum of peripheral nerve injury (PNI) patients, serum samples were collected on day 3 postsurgery. The inclusion criterion was age between 18 and 60 years. Patients with pure peripheral nerve injury, primarily median or ulnar nerve transection due to forearm trauma. The exclusion criteria were a history of central nervous system disorders, diabetes mellitus, autoimmune diseases, malignant tumors, or other underlying metabolic and immune-related diseases. Patients with severe infection or hepatic/renal insufficiency. Recent use of immunosuppressive agents or drugs that affect nerve regeneration. Pregnant or lactating females. Patients unable to cooperate with the study protocol. Twenty healthy volunteers, matched for sex and age, were recruited concurrently as the control group. 2.2.2 Serum CXCL12 Level Measurement in Clinical Patients Serum samples were obtained via venipuncture of the cubital vein and isolated after centrifugation at 3000×g for 15 minutes. The samples were then used for the quantitative detection of CXCL12 via an ELISA kit (Elabscience, China). The experiment was conducted according to the manufacturer’s instructions, with three technical replicates set for each sample. The absorbance was measured at a wavelength of 450 nm via a microplate reader (Bio-Rad Laboratories, USA). The results were calculated in pg/mL and subjected to statistical analysis. 2.3 Cell experiments 2.3.1 Cell culture and injury model The rat Schwann cell line (Procell, Wuhan, China) was routinely cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a constant-temperature incubator at 37°C with 5% CO2. To establish the cellular injury model, the cells were treated with 10 μM LPS for 6 hours, followed by replacement with fresh culture medium for further incubation. 2.3.2 Cell Grouping and Treatment The cell experiments were divided into four parts. First, the effects of CXCL12 on SC ferroptosis and inflammation were explored, and SCs were divided into 3 groups: the control group, in which SCs were routinely cultured for 6 h; the LPS group, in which SCs were treated with LPS for 6 h; and the LPS + CXCL12 group, in which SCs were treated with 100 ng/mL CXCL12 for 2 h before the addition of LPS. The second part investigated whether CXCL12 regulates SC ferroptosis via the ERK/Nrf2 pathway, and SCs were divided into 4 groups: the LPS group; the LPS + CXCL12 group; the LPS + CXCL12 + U0126 group, which was pretreated with 100 ng/mL CXCL12 and U0126 (20 μM) [41] for 2 hours before the addition of LPS; and the LPS + U0126 group, which was pretreated with U0126 (20 μM) for 2 hours before the addition of LPS. The third part explored whether CXCL12 regulates SC inflammation via the NF-κB pathway, and SCs were divided into 4 groups: the LPS group; the LPS + CXCL12 group; the LPS + CXCL12 + NF-κB-OE group, which was transfected with an NF-κB overexpression plasmid and then treated with CXCL12 for 2 hours before the addition of LPS; and the LPS + CXCL12 + NF-κB-EV group, which was transfected with an empty vector plasmid and then treated with CXCL12 for 2 hours before the addition of LPS. Fourth, we explored whether CXCL12 reduces inflammation by inhibiting SC ferroptosis. SCs were divided into 3 groups: the LPS group, the LPS + CXCL12 group, and the LPS + CXCL12 + FAC group, which were treated with FAC for 6 hours [42] before CXCL12 treatment. 2.3.3 Cell viability assay SCs were seeded in 96-well plates at a density of 5000 cells/well and precultured for 24 hours. Following the corresponding drug treatments according to the experimental groups, 10 μL of CCK-8 reagent (Dojindo, Japan) was added to each well and incubated at 37°C in the dark for 1 hour. The absorbance value was measured at a wavelength of 450 nm via a microplate reader. The experiment was repeated three times for each group. 2.3.4 Western blot analysis The treated cells were harvested and lysed via RIPA lysis buffer (Solarbio, China) containing the protease inhibitor PMSF (Solarbio, China) and phosphatase inhibitors (Solarbio, China) to extract total protein. The protein concentration was determined via the BCA method (Beyotime, China). Protein samples (30 μg/lane) were separated via SDS‒PAGE and subsequently transferred to PVDF membranes via the wet transfer method. The membranes were blocked for 1 h at room temperature with TBST buffer containing 5% nonfat milk. The membranes were then incubated overnight at 4°C with the following primary antibodies: ACSL4 (1:1000, Proteintech, Cat#22401-1-AP, RRID: AB_2783458); GPX4 (1:2000, Proteintech, Cat#67763-1-Ig, RRID: AB_2799687); FSP1 (1:1000, Proteintech, Cat#18412-1-AP, RRID: AB_2810984); ERK (1:2000, Proteintech, Cat#16443-1-AP, RRID: AB_10694175); P-ERK (1:1000, Proteintech, Cat#16443-1-AP, RRID: AB_2799689); NRF2 (1:1000, Proteintech, Cat#16396-1-AP, RRID: AB_11006916); NF-κB (1:1000, Proteintech, Cat#10745-1-AP, RRID: AB_2276536); P-NF-κB (1:500, Proteintech, Cat#30363-1-AP, RRID: AB_2799690); and IκBα (1:1000, Proteintech, Cat#10268-2-AP, RRID: AB_2276481)；P-IKBA (1:1000, Proteintech, Cat#28591-1-AP, RRID: AB_2799691)；β-actin (1:5000, Proteintech, Cat#60008-1-Ig, RRID: AB_2106289). After the membranes were washed three times with TBST, they were incubated with the HRP-labeled secondary antibody goat anti-rabbit IgG (H+L) (1:5000, Proteintech, Cat# SA00001-2, RRID: AB2722555) for 1 hour at room temperature. The bands were visualized via enhanced chemiluminescence (ECL) reagent, and the gray values were analyzed via ImageJ software. The experiment was repeated three times for each group. 2.3.4 Immunofluorescence The cell samples were fixed with paraformaldehyde and then permeabilized with 0.1% Triton X-100 for 20 min. After blocking with an immunofluorescence quick blocking solution for 30 min, the membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-MBP (1:100, Proteintech, Cat#10458-1-AP, RRID: AB_2336123) and rabbit anti-NF200 (1:100, Proteintech, Cat#18934-1-AP, RRID: AB_10640801). After being washed with PBST, the membranes were incubated for 1 h at room temperature with the following secondary antibodies: goat anti-mouse Alexa Fluor 488 targeting MBP (1:200, Proteintech, Cat# SA00013-1, RRID: AB_2810983) and goat anti-rabbit Alexa Fluor 594 targeting NF-200 (1:200, Proteintech, Cat# SA00013-4, RRID: AB_2810984). After mounting with a DAPI-containing mounting medium, images were captured under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed via ImageJ software. The experiment was repeated three times for each group. 2.3.5 ROS measurement After the completion of treatment, the culture medium was removed, and a DCFH-DA probe (10 μmol/L) diluted in serum-free medium was added. The cells were incubated at 37°C in a 5% CO2 incubator in the dark for 20 minutes. The cells were subsequently washed three times with serum-free medium, and fluorescence images were captured under a fluorescence microscope. The fluorescence intensity was analyzed via ImageJ software. The experiment was repeated three times for each group. 2.3.6 Ferrous Ion Detection After the cells were treated, the original culture medium was discarded, and serum-free medium containing a 5 μM fluorescent probe (Solarbio, China) was added. The cells were incubated in the dark for 30 minutes and then rinsed five times with PBS. Images were acquired under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed via ImageJ software. The experiment was repeated three times for each group. 2.3.7 MDA measurement The content of malondialdehyde (MDA) in the cells was determined via the thiobarbituric acid (TBA) colorimetric method. The cell samples were homogenized in ice-cold physiological saline, and the supernatant was collected after centrifugation. The procedure was performed according to the manufacturer's instructions for the MDA assay kit (Solarbio, China). The experiment was repeated three times for each group. 2.3.8 GSH measurement The intracellular GSH content was measured via a GSH detection kit (Solarbio, China). A standard curve was generated on the basis of the absorbance values of different concentrations of the standard product. The total GSH content was calculated by comparing the sample values against the standard curve. The experiment was repeated three times for each group. 2.3.9 ELISA detection An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of the inflammatory factors IL-1β and TNF-α in the cell culture supernatant (centrifuged at 3000×g for 15 min). The procedure was performed according to the manufacturer's instructions for the ELISA kit (Elabscience, China). After a standard curve with standard products was established, the absorbance was read at a wavelength of 450 nm via a microplate reader (Bio-Rad Laboratories, USA). The results were subjected to statistical analysis in units of pg/mL. The experiment was repeated three times for each group. 2.4 Animal experimentation 2.4.1 Sciatic nerve crush model establishment The sciatic nerve crush injury model was established according to the method reported by Wandling et al [43] . After anesthesia via intraperitoneal injection of sodium pentobarbital (30 mg/kg), the middle segment of the sciatic nerve was exposed through blunt dissection along the gap of the biceps femoris muscle of the right hind limb. The nerve was then crushed continuously three times via a vascular clamp (10 s each time, with a 10 s interval between crushes). Postsurgery, the rats were housed until day 5 for sacrifice and collection of the sciatic nerve for Western blot and transmission electron microscopy (TEM) experiments; the rats were housed until day 14 and underwent footprint gait analysis before sacrifice and collection of the sciatic nerve for HE staining and immunofluorescence experiments. 2.4.2 Animal Grouping and Treatment The experiment was divided into three parts. In the first part, 45 SD rats were randomly divided into three groups (n=15 per group): the sham group (only the sciatic nerve was exposed without injury); peripheral nerve injury group (PNI) (nerve injury model was established, followed by daily intramuscular injection of 0.2 mL of saline); and the CXCL12 treatment group (PNI + CXCL12) (nerve injury model was established, followed by daily intramuscular injection of 4 μg of CXCL12 [44] ). The second part included 20 SD rats randomly divided into four groups (n=5 per group) to investigate the role of the ERK signaling pathway. The groups were the PNI group, PNI + CXCL12 group, PNI + CXCL12 + U0126 group, and PNI + U0126 group. U0126, an ERK inhibitor, was administered via daily intraperitoneal injection at a dose of 25 mg/kg postsurgery [45] . The third part included 15 SD rats randomly divided into three groups (n=5 per group) to assess the role of ferroptosis in the inflammatory response. The groups were the PNI group, PNI + CXCL12 group, and PNI + CXCL12 + FAC group. FAC was administered via daily local intramuscular injection at a dose of 2 μg/rat postsurgery [46] . 2.4.3 Western blot analysis Tissue from the central area of the injured sciatic nerve (approximately 2 cm in length) was collected and quickly ground into powder in liquid nitrogen. RIPA lysis buffer was added for lysis, and the supernatant was collected after centrifugation. The subsequent procedures were the same as those for the Western blot method described for the cell experiments. The experiment was repeated three times for each group. 2.4.4 Immunofluorescence Nerve tissue sections were baked and dewaxed. The subsequent procedures were the same as those for the immunofluorescence method described in the cell experiments. The experiment was repeated three times for each group. 2.4.5 Ferrous Ion Detection Fresh nerve tissue from the central area of injury was collected, and lysis buffer from the Ferrous Ion Assay Kit (Pulilai, China) was added. The supernatant was obtained after grinding and centrifugation, and subsequent procedures were performed according to the manufacturer’s instructions. Finally, the absorbance was read at a wavelength of 593 nm via a microplate reader, and the ferrous ion concentration in the samples was calculated on the basis of the standard curve. The experiment was repeated three times for each group. 2.4.6 MDA measurement Fresh nerve tissue from the central area of injury was collected, and lysis buffer from the MDA assay kit (Solarbio, China) was added. The supernatant was obtained after grinding and centrifugation. Subsequent procedures strictly followed the instructions. The experiment was repeated three times for each group. 2.4.7 GSH measurement Tissue from the central area of nerve injury was collected, and a mixed solution from the GSH assay kit (Solarbio, China) was added. The supernatant was obtained after grinding and centrifugation. Subsequent procedures strictly followed the instructions. The experiment was repeated three times for each group. 2.4.8 ELISA detection An enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of IL-1β and TNF-α in rat serum (centrifuged at 3000×g for 15 min). The procedure was performed according to the manufacturer’s instructions for the ELISA kit (Elabscience, China). The experiment was repeated three times for each group. 2.4.9 H&E Staining The tissue sections were baked and dewaxed, stained with hematoxylin for 5 minutes, differentiated, rinsed with water, and then stained with eosin for 2 minutes. The tissue was dehydrated through a graded series of ethanol, cleared with xylene, and mounted with neutral balsam. Images were collected under an optical microscope and analyzed via ImageJ software. The experiment was repeated three times for each group. 2.4.10 Electron microscopy sample preparation and mitochondrial observation Freshly collected sciatic nerve tissue (1 mm×1 mm×1 mm) was immediately immersed in electron microscopy fixative and fixed at 4°C for 4 hours. The tissue was then rinsed three times with PBS and postfixed with 1% osmium tetroxide solution for 2 hours at room temperature, followed by three more rinses. The tissue was subsequently dehydrated sequentially through a graded series of ethanol, infiltrated with epoxy resin, and embedded. Ultrathin sections were prepared via an ultramicrotome and counterstained with uranyl acetate and lead citrate. Finally, the sections were dried overnight at room temperature, and morphological changes in the mitochondria were observed via transmission electron microscopy (TEM). 2.5 Data analysis All experimental data were statistically analyzed via GraphPad Prism 9 software and are presented as the means ± standard deviations (means ± SDss). After checking for a normal distribution and homogeneity of variance, comparisons among multiple groups were performed via one-way ANOVA combined with Tukey's HSD post hoc test. Comparisons between two groups were performed via Student's t test. p<0.05 was considered to indicate a statistically significant difference. 3. Results 3.1 CXCL12 is significantly elevated in the serum of PNI patients Serum samples were collected from patients with PNI and healthy controls to quantitatively analyze the CXCL12 concentration via ELISA. The results revealed that the serum CXCL12 levels in PNI patients were significantly greater than those in healthy individuals (Figure 1). 3.2 CXCL12 Inhibits SC Ferroptosis and Inflammation 3.2.1 CXCL12 Significantly Ameliorates LPS-Induced SC Injury by Regulating Key Ferroptosis Proteins and Oxidative Stress To investigate the protective effect of CXCL12 against LPS-induced SC injury, an LPS injury model was first established, and the optimal dose was determined. Treatment of SCs with 10 μM LPS led to a stable decrease in cell viability to approximately 50% (Figure 2A) while also inducing typical characteristics of ferroptosis (Figure 2C‒P). The results of the subsequent dose‒response screening revealed that CXCL12 could enhance SC viability in a dose-dependent manner. When the concentration reached 200 ng/mL, the cell survival rate significantly recovered to 91% (P<0.0001) (Figure 2B). This concentration was selected as the optimal intervention dose for subsequent mechanistic studies. We then further explored the protective mechanism of CXCL12 and found that its effect was due to the significant inhibition of ferroptosis. Compared with LPS treatment, CXCL12 treatment significantly reversed the LPS-induced increase in the expression of ferroptosis-related proteins, specifically the downregulation of the proferroptotic protein ACSL4 and the simultaneous upregulation of the antiferroptotic proteins GPX4 and FSP1 (Figure 2C-J). The results of the functional assays were highly consistent: CXCL12 intervention significantly reduced the levels of ROS, Fe2+, and MDA within the SCs while simultaneously restoring the content of the crucial antioxidant GSH (Figure 2K-P). In summary, CXCL12 effectively antagonized LPS-induced SC injury by regulating the expression of ferroptosis-related proteins and alleviating oxidative stress. 3.2.2 CXCL12 Alleviates LPS-Induced SC Ferroptosis by Activating the ERK/Nrf2 Pathway and Regulating Key Ferroptosis Proteins Nrf2 is a crucial regulator of cellular antioxidant responses and plays an important role in regulating cellular ferroptosis [47] . To investigate the potential mechanism by which CXCL12 inhibits SC ferroptosis, we first detected the expression of proteins related to the ERK/Nrf2 signaling pathway. Compared with LPS treatment, CXCL12 treatment significantly increased the p-ERK/ERK ratio and increased the protein level of Nrf2 (Figure 3A-C), suggesting that CXCL12 activates the ERK/Nrf2 signaling pathway in SCs. To verify the necessity of this pathway, we used the ERK inhibitor U0126. The CCK-8 assay confirmed that 20 μM U0126 alone did not significantly affect the viability of CXCL12-treated SCs (Figure 4A). However, the Western blot results indicated that U0126 significantly blocked the antiferroptotic effect of CXCL12. Compared with the LPS + CXCL12 group, the LPS + CXCL12 + U0126 group presented increased expression of the proferroptotic protein ACSL4, while the expression of the antiferroptotic proteins GPX4 and FSP1 was significantly reduced. Furthermore, U0126 alone aggravated LPS-induced ferroptotic injury, as evidenced by decreased Nrf2 expression, increased ACSL4 expression, and decreased GPX4 and FSP1 expression (Figure 4B-G). Collectively, these results demonstrate that CXCL12 inhibits SC ferroptosis by activating the ERK/Nrf2 signaling pathway. 3.2.3 CXCL12 Alleviates LPS-Induced SC Inflammation by Inhibiting the NF-κB Pathway and the Secretion of Inflammatory Factors We further investigated the role of CXCL12 in the inflammatory response. Compared with the LPS group, the CXCL12 treatment group presented significantly lower p-NF-κB/NF-κB and p-IκBα/IκBα ratios (Figure 5A-C), suggesting that CXCL12 can inhibit the activation of the NF-κB pathway. Concurrently, the ELISA results demonstrated that CXCL12 significantly reduced the secretion of the proinflammatory cytokines TNF-α and IL-1β (Figure 5D-E). Collectively, these results indicate that CXCL12 effectively mitigates the inflammatory response in Schwann cells (SCs) by inhibiting the NF-κB signaling pathway. To verify the critical role of the NF-κB pathway in this process, we intervened by transfecting an NF-κB overexpression plasmid. Compared with the CXCL12 + LPS group, NF-κB overexpression significantly reversed the anti-inflammatory effect of CXCL12, as evidenced by significantly increased ratios of p-NF-κB/NF-κB and elevated secretion levels of TNF-α and IL-1β (Figure 6A-E). These results confirm that the NF-κB pathway is a key mediator of the anti-inflammatory function of CXCL12. 3.2.4 CXCL12 Suppresses NF-κB Signaling Activation and Alleviates LPS-Induced Inflammation via the Ferroptosis Mechanism To clarify the relationship between ferroptosis and CXCL12 in the regulation of inflammation, we used the ferroptosis inducer ferric ammonium citrate (FAC). The CCK-8 results revealed that 10 μM FAC alone did not significantly affect the viability of CXCL12-treated SCs (Figure 7A). However, molecular and functional analyses confirmed that FAC reversed the antiferroptotic effect of CXCL12. Compared with the CXCL12 treatment, FAC treatment significantly upregulated the expression of the proferroptotic protein ACSL4 and downregulated the expression of the antiferroptotic proteins GPX4 and FSP1 (Figure 7B-E). Concurrently, FAC treatment significantly increased the accumulation of Fe 2+ and elevated the levels of ROS and MDA, while the GSH content was markedly reduced (Figure 7F-K). More importantly, these changes in ferroptosis indicators directly aggravated the inflammatory response. Compared with those in the CXCL12 treatment group, the ratios of p-NF-κB/NF-κB and p-IκBα/IκBα in the LPS + CXCL12 + FAC group were significantly greater, and the secretion of IL-1β and TNF-α was also clearly increased (Figure 7L‒P). These results demonstrate that CXCL12 suppresses the NF-κB pathway-mediated inflammatory response by inhibiting SC ferroptosis. 3.3 CXCL12 promotes nerve repair in rats following PNI 3.3.1 CXCL12 Alleviates PNI-Induced Ferroptosis in Nerve Tissue and Improves Mitochondrial Structure and Oxidative Stress Status To evaluate the effect of CXCL12 on ferroptosis in rats after peripheral nerve injury (PNI), we first established a sciatic nerve crush injury model. Both the western blot and immunofluorescence results confirmed that PNI significantly induced ferroptosis in nerve tissue. Compared with that in the sham group, the expression of the proferroptotic protein ACSL4 was significantly upregulated, while the expression of the antiferroptotic proteins GPX4 and FSP1 was inhibited in the sciatic nerves of the PNI group (Figure 8A-H). The results of functional assays corroborated this finding: the contents of Fe2+ and the lipid peroxidation product MDA were significantly elevated, whereas the level of the crucial antioxidant GSH was markedly reduced in the sciatic nerves of PNI group rats (Figure 8I-K). CXCL12 intervention significantly attenuated PNI-induced ferroptosis. Compared with those in the PNI group, the expression of ACSL4 was significantly lower, and the expression of GPX4 and FSP1 was restored in the sciatic nerve after CXCL12 treatment (Figure 8A-H). The functional indicators also displayed a similar trend: CXCL12 intervention effectively reduced the levels of Fe2+ and MDA in nerve tissue and significantly increased the GSH content (Figure 8I-K). Furthermore, transmission electron microscopy (TEM) revealed that the mitochondria within the Schwann cells (SCs) of the PNI group exhibited typical ferroptotic morphological features, characterized by a significant volume reduction, a decreased number of cristae, and a blurred structure. Following CXCL12 intervention, mitochondrial morphology was restored, resulting in an increased number of cristae and an intact structure (Figure 8L). Collectively, these results demonstrate that CXCL12 significantly alleviates PNI-induced ferroptosis by inhibiting lipid peroxidation, enhancing antioxidant capacity, and improving mitochondrial morphology. 3.3.2 CXCL12 Inhibits PNI-Induced Ferroptosis in Nerve Tissue by Activating the ERK/Nrf2 Signaling Pathway To clarify whether CXCL12 regulates the ERK/Nrf2 signaling pathway, we detected the levels of related proteins in the rat sciatic nerve. Compared with those of the control group, both Nrf2 expression and the p-ERK/ERK ratio were lower in the sciatic nerves of the PNI group. In contrast, compared with PNI alone, CXCL12 intervention significantly increased Nrf2 levels and elevated the p-ERK/ERK ratio (Figure 9A-C). These results suggest that CXCL12 can activate the ERK/Nrf2 signaling pathway in vivo. To verify the importance of the ERK/Nrf2 pathway in the in vivo effects of CXCL12, we treated the cells with U0126. Compared with the PNI group, the PNI + U0126 group presented significantly lower Nrf2 protein levels, which was accompanied by the upregulation of ACSL4 and the downregulation of GPX4 and FSP1. Furthermore, compared with the CXCL12 + PNI group, the CXCL12 + PNI + U0126 group displayed the same trend: decreased Nrf2 expression, increased ACSL4 expression, and decreased GPX4 and FSP1 expression (Figure 10A-F). These results indicate that CXCL12 inhibits SC ferroptosis in vivo by activating the ERK/Nrf2 pathway and that blocking this pathway reverses the protective effect of CXCL12. 3.3.3 CXCL12 Suppresses NF-κB Signaling Activation and Alleviates PNI-Induced Inflammation via the Ferroptosis Mechanism To investigate whether CXCL12 regulates the inflammatory response in vivo, we detected the expression of related proteins. Compared with PNI, CXCL12 treatment significantly reduced the p-NF-κB/NF-κB and p-IκBα/IκBα ratios. Concurrently, the ELISA results revealed that the secretion of TNF-α and IL-1β was decreased after CXCL12 treatment compared with that in the PNI group (Figure 11A-E). These results suggest that CXCL12 can attenuate PNI-induced inflammation by inhibiting the activation of the NF-κB pathway. To explore whether CXCL12 regulates inflammation via ferroptosis in vivo, we treated rats with the ferroptosis inducer FAC. Compared with the CXCL12 treatment, FAC treatment significantly upregulated ACSL4, downregulated GPX4 and FSP1, and simultaneously increased Fe 2+ and MDA while decreasing GSH (Figure 12A-G). FAC treatment significantly reversed the antiferroptotic effect of CXCL12. Compared with those in the CXCL12 treatment group, the p-NF-κB/NF-κB and p-IκBα/IκBα ratios were greater, and the secretion of IL-1β and TNF-α was greater in the FAC treatment group (Figure 12H-L). These results indicate that CXCL12 can weaken the inflammatory response in vivo by inhibiting ferroptosis, and in vivo validation confirmed that the NF-κB pathway is a critical pathway by which ferroptosis regulates the inflammatory response. To investigate whether ferroptosis is the core mechanism by which CXCL12 regulates inflammation, we treated rats with the ferroptosis inducer FAC. Western blot and functional assay results revealed that FAC treatment significantly reversed the antiferroptotic effect of CXCL12: the expression of the proferroptotic protein ACSL4 was upregulated, whereas the expression of the antiferroptotic proteins GPX4 and FSP1 was downregulated. Concurrently, the Fe 2+ and MDA levels were significantly elevated, and the GSH content was markedly reduced (Figure 12A-G). More importantly, ferroptosis exacerbation caused by FAC directly led to worsening of the inflammatory response. Compared with those in the CXCL12 treatment group, the p-NF-κB/NF-κB and p-IκBα/IκBα ratios were significantly greater, and the secretion levels of IL-1β and TNF-α were also clearly greater in the FAC treatment group (Figure 12H-L). These in vivo data strongly prove that CXCL12 mitigates PNI-induced inflammation by inhibiting ferroptosis and that the NF-κB pathway is the key downstream pathway by which ferroptosis regulates inflammation. 3.3.4 CXCL12 Improves Nerve Tissue Structure and Promotes Axon and Myelin Regeneration after PNI To further evaluate the role of CXCL12 in the functional and structural recovery of nerves after PNI, we assessed nerve regeneration. H&E staining revealed that, compared with those in the PNI group, the sciatic nerve fibers were more orderly and that the pathological structure was significantly improved after CXCL12 intervention. Immunofluorescence analysis further confirmed these findings, showing that CXCL12 treatment significantly increased the expression of the axonal marker NF-200 and the myelin marker MBP in injured nerves, indicating that it promoted axon regeneration and myelination (Figure 13A-C). Collectively, these results demonstrate that CXCL12 can promote structural and functional recovery after PNI by improving the pathological structure of nerves and restoring the continuity of axons and myelin. 4. Discussion In this study, we systematically investigated the role of CXCL12 in PNI repair by combining clinical sample analysis, an in vitro SC model, and an in vivo rat sciatic nerve injury model. Our main findings are as follows: (1) Our clinical data revealed a significant increase in serum CXCL12 levels in PNI patients, suggesting its potential involvement in the nerve repair process. Building on this observation, we further explored its mechanism of action through in vitro and in vivo experiments. (2) Our in vitro experiments demonstrated that CXCL12 can significantly inhibit LPS-induced ferroptosis in SCs. (3) We found that CXCL12 exerts its antiferroptotic effect by activating the ERK/Nrf2 signaling pathway. (4) Through this ferroptosis-inhibiting mechanism, CXCL12 reduces the production of ROS, which in turn blocks the activation of the NF-κB signaling pathway, ultimately leading to a significant reduction in the ongoing inflammatory response. (5) In our in vivo model, CXCL12 treatment improved mitochondrial morphology, promoted axon regeneration and myelin sheath structure repair, and significantly enhanced the recovery of nerve function. In summary, this study begins with clinical observations and reveals the protective mechanism of CXCL12 in PNI repair through the ERK/Nrf2-ferroptosis-NF-κB axis. These findings provide a new theoretical basis and potential therapeutic target for the clinical treatment of PNI. Functional recovery after PNI is a complex and limited process, with ferroptosis and the inflammatory response triggering key pathological factors that hinder axon regeneration and myelin repair [48-50] . Ferroptosis generates a large amount of reactive oxygen species (ROS) through iron-dependent lipid peroxidation, which not only directly damages SCs but also activates the NF-κB signaling pathway, promoting the release of proinflammatory factors such as IL-1β and TNF-α [51,52] . Our experimental results revealed that PNI leads to SC ferroptosis and excessive release of inflammatory factors, which impedes nerve regeneration. Therefore, finding an effective way to inhibit SC ferroptosis after PNI is a promising strategy for enhancing nerve repair. Studies have shown a close relationship between ferroptosis and the inflammatory response, as they form a vicious cycle after PNI that collectively impairs nerve regeneration [53] . On the one hand, ferroptosis generates many ROS and lipid peroxidation products (such as 4-HNE and MDA). These molecules act as damage-associated molecular patterns (DAMPs) that activate pattern recognition receptors such as TLR4, triggering the phosphorylation and nuclear translocation of the NF-κB signaling pathway. This then promotes the assembly and activation of the NLRP3 inflammasome, ultimately leading to the massive release of proinflammatory factors such as IL-1β and TNF-α [54-57] . On the other hand, the released inflammatory factors amplify damage by regulating ferroptosis-related proteins. TNF-α can significantly upregulate ACSL4 expression, which catalyzes the esterification of polyunsaturated fatty acids into lipid peroxidation substrates, exacerbating membrane lipid oxidative damage [58,59] . Simultaneously, IL-1β can inhibit the Nrf2 antioxidant pathway, leading to the degradation and loss of activity of the GPX4 protein, which weakens the ability of the cell to clear lipid ROS [60-62] . This amplifying “ferroptosis-ROS-inflammation” effect not only induces SC death but also inhibits axon growth and myelin regeneration. Currently, the inhibition of ferroptosis relies primarily on the use of selective inhibitors, which have limitations in research. Our study revealed that PNI induces the abnormal accumulation of intracellular ROS and iron, which enhances the secretion of inflammatory factors such as TNF-α and IL-1β, ultimately leading to cellular dysfunction. CXCL12 is widely considered a key signaling molecule that promotes nerve repair. It exerts its neurotrophic effects by increasing the expression of neurofilament light chain (NF-L), promoting neuronal differentiation, and encouraging axon growth [63,64] . After nerve injury, CXCL12 can act on nerve tissue cells via the ERK1/2 and p38 MAPK pathways to regulate the speed of cell migration and facilitate nerve repair [65,66] . Moreover, CXCL12 can indirectly promote nerve repair by recruiting various cell types that highly express CXCR4. For example, CXCL12 promotes SC migration and autophagy through the PI3K/AKT/mTOR pathway without affecting proliferation or apoptosis [67] . Zhang et al. reported that neururin can increase CXCL12 expression in nerve tissue, which in turn regulates the migration of bone marrow mesenchymal stem cells (MSCs) via the CXCL12/CXCR4-PI3K/Akt signaling pathway, thereby promoting nerve repair [68] . Additionally, studies have shown that CXCL12 can inhibit endothelial cell ferroptosis in age-related macular degeneration (AMD) by promoting the transport and maturation of sterol regulatory element-binding protein 1 (SREBP1) from the endoplasmic reticulum (ER) to the Golgi apparatus [69] . On the basis of these findings, we hypothesized that CXCL12 might inhibit ferroptosis and accelerate nerve repair. In our clinical study, we observed that serum CXCL12 levels were significantly elevated in PNI patients on the third day after surgery. Next, we locally injected CXCL12 into a sciatic nerve injury model in rats and reported a marked improvement in the continuity of axons and the integrity of myelin sheaths. This finding is consistent with previous research indicating that CXCL12 effectively promotes nerve repair. Furthermore, using both in vitro and in vivo PNI models, we observed that CXCL12 effectively reduced PNI-induced SC ferroptosis. This was evidenced by a decrease in intracellular Fe 2+ accumulation, lower levels of ROS and the lipid peroxidation product MDA, and an increase in the antioxidant GSH. The expression of the key ferroptosis execution protein ACSL4 was downregulated, whereas the expression of the antiferroptotic proteins GPX4 and FSP1 was significantly upregulated. Moreover, our study revealed that this protective effect of CXCL12 is dependent on the activation of the ERK/Nrf2 signaling pathway. CXCL12 treatment significantly increased ERK phosphorylation levels and promoted Nrf2 protein expression in SCs. The ERK inhibitor U0126 partially blocked CXCL12-mediated activation of Nrf2 and its subsequent inhibition of ferroptosis, confirming that the ERK/Nrf2 axis is a crucial signaling pathway involved in the antiferroptotic effect of CXCL12 on SCs. This finding is consistent with previous research showing that ERK activation promotes Nrf2 nuclear translocation, which then induces the expression of antioxidant and ferroptosis-inhibiting genes such as GPX4 and FSP1 [70,71] . Moreover, our research revealed that CXCL12 significantly reduces the inflammatory response in SCs by inhibiting ferroptosis and that ferroptosis is a crucial intermediary mechanism for the anti-inflammatory effect of CXCL12. CXCL12 treatment effectively inhibits PNI-induced activation of the NF-κB pathway and reduces the secretion of IL-1β and TNF-α. However, when we used the iron chelator FAC to induce iron overload, the anti-inflammatory effect of CXCL12 was significantly reversed. FAC treatment not only worsened ferroptosis indicators but also reactivated the NF-κB pathway and increased the release of inflammatory factors. This result proves that the anti-inflammatory action of CXCL12 is achieved by inhibiting ferroptosis, which is upstream of the NF-κB signaling pathway. The excessive ROS produced during ferroptosis are key factors in activating NF-κB, and CXCL12 effectively blocks this process by enhancing the cell's antioxidant capacity through the ERK/Nrf2 pathway. In conclusion, our study clarifies the dual protective mechanism of CXCL12 in PNI repair. First, by activating the ERK/Nrf2 signaling pathway, key antiferroptotic proteins, such as GPX4 and FSP1, are upregulated, effectively inhibiting SC ferroptosis. Second, inhibiting ferroptosis reduces ROS production, which in turn blocks the activation of the NF-κB pathway, thereby significantly mitigating the inflammatory response. This study revealed that ferroptosis is a key link in the regulation of SC inflammation by CXCL12, providing a new perspective on the neuroprotective role of CXCL12. This finding not only deepens our understanding of the interaction between ferroptosis and inflammation after PNI but also provides a theoretical basis for the development of new therapeutic strategies for PNI, such as CXCL12 analogs, ERK/Nrf2 activators, or ferroptosis inhibitors. Despite these important advances, our study has several limitations. First, while we confirmed that the ERK/Nrf2 pathway mediates the effect of CXCL12 and observed changes in GPX4 and FSP1 expression, whether Nrf2 directly transcriptionally regulates the expression of GPX4 and FSP1 remains to be determined. We also need to investigate whether other downstream targets are involved, which could be clarified via techniques such as chromatin immunoprecipitation (ChIP), gene knockdown (siRNA/shRNA), or gene editing (CRISPR-Cas9) [72,73] . Second, this study focused primarily on early-stage SC ferroptosis and the inflammatory response. PNI repair is a complex process involving other cells, such as immune cells (e.g., macrophage polarization) [74] , angiogenesis [75] , and axon guidance [76] . Future research should explore whether and how CXCL12 regulates these subsequent events. 5. Conclusion Our study revealed that CXCL12 promotes nerve regeneration by activating the ERK‒Nrf2 pathway to inhibit SC ferroptosis and suppressing the NF-κB signaling pathway to mitigate inflammation. These findings provide a new theoretical basis and potential therapeutic target for the treatment of PNI. Declarations Funding: Directive Project of Hebei Provincial Department of Science and Technology 142777105D (Pei Wang); Hebei Key Laboratory of Nerve Injury and Repair SZX2020020 Conflict of interest : The authors declare no conflicts of interest. Author Contributions: Y.Y. performed the experimental implementation, technical support, data analysis, and figure preparation and drafted the manuscript. P.W. contributed to the study design. Y.J. and S.D. provided technical support and assisted with experimental implementation. G.Y. and Z.Y. provided experimental supervision. Ethics approval and consent to participate: All patients provided informed consent, and the collection and use of human samples were approved by the Ethics Committee of the Affiliated Hospital of Chengde Medical University. The submission and approval of the ethical guidelines comply with the Declaration of Helsinki. The ethical approval number is CYFYLL2024077. The animal use and care plan adheres to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All animal experiments in this study were approved by the Animal Care and Use Committee of the Affiliated Hospital of Chengde Medical University. The ethical approval number is CYFYLL2025006. Clinical trial number: not applicable. Acknowledgments: All the authors read and approved the final manuscript. 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Supplementary Files figure2GPX4ACTIN.tif figure2ACSL4ACTIN.tif figure2FSP1ACTINa.tif figure2ACSL4.tif figure2GPX4.tif figure2FSP1.tif figure3ERK.tif figure3ERKACTIN.tif figure3PERKACTIN.tif figure3PERK.tif figure3NRF2.tif figure3NRF2ACTIN.tif figure4ACSL4ACTIN.tif figure4FSP1ACTIN.tif figure4GPX4ACTIN.tif figure4ACSL4.tif figure4GPX4.tif figure4FSP1.tif figure4NRF2.tif figure4NRF2ACTIN.tif figure5IKBA.tif figure5NFKBACTIN.tif figure5IKBAACTIN.tif figure5PIKBA.tif figure5NFKB.tif figure5PIKBAACTIN.tif figure5PNFKBACTIN.tif figure5PNFKB.tif figure6IKBAACTIN.tif figure6IKBA.tif figure6NFKBACTIN.tif figure6PIKBAACTIN.tif figure6NFKB.tif figure6PIKBA.tif figure6PNFKB.tif figure6PNFKBACTIN.tif figure7ACSL4.tif figure7FSP1ACTIN.tif figure7FSP1.tif figure7GPX4ACTIN.tif figure7ACSL4ACTIN.tif figure7GPX4.tif figure7NFKBACTIN.tif figure7IKBA.tif figure7PIKBA.tif figure7NFKB.tif figure7PNFKBACTIN.tif figure7IKBAACTIN.tif figure7PIKBAACTIN.tif figure7PNFKB.tif figure8ACSL4.tif figure8FSP1.tif figure8ACSL4ACTIN.tif figure8FSP1ACTIN.tif figure8GPX4ACTIN.tif figure8GPX4.tif figure9Nrf2.tif figure9ERKACTIN.tif figure9ERK.tif figure9Nrf2ACTIN.tif figure9pERK.tif figure9pERKACTIN.tif figure10FSP1ACTIN.tif figure10ACSL4.tif figure10ACSL4ACTIN.tif figure10GPX4ACTIN.tif figure10GPX4.tif figure10FSP1.tif figure10NRF2.tif figure10NRF2ACTIN.tif figure11IKBAACTIN.tif figure11NFKB.tif figure11IKBA.tif figure11NFKBACTIN.tif figure11PIKBAACTIN.tif figure11PIKBA.tif figure11PNFKB.tif figure11PNFKBACTIN.tif figure12ACSL4.tif figure12GPX4.tif figure12FSP1ACTIN.tif figure12GPX4ACTIN.tif figure12ACSL4ACTIN.tif figure12FSP1.tif figure12IKBA.tif figure12NFKBACTIN.tif figure12PIKBA.tif figure12PNFKB.tif figure12PNFKBACTIN.tif figure12NFKB.tif figure12PIKBAACTIN.tif figure12IKBAACTIN.tif Cite Share Download PDF Status: Published Journal Publication published 12 Mar, 2026 Read the published version in Inflammation → Version 1 posted Editorial decision: Revision requested 05 Nov, 2025 Reviews received at journal 05 Nov, 2025 Reviews received at journal 16 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Editor assigned by journal 13 Oct, 2025 Submission checks completed at journal 13 Oct, 2025 First submitted to journal 13 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7846278","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":534915653,"identity":"306f67a3-436d-4a49-9566-d67a2b5f6281","order_by":0,"name":"Ye Yuan","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Yuan","suffix":""},{"id":534915654,"identity":"9c1c6563-bbb5-41b7-9b07-98d600d39f4b","order_by":1,"name":"Yu Jiang","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Jiang","suffix":""},{"id":534915655,"identity":"e19e4281-dcfc-47a3-8567-5c04ad447248","order_by":2,"name":"Saisai Du","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Saisai","middleName":"","lastName":"Du","suffix":""},{"id":534915656,"identity":"21e68663-3718-4e99-a4ad-a260a50ac7cd","order_by":3,"name":"Guohong Yuan","email":"","orcid":"","institution":"National Health Commission of the People’s Republic of China","correspondingAuthor":false,"prefix":"","firstName":"Guohong","middleName":"","lastName":"Yuan","suffix":""},{"id":534915657,"identity":"de88ec48-8abe-42ad-babc-35be04aaf538","order_by":4,"name":"Zhenjun Yang","email":"","orcid":"","institution":"Chengde Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenjun","middleName":"","lastName":"Yang","suffix":""},{"id":534915658,"identity":"548143f3-71da-4c5b-8122-60ad3a4403ff","order_by":5,"name":"Pei Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYFCCBGYgYcPARqqWNNK1HCZBA397+mVj3h3n7fmkmx8w/KjYRliLxJk3xcm8Z24ntskcM2DsOXObsBYDiZzkw7lttxPYJBIMmBnbiNdyzp5NIv0DsVrSDyfnth1gbJPIIdIWoF+Yjf+eSU4Eaik4SJRfgCH2WHLmDjt7+RnpGx/8qCBCCwMDjwEDYwOEeYAY9UDA/gCuZRSMglEwCkYBVgAAqzk6SMR5+7AAAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Chengde Medical University","correspondingAuthor":true,"prefix":"","firstName":"Pei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-10-13 08:26:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7846278/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7846278/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10753-026-02453-2","type":"published","date":"2026-03-12T15:58:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94665731,"identity":"184aa46b-ed5c-4bd6-b110-e4a79fc465a0","added_by":"auto","created_at":"2025-10-29 12:29:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":326487,"visible":true,"origin":"","legend":"\u003cp\u003eSerum CXCL12 is significantly elevated in PNI patients. The expression level of CXCL12 in the serum was analyzed via ELISA. The data are presented as the means ± SDs, n=20, ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/9a68b94893304a3fda590c1e.png"},{"id":94665715,"identity":"7ef37bb3-89b3-4d1f-a538-292c7d1bfe2e","added_by":"auto","created_at":"2025-10-29 12:29:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1708506,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 Inhibits LPS-Induced SC Ferroptosis. (A) Effects of different concentrations of LPS on SC viability. (B) Effects of different concentrations of CXCL12 on SC viability following LPS-induced SC injury. (C) Western blot analysis of the expression levels of the ferroptosis-related proteins ACSL4, GPX4, and FSP1 in SCs after CXCL12 treatment. (D-F) Densitometric analysis of ACSL4, GPX4, and FSP1 protein levels. (G, H) Immunofluorescence analysis of the expression levels of the ferroptosis-related proteins ACSL4 and GPX4 in SCs after CXCL12 treatment. Scale bar = 50\u0026nbsp;μm. (I, J) Quantification of the relative fluorescence intensity of ACSL4 and GPX4. (K, L) Fluorescence images of ROS and Fe\u003csup\u003e2+\u003c/sup\u003e in SCs after CXCL12 treatment. Scale bar = 50\u0026nbsp;μm. (M, N) Quantification of the relative fluorescence intensity of ROS and Fe2+. (O, P) Expression levels of MDA and GSH in SCs after CXCL12 treatment. The data are presented as the means±SDs, n=3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/e21bfb6e8405a35c6df3b6c5.png"},{"id":94665714,"identity":"e86de529-0b5e-4c4c-bf57-78f2e94d2c5b","added_by":"auto","created_at":"2025-10-29 12:29:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":815330,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 activates the ERK/Nrf2 signaling pathway in SCs. (A) Western blot analysis of ERK, p-ERK, and Nrf2 protein expression levels in SCs after CXCL12 treatment. (B-C) Quantification of the gray values for the ERK, p-ERK, and Nrf2 proteins. The data are presented as the means ± SDs, n=3, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/60de5456505554431b9afb60.png"},{"id":94665732,"identity":"4445b26b-ff11-47ee-8bc6-3c05184f3470","added_by":"auto","created_at":"2025-10-29 12:29:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":853493,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits SC ferroptosis via the ERK/Nrf2 signaling pathway. (A) Western blot analysis of Nrf2 protein expression levels in SCs after treatment with the ERK inhibitor U0126. (B) Quantification of the gray values for the Nrf2 protein. (C) Western blot analysis of ferroptosis-related proteins (ACSL4, GPX4, FSP1) in SCs after treatment with the ERK inhibitor U0126. (D-F) Quantification of the gray values for the ACSL4, GPX4, and FSP1 proteins. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/3fa13fc0f59d4e1e7b0c088f.png"},{"id":94672845,"identity":"fbb1c03a-dbc4-4ed4-8291-36d8d79aa63d","added_by":"auto","created_at":"2025-10-29 13:41:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":661023,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits NF-κB signaling pathway activation and inflammatory factor secretion in SCs. (A) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in SCs after CXCL12 treatment. (B-C) Quantification of gray values for the NF-κB, p-NF-κB, κBα, and p-IκBα proteins. (D-E) Levels of IL-1β and TNF-α in SCs after CXCL12 treatment. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/355df2a9b4d1909ab7c4b4b4.png"},{"id":94665723,"identity":"0d988f43-87fc-4a0b-9be1-1cf04dd4b555","added_by":"auto","created_at":"2025-10-29 12:29:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1043990,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits the inflammatory response in SCs via the NF-κB signaling pathway. (A) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in SCs after NF-κB overexpression. (B-C) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα, and p-IκBα proteins. (D-E) Levels of IL-1β and TNF-α in SCs after NF-κB overexpression. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ns, not significant.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/3a90c0c7568b6cdd1123da18.png"},{"id":94665718,"identity":"f95dc24c-7b2c-4652-a109-9765a6d7eb0b","added_by":"auto","created_at":"2025-10-29 12:29:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1568573,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits SC ferroptosis, thereby mitigating the inflammatory response. (A) Western blot analysis of ferroptosis-related protein expression levels (ACSL4, GPX4, FSP1) in SCs after FAC treatment. (B-D) Quantification of the gray values for the ACSL4, GPX4, and FSP1 proteins. (E-F) Representative fluorescence images of ROS and Fe\u003csup\u003e2+\u003c/sup\u003e in SCs after FAC treatment. Scale bar = 50 µm. (G, H) Quantification of the relative fluorescence intensities of ROS and Fe\u003csup\u003e2+\u003c/sup\u003e. (I, J) Levels of MDA and GSH in SCs after FAC treatment. (K) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in SCs after FAC treatment. (L, M) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα, and p-IκBα proteins. (N, O) Levels of IL-1β and TNF-α in SCs after FAC treatment. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, ns, not significant.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/a097ce3d155481efaec45429.png"},{"id":94665755,"identity":"847a1f59-5058-4444-b9d4-9deccec77c3f","added_by":"auto","created_at":"2025-10-29 12:29:21","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":851372,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits PNI-induced ferroptosis in SCs. (A) Western blot analysis of ferroptosis-related protein expression levels (ACSL4, GPX4, FSP1) in the sciatic nerve after CXCL12 treatment. (B-D) Quantification of the gray values for the ACSL4, GPX4, and FSP1 proteins. (E-F) Representative immunofluorescence images of the ferroptosis-related proteins ACSL4 and GPX4 in the sciatic nerve after CXCL12 treatment. Scale bar = 50 µm. (G, H) Quantification of the relative fluorescence intensities of ACSL4 and GPX4. (I-K) Levels of Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, and GSH in the sciatic nerve after CXCL12 treatment. (L) Transmission electron microscopy images showing the condition of the mitochondria in the SCs of the sciatic nerve after CXCL12 treatment. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/db6ce742be469afe1cc6417f.png"},{"id":94728382,"identity":"a7b3c364-1a38-41ed-a527-256f50c98079","added_by":"auto","created_at":"2025-10-30 07:03:41","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":839659,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 activates the ERK/Nrf2 signaling pathway in the sciatic nerve. (A) Western blot analysis of ERK, p-ERK, and Nrf2 protein expression levels in the sciatic nerve after CXCL12 treatment. (B-C) Quantification of the gray values for the ERK, p-ERK, and Nrf2 proteins. The data are presented as the means ± SDs, n=3, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/0ffcd51ac46d7bb569890850.png"},{"id":94665735,"identity":"8145237f-652f-4e8a-af2b-e6e267a64ad7","added_by":"auto","created_at":"2025-10-29 12:29:20","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":972765,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits ferroptosis in the sciatic nerve via the ERK/Nrf2 signaling pathway. (A) Western blot analysis of Nrf2 protein expression levels in the sciatic nerve after treatment with the ERK inhibitor U0126. (B) Quantification of the gray values for the Nrf2 protein. (C) Western blot analysis of ferroptosis-related protein expression levels (ACSL4, GPX4, FSP1) in the sciatic nerve after treatment with the ERK inhibitor U0126. (D-F) Quantification of the gray values for the ACSL4, GPX4, and FSP1 proteins. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/6e188f6dccb7a3d80b37c9d4.png"},{"id":94672828,"identity":"e1ae8dc5-8ccd-4ca6-8199-66f575ea527f","added_by":"auto","created_at":"2025-10-29 13:41:01","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":909011,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits NF-κB signaling pathway activation and inflammatory factor secretion in the sciatic nerve. (A) Western blot analysis of NF-κB, p-NF-κB, IκBα, and p-IκBα protein expression levels in the sciatic nerve after CXCL12 treatment. (B-C) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα, and p-IκBαproteins. (D-E) Levels of IL-1β and TNF-α in the sciatic nerve after CXCL12 treatment. The data are presented as the means ± SDs, n=3, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/780c2770c6e42a60d7c1f7c5.png"},{"id":94665739,"identity":"693654de-1cb3-4262-8cfb-f42b6ff408b5","added_by":"auto","created_at":"2025-10-29 12:29:20","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1026944,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 inhibits sciatic nerve ferroptosis, thereby mitigating the inflammatory response. (A) Western blot analysis of ferroptosis-related protein expression levels (ACSL4, GPX4, FSP1) in the sciatic nerve after FAC treatment. (B-D) Quantification of the gray values for the ACSL4, GPX4, and FSP1 proteins. (E‒G) Levels of Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, and GSH in the sciatic nerve after FAC treatment. (H) Western blot analysis of NF-κB, p-NF-κB, IκBα and p-IκBα protein expression levels in the sciatic nerve after FAC treatment. (I, J) Quantification of the gray values for the NF-κB, p-NF-κB, IκBα and p-IκBα proteins. (K, L) Levels of IL-1β and TNF-α in the sciatic nerve after FAC treatment. The data are presented as the means ± SDs, n=3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-7846278/v1/a433aa227492a5fec0878e24.png"},{"id":94665751,"identity":"c38e4b54-82bc-4b68-b220-c32a3169cb96","added_by":"auto","created_at":"2025-10-29 12:29:21","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":3777449,"visible":true,"origin":"","legend":"\u003cp\u003eCXCL12 promotes nerve regeneration after PNI. (A) Changes in axons and myelin sheaths after CXCL12 treatment. (B-C) Quantification of the relative fluorescence intensity of the MBP and NF200 proteins. 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Introduction","content":"\u003cp\u003ePeripheral nerve injury (PNI), often caused by trauma, ischemia, or metabolic disorders, leads to impaired motor and sensory function\u003csup\u003e[1,2]\u003c/sup\u003e. Despite continuous advancements in treatments such as surgical suturing and nerve transplantation\u003csup\u003e[3]\u003c/sup\u003e, the process of nerve regeneration remains limited by challenges, including slow axon regrowth, persistent inflammatory responses, and inadequate reconstruction of myelin sheaths\u003csup\u003e[4,5]\u003c/sup\u003e. Recent studies have shown that ferroptosis, a form of iron-dependent programmed cell death, plays a critical role in various neurological disorders, such as Alzheimer's disease and spinal cord injury. Furthermore, ferroptosis is closely linked to neuroinflammation, suggesting that it may be a significant mechanism hindering nerve regeneration.\u003c/p\u003e\n\u003cp\u003eIn a clinical context, PNI is often accompanied by dysregulated iron metabolism and increased oxidative stress. This leads to the abnormal accumulation of iron ions within Schwann cells (SCs), which then catalyze the production of large amounts of highly reactive hydroxyl radicals (·OH) via the Fenton reaction. This process can induce lipid peroxidation, thereby triggering ferroptosis in SCs\u003csup\u003e[6-8]\u003c/sup\u003e. However, ferroptosis is not just a passive result of ROS; it is also a significant source.\u0026nbsp;Continuous\u0026nbsp;ROS production during ferroptosis can create a positive feedback loop, further aggravating SC damage\u003csup\u003e[9,10]\u003c/sup\u003e. Additionally, ROS can oxidatively modify and enhance the activity of NF-κB subunits, promoting the nuclear translocation of the p65/p50 dimer. This, in turn, upregulates the expression of IL-1β and TNF-α, exacerbating local inflammation and hindering nerve regeneration\u003csup\u003e[11-13]\u003c/sup\u003e. Therefore, the mechanism by which ferroptosis regulates SC inflammation through iron-dependent ROS production requires further investigation.\u003c/p\u003e\n\u003cp\u003eCXCL12 (C-X-C motif chemokine ligand 12), also known as stromal cell-derived factor-1 (SDF-1), belongs to the CXC chemokine family and was first cloned and identified in the 1990s\u003csup\u003e[14,15]\u003c/sup\u003e. By binding to its two main receptors, CXCR4 and CXCR7, CXCL12 participates in a variety of biological processes, including cell migration, survival, proliferation, and tissue homeostasis\u003csup\u003e[16-18]\u003c/sup\u003e. This molecule has several splice variants, with CXCL12α being the most common and widely studied isoform\u003csup\u003e[19,20]\u003c/sup\u003e. The protein structure of CXCL12 includes a typical chemokine core fold (a three-stranded β-sheet and a C-terminal α-helix). Its N-terminal region is responsible for receptor activation (entering the binding pocket of CXCR4 and triggering the signal),\u0026nbsp;whereas\u0026nbsp;the C-terminus binds to glycosaminoglycans/heparan sulfate proteoglycans, which helps with its localization and the formation of concentration gradients within the tissue microenvironment\u003csup\u003e[21-23]\u003c/sup\u003e.\u0026nbsp;Upon binding to CXCL12,\u0026nbsp;CXCR4, a G protein-coupled receptor, can activate multiple downstream signaling pathways, including\u0026nbsp;the\u0026nbsp;PI3K/Akt, ERK/MAPK, and JAK/STAT\u0026nbsp;pathways, which then regulate gene expression and cellular responses\u003csup\u003e[24-26]\u003c/sup\u003e. In contrast, CXCR7 mainly regulates the availability and distribution of CXCL12 through β-arrestin-mediated signal transduction and endocytosis\u003csup\u003e[27-29]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn the central nervous system, the CXCL12/CXCR4 axis is involved in the migration of neuroblasts and the formation of brain regions during embryonic development\u003csup\u003e[30,31]\u003c/sup\u003e. In adulthood, it plays crucial roles in hippocampal neurogenesis, neuronal survival, and maintenance of the blood‒brain barrier\u003csup\u003e[32,33]\u003c/sup\u003e. Studies\u0026nbsp;have shown\u0026nbsp;that CXCL12\u0026nbsp;plays\u0026nbsp;dual\u0026nbsp;roles\u0026nbsp;in diseases such as ischemic stroke, Alzheimer's disease, and multiple sclerosis. For\u0026nbsp;example, in a model of cerebral ischemia, CXCL12 can promote vascular regeneration and the recovery of behavioral function\u003csup\u003e[34]\u003c/sup\u003e. However, during the acute phase of stroke, its signaling activity may also exacerbate the inflammatory response\u003csup\u003e[35]\u003c/sup\u003e, and some reports link its elevated expression to inflammatory cell infiltration and neuronal apoptosis in these diseases\u003csup\u003e[36,37]\u003c/sup\u003e. In recent years, the function of CXCL12 in the process of peripheral nerve injury and repair has garnered increasing attention. Research indicates that the CXCL12/CXCR4 axis can promote both axon growth and angiogenesis and is also involved in the migration and myelin repair of SCs\u003csup\u003e[38,39]\u003c/sup\u003e. Furthermore, stimuli\u0026nbsp;such as\u0026nbsp;oxidative stress and inflammatory factors released after tissue injury can also induce CXCL12 expression\u003csup\u003e[40]\u003c/sup\u003e. However, it remains unclear whether CXCL12 is involved in the regulation of ferroptosis following PNI, particularly its role in the\u0026nbsp;ferroptosis‒inflammation\u0026nbsp;axis.\u003c/p\u003e\n\u003cp\u003eTherefore, our study utilized clinical, in vitro, and in vivo experiments to investigate the mechanism by which CXCL12 promotes nerve regeneration after PNI by regulating SC ferroptosis and mitigating the inflammatory response. This research aims to provide new insights and potential therapeutic strategies for the clinical treatment of PNI.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Ethical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eapproval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.1.1 Clinical Sample Collection and Ethics\u003c/p\u003e\n\u003cp\u003eA total of 20 patients with peripheral nerve injury (PNI) who presented to the Affiliated Hospital of Chengde Medical University between November 2024 and May 2025 were enrolled in this study. Concurrently, 20 healthy volunteers matched for sex and age were recruited. All participants provided informed consent for their participation. The collection and use of human samples were approved by the Ethics Committee of The Affiliated Hospital of Chengde Medical University. The submission and approval of ethical standards adhered to the Declaration of Helsinki. The ethical approval number is CYFYLL2024077.\u003c/p\u003e\n\u003cp\u003e2.1.2 Animal Subjects and Ethics\u003c/p\u003e\n\u003cp\u003eHealthy adult male Sprague–Dawley rats weighing approximately 200 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experimental rats were housed under standard barrier conditions (temperature 23±1°C, 12-h light/dark cycle). All animal procedures were approved by the Animal Care and Use Committee of The Affiliated Hospital of Chengde Medical University. The ethical approval number is CYFYLL2025006.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Clinical Study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.2.1 Inclusion and exclusion criteria for peripheral nerve injury patients\u003c/p\u003e\n\u003cp\u003eTo observe the changes in the expression of CXCL12 in the serum of peripheral nerve injury (PNI) patients, serum samples were collected on day 3 postsurgery. The inclusion criterion was age between 18 and 60 years. Patients with pure peripheral nerve injury, primarily median or ulnar nerve transection due to forearm trauma. The exclusion criteria were a history of central nervous system disorders, diabetes mellitus, autoimmune diseases, malignant tumors, or other underlying metabolic and immune-related diseases. Patients with severe infection or hepatic/renal insufficiency. Recent use of immunosuppressive agents or drugs that affect nerve regeneration. Pregnant or lactating females. Patients unable to cooperate with the study protocol. Twenty healthy volunteers, matched for sex and age, were recruited concurrently as the control group.\u003c/p\u003e\n\u003cp\u003e2.2.2 Serum CXCL12 Level Measurement in Clinical Patients\u003c/p\u003e\n\u003cp\u003eSerum samples were obtained via venipuncture of the cubital vein and isolated after centrifugation at 3000×g for 15 minutes. The samples were then used for the quantitative detection of CXCL12 via an ELISA kit (Elabscience, China). The experiment was conducted according to the manufacturer’s instructions, with three technical replicates set for each sample. The absorbance was measured at a wavelength of 450 nm via a microplate reader (Bio-Rad Laboratories, USA). The results were calculated in pg/mL and subjected to statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Cell\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexperiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.3.1 Cell culture and injury model\u003c/p\u003e\n\u003cp\u003eThe rat Schwann cell line (Procell, Wuhan, China) was routinely cultured in DMEM supplemented with 10% fetal bovine serum (FBS) in a constant-temperature incubator at 37°C with 5%\u0026nbsp;CO2. To establish the cellular injury model, the cells were treated with 10\u0026nbsp;μM LPS for 6 hours, followed by replacement with fresh culture medium for further incubation.\u003c/p\u003e\n\u003cp\u003e2.3.2 Cell Grouping and Treatment\u003c/p\u003e\n\u003cp\u003eThe cell experiments were divided into four parts. First, the effects of CXCL12 on SC ferroptosis and inflammation were explored, and SCs were divided into 3 groups: the control group, in which SCs were routinely cultured for 6 h; the LPS group, in which SCs were treated with LPS for 6 h; and the LPS + CXCL12 group, in which SCs were treated with 100\u0026nbsp;ng/mL CXCL12 for 2 h before the addition of LPS. The second part investigated whether CXCL12 regulates SC ferroptosis via the ERK/Nrf2 pathway, and SCs were divided into 4 groups: the LPS group; the LPS + CXCL12 group; the LPS + CXCL12 + U0126 group, which was pretreated with 100\u0026nbsp;ng/mL CXCL12 and U0126 (20\u0026nbsp;μM)\u003csup\u003e[41]\u003c/sup\u003e for 2 hours before the addition of LPS;\u0026nbsp;and the\u0026nbsp;LPS + U0126\u0026nbsp;group, which was pretreated\u0026nbsp;with U0126 (20 μM) for 2 hours before the addition of LPS. The third part explored whether CXCL12 regulates SC inflammation via the NF-κB pathway, and SCs were divided into 4 groups:\u0026nbsp;the\u0026nbsp;LPS\u0026nbsp;group;\u0026nbsp;the\u0026nbsp;LPS + CXCL12\u0026nbsp;group;\u0026nbsp;the\u0026nbsp;LPS + CXCL12 + NF-κB-OE\u0026nbsp;group, which was\u0026nbsp;transfected with an NF-κB overexpression plasmid and then treated with CXCL12 for 2 hours before the addition of LPS;\u0026nbsp;and the\u0026nbsp;LPS + CXCL12 + NF-κB-EV\u0026nbsp;group, which was\u0026nbsp;transfected with an empty vector plasmid and then treated with CXCL12 for 2 hours before the addition of LPS.\u0026nbsp;Fourth, we\u0026nbsp;explored whether CXCL12 reduces inflammation by inhibiting SC ferroptosis.\u0026nbsp;SCs were divided into 3 groups:\u0026nbsp;the\u0026nbsp;LPS\u0026nbsp;group, the\u0026nbsp;LPS + CXCL12\u0026nbsp;group, and the\u0026nbsp;LPS + CXCL12 + FAC\u0026nbsp;group, which were\u0026nbsp;treated with FAC for 6 hours\u003csup\u003e[42]\u003c/sup\u003e before CXCL12 treatment.\u003c/p\u003e\n\u003cp\u003e2.3.3 Cell viability assay\u003c/p\u003e\n\u003cp\u003eSCs were seeded in 96-well plates at a density of 5000 cells/well and precultured for 24 hours. Following the corresponding drug treatments according to the experimental groups, 10\u0026nbsp;μL of CCK-8 reagent (Dojindo, Japan) was added to each well and incubated at 37°C in the dark for 1 hour. The absorbance value was measured at a wavelength of 450 nm via a microplate reader. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.4 Western blot analysis\u003c/p\u003e\n\u003cp\u003eThe treated cells were harvested and lysed via RIPA lysis buffer (Solarbio, China) containing the protease inhibitor PMSF (Solarbio, China) and phosphatase inhibitors (Solarbio, China) to extract total protein. The protein concentration was determined via the BCA method (Beyotime, China). Protein samples (30\u0026nbsp;μg/lane) were separated via SDS‒PAGE and subsequently transferred to PVDF membranes via the wet transfer method. The membranes were blocked for 1 h at room temperature with TBST buffer containing 5% nonfat milk. The membranes were then incubated overnight at 4°C with the following primary antibodies: ACSL4 (1:1000, Proteintech, Cat#22401-1-AP, RRID: AB_2783458); GPX4 (1:2000, Proteintech, Cat#67763-1-Ig, RRID: AB_2799687); FSP1 (1:1000, Proteintech, Cat#18412-1-AP, RRID: AB_2810984); ERK (1:2000, Proteintech, Cat#16443-1-AP, RRID: AB_10694175); P-ERK (1:1000, Proteintech, Cat#16443-1-AP, RRID: AB_2799689); NRF2 (1:1000, Proteintech, Cat#16396-1-AP, RRID: AB_11006916); NF-κB (1:1000, Proteintech, Cat#10745-1-AP, RRID: AB_2276536); P-NF-κB (1:500, Proteintech, Cat#30363-1-AP, RRID: AB_2799690); and IκBα (1:1000, Proteintech, Cat#10268-2-AP, RRID: AB_2276481)；P-IKBA (1:1000, Proteintech, Cat#28591-1-AP, RRID: AB_2799691)；β-actin (1:5000, Proteintech, Cat#60008-1-Ig, RRID: AB_2106289). After the membranes were washed three times with TBST, they were incubated with the HRP-labeled secondary antibody\u0026nbsp;goat anti-rabbit\u0026nbsp;IgG (H+L) (1:5000, Proteintech,\u0026nbsp;Cat# SA00001-2, RRID: AB2722555) for 1 hour at room temperature.\u0026nbsp;The bands\u0026nbsp;were visualized\u0026nbsp;via enhanced\u0026nbsp;chemiluminescence\u0026nbsp;(ECL)\u0026nbsp;reagent, and the gray values were analyzed\u0026nbsp;via ImageJ\u0026nbsp;software. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.4 Immunofluorescence\u003c/p\u003e\n\u003cp\u003eThe cell samples were fixed with paraformaldehyde and then permeabilized with 0.1% Triton X-100 for 20 min. After blocking with an immunofluorescence quick blocking solution for 30 min, the membranes were incubated overnight at 4°C with the following primary antibodies: mouse anti-MBP (1:100, Proteintech, Cat#10458-1-AP, RRID: AB_2336123) and rabbit anti-NF200 (1:100, Proteintech, Cat#18934-1-AP, RRID: AB_10640801). After being washed with PBST, the membranes were incubated for 1 h at room temperature with the following secondary antibodies: goat anti-mouse Alexa Fluor 488 targeting MBP (1:200, Proteintech, Cat# SA00013-1, RRID: AB_2810983) and goat anti-rabbit Alexa Fluor 594 targeting NF-200 (1:200, Proteintech, Cat# SA00013-4, RRID: AB_2810984). After mounting with a DAPI-containing mounting medium, images were captured under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed via ImageJ software. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.5 ROS measurement\u003c/p\u003e\n\u003cp\u003eAfter the completion of treatment, the culture medium was removed, and a DCFH-DA probe (10\u0026nbsp;μmol/L) diluted in serum-free medium was added. The cells were incubated at 37°C in a 5%\u0026nbsp;CO2 incubator in the dark for 20 minutes. The cells were subsequently washed three times with serum-free medium, and fluorescence images were captured under a fluorescence microscope. The fluorescence intensity was analyzed via ImageJ software. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.6 Ferrous Ion Detection\u003c/p\u003e\n\u003cp\u003eAfter the cells were treated, the original culture medium was discarded, and serum-free medium containing a 5\u0026nbsp;μM fluorescent probe (Solarbio, China) was added. The cells were incubated in the dark for 30 minutes and then rinsed five times with PBS. Images were acquired under a fluorescence microscope, and the fluorescence intensity was quantitatively analyzed via ImageJ software. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.7 MDA measurement\u003c/p\u003e\n\u003cp\u003eThe content of malondialdehyde (MDA) in the cells was determined via the thiobarbituric acid (TBA) colorimetric method. The cell samples were homogenized in ice-cold physiological saline, and the supernatant was collected after centrifugation. The procedure was performed according to the manufacturer's instructions for the MDA assay kit (Solarbio, China). The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.8 GSH measurement\u003c/p\u003e\n\u003cp\u003eThe intracellular GSH content was measured via a GSH detection kit (Solarbio, China). A standard curve was generated on the basis of the absorbance values of different concentrations of the standard product. The total GSH content was calculated by comparing the sample values against the standard curve. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.3.9 ELISA detection\u003c/p\u003e\n\u003cp\u003eAn enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of the inflammatory factors IL-1β and TNF-α in the cell culture supernatant (centrifuged at 3000×g for 15 min). The procedure was performed according to the manufacturer's instructions for the ELISA kit (Elabscience, China). After a standard curve with standard products was established, the absorbance was read at a wavelength of 450 nm via a microplate reader (Bio-Rad Laboratories, USA). The results were subjected to statistical analysis in units of pg/mL. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4 Animal experimentation\u003c/p\u003e\n\u003cp\u003e2.4.1 Sciatic nerve crush model establishment\u003c/p\u003e\n\u003cp\u003eThe sciatic nerve crush injury model was established according to the method reported by Wandling et al\u003csup\u003e[43]\u003c/sup\u003e. After anesthesia via intraperitoneal injection of sodium pentobarbital (30\u0026nbsp;mg/kg), the middle segment of the sciatic nerve was exposed through blunt dissection along the gap of the biceps femoris muscle of the right hind limb. The nerve was then crushed continuously three times via a vascular clamp (10 s each time, with a 10 s interval between crushes). Postsurgery, the rats were housed until day 5 for sacrifice and collection of the sciatic nerve for Western blot and transmission electron microscopy (TEM) experiments; the rats were housed until day 14 and underwent footprint gait analysis before sacrifice and collection of the sciatic nerve for HE staining and immunofluorescence experiments.\u003c/p\u003e\n\u003cp\u003e2.4.2 Animal Grouping and Treatment\u003c/p\u003e\n\u003cp\u003eThe experiment was divided into three parts. In the first part, 45 SD rats were randomly divided into three groups (n=15 per group): the sham group (only the sciatic nerve was exposed without injury); peripheral nerve injury group (PNI) (nerve injury model was established, followed by daily intramuscular injection of 0.2\u0026nbsp;mL of saline); and the CXCL12 treatment group (PNI + CXCL12) (nerve injury model was established, followed by daily intramuscular injection of 4\u0026nbsp;μg of CXCL12\u003csup\u003e[44]\u003c/sup\u003e). The second part included 20 SD rats randomly divided into four groups (n=5 per group) to investigate the role of the ERK signaling pathway. The groups were\u0026nbsp;the\u0026nbsp;PNI\u0026nbsp;group, PNI + CXCL12\u0026nbsp;group, PNI + CXCL12 + U0126\u0026nbsp;group, and PNI + U0126\u0026nbsp;group. U0126, an ERK inhibitor, was administered via daily intraperitoneal injection at a dose of 25 mg/kg\u0026nbsp;postsurgery\u003csup\u003e[45]\u003c/sup\u003e. The third part included 15 SD rats randomly divided into three groups (n=5 per group) to assess the role of ferroptosis in the inflammatory response. The groups were\u0026nbsp;the\u0026nbsp;PNI\u0026nbsp;group, PNI + CXCL12\u0026nbsp;group, and PNI + CXCL12 + FAC\u0026nbsp;group. FAC was administered via daily local intramuscular injection at a dose of 2\u0026nbsp;μg/rat postsurgery\u003csup\u003e[46]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e2.4.3 Western blot analysis\u003c/p\u003e\n\u003cp\u003eTissue from the central area of the injured sciatic nerve (approximately 2\u0026nbsp;cm in length) was collected and quickly ground into powder in liquid nitrogen. RIPA lysis buffer was added for lysis, and the supernatant was collected after centrifugation. The subsequent procedures were the same as those for the Western blot method described for the cell experiments. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.4 Immunofluorescence\u003c/p\u003e\n\u003cp\u003eNerve tissue sections were baked and dewaxed. The subsequent procedures were the same as those for the immunofluorescence method described in the cell experiments. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.5 Ferrous Ion Detection\u003c/p\u003e\n\u003cp\u003eFresh nerve tissue from the central area of injury was collected, and lysis buffer from the Ferrous Ion Assay Kit (Pulilai, China) was added. The supernatant was obtained after grinding and centrifugation, and subsequent procedures were performed according to the manufacturer’s instructions. Finally, the absorbance was read at a wavelength of 593 nm via a microplate reader, and the ferrous ion concentration in the samples was calculated on the basis of the standard curve. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.6 MDA measurement\u003c/p\u003e\n\u003cp\u003eFresh nerve tissue from the central area of injury was collected, and lysis buffer from the MDA assay kit (Solarbio, China) was added. The supernatant was obtained after grinding and centrifugation. Subsequent procedures strictly followed the instructions. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.7 GSH measurement\u003c/p\u003e\n\u003cp\u003eTissue from the central area of nerve injury was collected, and a mixed solution from the GSH assay kit (Solarbio, China) was added. The supernatant was obtained after grinding and centrifugation. Subsequent procedures strictly followed the instructions. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.8 ELISA detection\u003c/p\u003e\n\u003cp\u003eAn enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of IL-1β and TNF-α in rat serum (centrifuged at 3000×g for 15 min). The procedure was performed according to the manufacturer’s instructions for the ELISA kit (Elabscience, China). The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.9 H\u0026amp;E Staining\u003c/p\u003e\n\u003cp\u003eThe tissue sections were baked and dewaxed, stained with hematoxylin for 5 minutes, differentiated, rinsed with water, and then stained with eosin for 2 minutes. The tissue was dehydrated through a graded series of ethanol, cleared with xylene, and mounted with neutral balsam. Images were collected under an optical microscope and analyzed via ImageJ software. The experiment was repeated three times for each group.\u003c/p\u003e\n\u003cp\u003e2.4.10 Electron microscopy sample preparation and mitochondrial observation\u003c/p\u003e\n\u003cp\u003eFreshly collected sciatic nerve tissue (1\u0026nbsp;mm×1\u0026nbsp;mm×1\u0026nbsp;mm) was immediately immersed in electron microscopy fixative and fixed at 4°C for 4 hours. The tissue was then rinsed three times with PBS and postfixed with 1% osmium tetroxide solution for 2 hours at room temperature, followed by three more rinses. The tissue was subsequently dehydrated sequentially through a graded series of ethanol, infiltrated with epoxy resin, and embedded. Ultrathin sections were prepared via an ultramicrotome and counterstained with uranyl acetate and lead citrate. Finally, the sections were dried overnight at room temperature, and morphological changes in the mitochondria were observed via transmission electron microscopy (TEM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Data\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental data were statistically analyzed via GraphPad Prism 9 software and are presented as the means ± standard deviations (means ± SDss). After checking for a normal distribution and homogeneity of variance, comparisons among multiple groups were performed via one-way ANOVA combined with Tukey's HSD post hoc test. Comparisons between two groups were performed via Student's t test. p\u0026lt;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 CXCL12 is significantly elevated in the serum of PNI patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum samples were collected from patients with PNI and healthy controls to quantitatively analyze the CXCL12 concentration via ELISA. The results revealed that the serum CXCL12 levels in PNI patients were significantly greater than those in healthy individuals (Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 CXCL12 Inhibits SC Ferroptosis and Inflammation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.2.1 CXCL12 Significantly Ameliorates LPS-Induced SC Injury by Regulating Key Ferroptosis Proteins and Oxidative Stress\u003c/p\u003e\n\u003cp\u003eTo investigate the protective effect of CXCL12 against LPS-induced SC injury, an LPS injury model was first established, and the optimal dose was determined. Treatment of SCs with 10\u0026nbsp;μM LPS led to a stable decrease in cell viability to approximately 50% (Figure 2A) while also inducing typical characteristics of ferroptosis (Figure 2C‒P). The results of the subsequent dose‒response screening revealed that CXCL12 could enhance SC viability in a dose-dependent manner. When the concentration reached 200\u0026nbsp;ng/mL, the cell survival rate significantly recovered to 91% (P\u0026lt;0.0001) (Figure 2B). This concentration was selected as the optimal intervention dose for subsequent mechanistic studies.\u003c/p\u003e\n\u003cp\u003eWe then further explored the protective mechanism of CXCL12 and found that its effect was due to the significant inhibition of ferroptosis. Compared with LPS treatment, CXCL12 treatment significantly reversed the LPS-induced increase in the expression of ferroptosis-related proteins, specifically the downregulation of the proferroptotic protein ACSL4 and the simultaneous upregulation of the antiferroptotic proteins GPX4 and FSP1 (Figure 2C-J). The results of the functional assays were highly consistent: CXCL12 intervention significantly reduced the levels of ROS, Fe2+, and MDA within the SCs while simultaneously restoring the content of the crucial antioxidant GSH (Figure 2K-P). In summary, CXCL12 effectively antagonized LPS-induced SC injury by regulating the expression of ferroptosis-related proteins and alleviating oxidative stress.\u003c/p\u003e\n\u003cp\u003e3.2.2 CXCL12 Alleviates LPS-Induced SC Ferroptosis by Activating the ERK/Nrf2 Pathway and Regulating Key Ferroptosis Proteins\u003c/p\u003e\n\u003cp\u003eNrf2 is a crucial regulator of cellular antioxidant responses and plays an important role in regulating cellular ferroptosis\u003csup\u003e[47]\u003c/sup\u003e. To investigate the potential mechanism by which CXCL12 inhibits SC ferroptosis, we first detected the expression of proteins related to the ERK/Nrf2 signaling pathway. Compared with LPS treatment, CXCL12 treatment significantly increased the p-ERK/ERK ratio and increased the protein level of Nrf2 (Figure 3A-C), suggesting that CXCL12 activates the ERK/Nrf2 signaling pathway in SCs.\u003c/p\u003e\n\u003cp\u003eTo verify the necessity of this pathway, we used the ERK inhibitor U0126. The CCK-8 assay confirmed that 20\u0026nbsp;μM U0126 alone did not significantly affect the viability of CXCL12-treated SCs (Figure 4A). However, the Western blot results indicated that U0126 significantly blocked the antiferroptotic effect of CXCL12. Compared with the LPS + CXCL12 group, the LPS + CXCL12 + U0126 group presented increased expression of the proferroptotic protein ACSL4, while the expression of the antiferroptotic proteins GPX4 and FSP1 was significantly reduced. Furthermore, U0126 alone aggravated LPS-induced ferroptotic injury, as evidenced by decreased Nrf2 expression, increased ACSL4 expression, and decreased GPX4 and FSP1 expression (Figure 4B-G). Collectively, these results demonstrate that CXCL12 inhibits SC ferroptosis by activating the ERK/Nrf2 signaling pathway.\u003c/p\u003e\n\u003cp\u003e3.2.3 CXCL12 Alleviates LPS-Induced SC Inflammation by Inhibiting the NF-κB Pathway and the Secretion of Inflammatory Factors\u003c/p\u003e\n\u003cp\u003eWe further investigated the role of CXCL12 in the inflammatory response. Compared with the LPS group, the CXCL12 treatment group presented significantly lower p-NF-κB/NF-κB and p-IκBα/IκBα ratios (Figure 5A-C), suggesting that CXCL12 can inhibit the activation of the NF-κB pathway. Concurrently, the ELISA results demonstrated that CXCL12 significantly reduced the secretion of the proinflammatory cytokines TNF-α and IL-1β (Figure 5D-E). Collectively, these results indicate that CXCL12 effectively mitigates the inflammatory response in Schwann cells (SCs) by inhibiting the NF-κB signaling pathway.\u003c/p\u003e\n\u003cp\u003eTo verify the critical role of the NF-κB pathway in this process, we intervened by transfecting an NF-κB overexpression plasmid. Compared with the CXCL12 + LPS group, NF-κB overexpression significantly reversed the anti-inflammatory effect of CXCL12, as evidenced by significantly increased ratios of p-NF-κB/NF-κB and elevated secretion levels of TNF-α and IL-1β (Figure 6A-E). These results confirm that the NF-κB pathway is a key mediator of the anti-inflammatory function of CXCL12.\u003c/p\u003e\n\u003cp\u003e3.2.4 CXCL12 Suppresses NF-κB Signaling Activation and Alleviates LPS-Induced Inflammation via the Ferroptosis Mechanism\u003c/p\u003e\n\u003cp\u003eTo clarify the relationship between ferroptosis and CXCL12 in the regulation of inflammation, we used the ferroptosis inducer ferric ammonium citrate (FAC). The CCK-8 results revealed that 10\u0026nbsp;μM FAC alone did not significantly affect the viability of CXCL12-treated SCs (Figure 7A). However, molecular and functional analyses confirmed that FAC reversed the antiferroptotic effect of CXCL12. Compared with the CXCL12 treatment, FAC treatment significantly upregulated the expression of the proferroptotic protein ACSL4 and downregulated the expression of the antiferroptotic proteins GPX4 and FSP1 (Figure 7B-E). Concurrently, FAC treatment significantly increased the accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e and elevated the levels of ROS and MDA, while the GSH content was markedly reduced (Figure 7F-K).\u003c/p\u003e\n\u003cp\u003eMore importantly, these changes in ferroptosis indicators directly aggravated the inflammatory response. Compared with those in the CXCL12 treatment group, the ratios of p-NF-κB/NF-κB and p-IκBα/IκBα in the LPS + CXCL12 + FAC group were significantly greater, and the secretion of IL-1β and TNF-α was also clearly\u0026nbsp;increased\u0026nbsp;(Figure\u0026nbsp;7L‒P). These results demonstrate that CXCL12 suppresses the NF-κB pathway-mediated inflammatory response by inhibiting SC ferroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 CXCL12\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epromotes nerve repair in rats following\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;PNI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.3.1 CXCL12 Alleviates PNI-Induced Ferroptosis in Nerve Tissue and Improves Mitochondrial Structure and Oxidative Stress Status\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of CXCL12 on ferroptosis in rats after peripheral nerve injury (PNI), we first established a sciatic nerve crush injury model. Both the western blot and immunofluorescence results confirmed that PNI significantly induced ferroptosis in nerve tissue. Compared with that in the sham group, the expression of the proferroptotic protein ACSL4 was significantly upregulated, while the expression of the antiferroptotic proteins GPX4 and FSP1 was inhibited in the sciatic nerves of the PNI group (Figure 8A-H). The results of functional assays corroborated this finding: the contents of Fe2+ and the lipid peroxidation product MDA were significantly elevated, whereas the level of the crucial antioxidant GSH was markedly reduced in the sciatic nerves of PNI group rats (Figure 8I-K). CXCL12 intervention significantly attenuated PNI-induced ferroptosis. Compared with those in the PNI group, the expression of ACSL4 was significantly lower, and the expression of GPX4 and FSP1 was restored in the sciatic nerve after CXCL12 treatment (Figure 8A-H). The functional indicators also displayed a similar trend: CXCL12 intervention effectively reduced the levels of Fe2+ and MDA in nerve tissue and significantly increased the GSH content (Figure 8I-K). Furthermore, transmission electron microscopy (TEM) revealed that the mitochondria within the Schwann cells (SCs) of the PNI group exhibited typical ferroptotic morphological features, characterized by a significant volume reduction, a decreased number of cristae, and a blurred structure. Following CXCL12 intervention, mitochondrial morphology was restored, resulting in an increased number of cristae and an intact structure (Figure 8L). Collectively, these results demonstrate that CXCL12 significantly alleviates PNI-induced ferroptosis by inhibiting lipid peroxidation, enhancing antioxidant capacity, and improving mitochondrial morphology.\u003c/p\u003e\n\u003cp\u003e3.3.2 CXCL12 Inhibits PNI-Induced Ferroptosis in Nerve Tissue by Activating the ERK/Nrf2 Signaling Pathway\u003c/p\u003e\n\u003cp\u003eTo clarify whether CXCL12 regulates the ERK/Nrf2 signaling pathway, we detected the levels of related proteins in the rat sciatic nerve. Compared with those of the control group, both Nrf2 expression and the p-ERK/ERK ratio were lower in the sciatic nerves of the PNI group. In contrast, compared with PNI alone, CXCL12 intervention significantly increased Nrf2 levels and elevated the p-ERK/ERK ratio (Figure 9A-C). These results suggest that CXCL12 can activate the ERK/Nrf2 signaling pathway in vivo.\u003c/p\u003e\n\u003cp\u003eTo verify the importance of the ERK/Nrf2 pathway in the in vivo effects of CXCL12, we treated the cells with U0126. Compared with the PNI group, the PNI + U0126 group presented significantly lower Nrf2 protein levels, which was accompanied by the upregulation of ACSL4 and the downregulation of GPX4 and FSP1. Furthermore, compared with the CXCL12 + PNI group, the CXCL12 + PNI + U0126 group displayed the same trend: decreased Nrf2 expression, increased ACSL4 expression, and decreased GPX4 and FSP1 expression (Figure 10A-F). These results indicate that CXCL12 inhibits SC ferroptosis in vivo by activating the ERK/Nrf2 pathway and that blocking this pathway reverses the protective effect of CXCL12.\u003c/p\u003e\n\u003cp\u003e3.3.3 CXCL12 Suppresses NF-κB Signaling Activation and Alleviates PNI-Induced Inflammation via the Ferroptosis Mechanism\u003c/p\u003e\n\u003cp\u003eTo investigate whether CXCL12 regulates the inflammatory response in vivo, we detected the expression of related proteins. Compared with PNI, CXCL12 treatment significantly reduced the p-NF-κB/NF-κB and p-IκBα/IκBα ratios. Concurrently, the ELISA results revealed that the secretion of TNF-α and IL-1β was decreased after CXCL12 treatment compared with that in the PNI group (Figure 11A-E). These results suggest that CXCL12 can attenuate PNI-induced inflammation by inhibiting the activation of the NF-κB pathway.\u003c/p\u003e\n\u003cp\u003eTo explore whether CXCL12 regulates inflammation via ferroptosis in vivo, we treated rats with the ferroptosis inducer FAC. Compared with the CXCL12 treatment, FAC treatment significantly upregulated ACSL4, downregulated GPX4 and FSP1, and simultaneously increased Fe\u003csup\u003e2+\u003c/sup\u003e and MDA while decreasing GSH (Figure 12A-G). FAC treatment significantly reversed the antiferroptotic effect of CXCL12. Compared with those in the CXCL12 treatment group, the p-NF-κB/NF-κB and p-IκBα/IκBα ratios were greater, and the secretion of IL-1β and TNF-α was greater in the FAC treatment group (Figure 12H-L). These results indicate that CXCL12 can weaken the inflammatory response in vivo by inhibiting ferroptosis, and in vivo validation confirmed that the NF-κB pathway is a critical pathway by which ferroptosis regulates the inflammatory response.\u003c/p\u003e\n\u003cp\u003eTo investigate whether ferroptosis is the core mechanism by which CXCL12 regulates inflammation, we treated rats with the ferroptosis inducer FAC. Western blot and functional assay results revealed that FAC treatment significantly reversed the antiferroptotic effect of CXCL12: the expression of the proferroptotic protein ACSL4 was upregulated, whereas the expression of the antiferroptotic proteins GPX4 and FSP1 was downregulated. Concurrently, the Fe\u003csup\u003e2+\u003c/sup\u003e and MDA levels were significantly elevated, and the GSH content was markedly reduced (Figure 12A-G). More importantly, ferroptosis exacerbation caused by FAC directly led to worsening of the inflammatory response. Compared with those in the CXCL12 treatment group, the p-NF-κB/NF-κB and p-IκBα/IκBα ratios were significantly greater, and the secretion levels of IL-1β and TNF-α were also clearly greater in the FAC treatment group (Figure 12H-L). These in vivo data strongly prove that CXCL12 mitigates PNI-induced inflammation by inhibiting ferroptosis and that the NF-κB pathway is the key downstream pathway by which ferroptosis regulates inflammation.\u003c/p\u003e\n\u003cp\u003e3.3.4 CXCL12 Improves Nerve Tissue Structure and Promotes Axon and Myelin Regeneration after PNI\u003c/p\u003e\n\u003cp\u003eTo further evaluate the role of CXCL12 in the functional and structural recovery of nerves after PNI, we assessed nerve regeneration. H\u0026amp;E staining revealed that, compared with those in the PNI group, the sciatic nerve fibers were more orderly and that the pathological structure was significantly improved after CXCL12 intervention. Immunofluorescence analysis further confirmed these findings, showing that CXCL12 treatment significantly increased the expression of the axonal marker NF-200 and the myelin marker MBP in injured nerves, indicating that it promoted axon regeneration and myelination (Figure 13A-C). Collectively, these results demonstrate that CXCL12 can promote structural and functional recovery after PNI by improving the pathological structure of nerves and restoring the continuity of axons and myelin.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we systematically investigated the role of CXCL12 in PNI repair by combining clinical sample analysis, an in vitro SC model, and an in vivo rat sciatic nerve injury model. Our main findings are as follows: (1) Our clinical data revealed a significant increase in serum CXCL12 levels in PNI patients, suggesting its potential involvement in the nerve repair process. Building on this observation, we further explored its mechanism of action through in vitro and in vivo experiments. (2) Our in vitro experiments demonstrated that CXCL12 can significantly inhibit LPS-induced ferroptosis in SCs. (3) We found that CXCL12 exerts its antiferroptotic effect by activating the ERK/Nrf2 signaling pathway. (4) Through this ferroptosis-inhibiting mechanism, CXCL12 reduces the production of ROS, which in turn blocks the activation of the NF-κB signaling pathway, ultimately leading to a significant reduction in the ongoing inflammatory response. (5) In our in vivo model, CXCL12 treatment improved mitochondrial morphology, promoted axon regeneration and myelin sheath structure repair, and significantly enhanced the recovery of nerve function. In summary, this study begins with clinical observations and reveals the protective mechanism of CXCL12 in PNI repair through the ERK/Nrf2-ferroptosis-NF-κB axis. These findings provide a new theoretical basis and potential therapeutic target for the clinical treatment of PNI.\u003c/p\u003e\n\u003cp\u003eFunctional recovery after PNI is a complex and limited process, with ferroptosis and the inflammatory response triggering key pathological factors that hinder axon regeneration and myelin repair\u003csup\u003e[48-50]\u003c/sup\u003e. Ferroptosis generates a large amount of reactive oxygen species (ROS) through iron-dependent lipid peroxidation, which not only directly damages SCs but also activates the NF-κB signaling pathway, promoting the release of\u0026nbsp;proinflammatory\u0026nbsp;factors such as IL-1β and TNF-α\u003csup\u003e[51,52]\u003c/sup\u003e. Our experimental results\u0026nbsp;revealed\u0026nbsp;that PNI leads to SC ferroptosis and excessive release of inflammatory factors, which impedes nerve regeneration. Therefore, finding an effective way to inhibit SC ferroptosis after PNI is a promising strategy for enhancing nerve repair.\u003c/p\u003e\n\u003cp\u003eStudies have shown a close relationship between ferroptosis and the inflammatory response, as they form a vicious cycle after PNI that collectively impairs nerve regeneration\u003csup\u003e[53]\u003c/sup\u003e. On\u0026nbsp;the\u0026nbsp;one hand, ferroptosis generates\u0026nbsp;many\u0026nbsp;ROS and lipid peroxidation products (such as 4-HNE and MDA). These molecules act as damage-associated molecular patterns (DAMPs) that activate pattern recognition receptors\u0026nbsp;such as\u0026nbsp;TLR4, triggering the phosphorylation and nuclear translocation of the NF-κB signaling pathway. This then promotes the assembly and activation of the NLRP3 inflammasome, ultimately leading to the massive release of\u0026nbsp;proinflammatory\u0026nbsp;factors\u0026nbsp;such as\u0026nbsp;IL-1β and TNF-α\u003csup\u003e[54-57]\u003c/sup\u003e. On the other hand, the released inflammatory factors amplify damage by regulating ferroptosis-related proteins. TNF-α can significantly upregulate ACSL4 expression, which catalyzes the esterification of polyunsaturated fatty acids into lipid peroxidation substrates, exacerbating membrane lipid oxidative damage\u003csup\u003e[58,59]\u003c/sup\u003e. Simultaneously, IL-1β can inhibit the Nrf2 antioxidant pathway, leading to the degradation and loss of activity of the GPX4 protein, which weakens the ability\u0026nbsp;of the cell\u0026nbsp;to clear lipid ROS\u003csup\u003e[60-62]\u003c/sup\u003e. This amplifying “ferroptosis-ROS-inflammation” effect not only induces SC death but also inhibits axon growth and myelin regeneration.\u003c/p\u003e\n\u003cp\u003eCurrently, the inhibition of ferroptosis relies primarily on the use of selective inhibitors, which have limitations in research. Our study revealed that PNI induces the abnormal accumulation of intracellular ROS and iron, which enhances the secretion of inflammatory factors such as TNF-α and IL-1β, ultimately leading to cellular dysfunction. CXCL12 is widely considered a key signaling molecule that promotes nerve repair. It exerts its neurotrophic effects by increasing the expression of neurofilament light chain (NF-L), promoting neuronal differentiation, and encouraging axon growth\u003csup\u003e[63,64]\u003c/sup\u003e. After nerve injury, CXCL12 can act on nerve tissue cells\u0026nbsp;via\u0026nbsp;the ERK1/2 and p38 MAPK pathways to regulate\u0026nbsp;the speed of\u0026nbsp;cell migration and facilitate nerve repair\u003csup\u003e[65,66]\u003c/sup\u003e.\u0026nbsp;Moreover, CXCL12 can indirectly promote nerve repair by recruiting various cell types that highly express CXCR4. For example, CXCL12 promotes SC migration and autophagy through the PI3K/AKT/mTOR pathway without affecting proliferation or apoptosis\u003csup\u003e[67]\u003c/sup\u003e. Zhang et al. reported that\u0026nbsp;neururin\u0026nbsp;can increase CXCL12 expression in nerve tissue, which in turn regulates the migration of bone marrow mesenchymal stem cells (MSCs) via the CXCL12/CXCR4-PI3K/Akt signaling pathway, thereby promoting nerve repair\u003csup\u003e[68]\u003c/sup\u003e. Additionally, studies have shown that CXCL12 can inhibit endothelial cell ferroptosis in age-related macular degeneration (AMD) by promoting the transport and maturation of sterol regulatory element-binding protein 1 (SREBP1) from the endoplasmic reticulum (ER) to the Golgi apparatus\u003csup\u003e[69]\u003c/sup\u003e.\u0026nbsp;On the basis of\u0026nbsp;these findings, we hypothesized that CXCL12 might inhibit ferroptosis and accelerate nerve repair.\u003c/p\u003e\n\u003cp\u003eIn our clinical study, we observed that serum CXCL12 levels were significantly elevated in PNI patients on the third day after surgery. Next, we locally injected CXCL12 into a sciatic nerve injury model in rats and reported a marked improvement in the continuity of axons and the integrity of myelin sheaths. This finding is consistent with previous research indicating that CXCL12 effectively promotes nerve repair. Furthermore, using both in vitro and in vivo PNI models, we observed that CXCL12 effectively reduced PNI-induced SC ferroptosis. This was evidenced by a decrease in intracellular Fe\u003csup\u003e2+\u003c/sup\u003e accumulation, lower levels of ROS and the lipid peroxidation product MDA, and an increase in the antioxidant GSH. The expression of the key ferroptosis execution protein ACSL4 was downregulated, whereas the expression of the antiferroptotic proteins GPX4 and FSP1 was significantly upregulated. Moreover, our study revealed that this protective effect of CXCL12 is dependent on the activation of the ERK/Nrf2 signaling pathway. CXCL12 treatment significantly increased ERK phosphorylation levels and promoted Nrf2 protein expression in SCs. The ERK inhibitor U0126 partially blocked CXCL12-mediated activation of Nrf2 and its subsequent inhibition of ferroptosis, confirming that the ERK/Nrf2 axis is a crucial signaling pathway involved in the antiferroptotic effect of CXCL12 on SCs. This finding is consistent with previous research showing that ERK activation promotes Nrf2 nuclear translocation, which then induces the expression of antioxidant and ferroptosis-inhibiting genes such as GPX4 and FSP1\u003csup\u003e[70,71]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMoreover, our research revealed that CXCL12 significantly reduces the inflammatory response in SCs by inhibiting ferroptosis and that ferroptosis is a crucial intermediary mechanism for the anti-inflammatory effect of CXCL12. CXCL12 treatment effectively inhibits PNI-induced activation of the NF-κB pathway and reduces the secretion of IL-1β and TNF-α. However, when we used the iron chelator FAC to induce iron overload, the anti-inflammatory effect of CXCL12 was significantly reversed. FAC treatment not only worsened ferroptosis indicators but also reactivated the NF-κB pathway and increased the release of inflammatory factors. This result proves that the anti-inflammatory action of CXCL12 is achieved by inhibiting ferroptosis, which is upstream of the NF-κB signaling pathway. The excessive ROS produced during ferroptosis are key factors in activating NF-κB, and CXCL12 effectively blocks this process by enhancing the cell's antioxidant capacity through the ERK/Nrf2 pathway.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study clarifies the dual protective mechanism of CXCL12 in PNI repair. First, by activating the ERK/Nrf2 signaling pathway, key antiferroptotic proteins, such as GPX4 and FSP1, are upregulated, effectively inhibiting SC ferroptosis. Second, inhibiting ferroptosis reduces ROS production, which in turn blocks the activation of the NF-κB pathway, thereby significantly mitigating the inflammatory response. This study revealed that ferroptosis is a key link in the regulation of SC inflammation by CXCL12, providing a new perspective on the neuroprotective role of CXCL12. This finding not only deepens our understanding of the interaction between ferroptosis and inflammation after PNI but also provides a theoretical basis for the development of new therapeutic strategies for PNI, such as CXCL12 analogs, ERK/Nrf2 activators, or ferroptosis inhibitors.\u003c/p\u003e\n\u003cp\u003eDespite these important advances, our study has several limitations. First, while we confirmed that the ERK/Nrf2 pathway mediates the effect of CXCL12 and observed changes in GPX4 and FSP1 expression, whether Nrf2 directly transcriptionally regulates the expression of GPX4 and FSP1 remains to be determined. We also need to investigate whether other downstream targets are involved, which could be clarified via techniques such as chromatin immunoprecipitation (ChIP), gene knockdown (siRNA/shRNA), or gene editing (CRISPR-Cas9)\u003csup\u003e[72,73]\u003c/sup\u003e. Second, this study focused\u0026nbsp;primarily\u0026nbsp;on early-stage SC ferroptosis and the inflammatory response. PNI repair is a complex process involving other cells, such as\u0026nbsp;immune cells (e.g., macrophage polarization)\u003csup\u003e[74]\u003c/sup\u003e, angiogenesis\u003csup\u003e[75]\u003c/sup\u003e, and axon guidance\u003csup\u003e[76]\u003c/sup\u003e. Future research should explore whether and how CXCL12 regulates these subsequent events.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study revealed that CXCL12 promotes nerve regeneration by activating the ERK‒Nrf2 pathway to inhibit SC ferroptosis and suppressing the NF-κB signaling pathway to mitigate inflammation. These findings provide a new theoretical basis and potential therapeutic target for the treatment of PNI.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eDirective Project of Hebei Provincial Department of Science and Technology 142777105D (Pei Wang); Hebei Key Laboratory of Nerve Injury and Repair SZX2020020\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einterest\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Y.Y. performed the experimental implementation, technical support, data analysis, and figure preparation and drafted the manuscript. P.W. contributed to the study design. Y.J. and S.D. provided technical support and assisted with experimental implementation. G.Y. and Z.Y. provided experimental supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eparticipate:\u0026nbsp;\u003c/strong\u003eAll patients provided informed consent, and the collection and use of human samples were approved by the Ethics Committee of the Affiliated Hospital of Chengde Medical University. The submission and approval of the ethical guidelines comply with the Declaration of Helsinki. The ethical approval number is CYFYLL2024077. The animal use and care plan adheres to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All animal experiments in this study were approved by the Animal Care and Use Committee of the Affiliated Hospital of Chengde Medical University. The ethical approval number is CYFYLL2025006.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eAll the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eavailability:\u003c/strong\u003e No datasets were generated or analyzed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLim T K Y, Shi X Q, Johnson J M, et al. Peripheral nerve injury induces persistent vascular dysfunction and endoneurial hypoxia, contributing to the genesis of neuropathic pain[J]. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 2015, 35(8): 3346-3359.\u003c/li\u003e\n\u003cli\u003eGriffin J W, Hogan M V, Chhabra A B, et al. Peripheral nerve repair and reconstruction[J]. 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Open Research Europe, 2024, 4: 80.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Nerve regeneration, Ferroptosis, Inflammatory response, CXCL12, ERK/Nrf2","lastPublishedDoi":"10.21203/rs.3.rs-7846278/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7846278/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Peripheral nerve injury (PNI) is often limited by the activation of the ferroptosis‒inflammation axis. Although CXCL12 is a crucial neuroregenerative factor, the precise mechanism by which it promotes nerve repair through regulating this axis remains unclear. This study systematically investigated the role and underlying mechanism of CXCL12 via clinical samples, cell models, and animal experiments. Clinical data revealed a significant increase in serum CXCL12 levels in PNI patients, suggesting its potential involvement in nerve injury regulation. In an LPS-induced Schwann cell (SC) injury model, CXCL12 attenuated ferroptosis and oxidative damage by activating the ERK/Nrf2 signaling pathway to upregulate GPX4 and FSP1 while suppressing ACSL4 expression. Concurrently, CXCL12 inhibited the activation of the NF-κB signaling pathway, thereby reducing the secretion of TNF-α and IL-1β and alleviating the inflammatory response. The antiferroptotic effect of CXCL12 was reversed by the ERK inhibitor U0126. Furthermore, ferrous ammonium citrate (FAC)-induced iron overload experiments confirmed that ferroptosis is a critical mechanism bridging CXCL12 regulation of inflammation and that NF-κB overexpression weakens its anti-inflammatory effects. In animal experiments further demonstrated that CXCL12 improved the mitochondrial structure, reduced the accumulation of Fe2+ and lipid peroxidation in injured nerve tissue, and promoted axon and myelin regeneration after PNI. Overall, CXCL12 promotes PNI repair by activating the ERK/Nrf2 pathway to inhibit SC ferroptosis, which subsequently downregulates the NF-κB-mediated inflammatory response. This study is the first to elucidate the bridging role of ferroptosis in CXCL12-mediated inflammation regulation, suggesting a new theoretical basis for targeting CXCL12 as a potential therapeutic strategy for PNI.","manuscriptTitle":"CXCL12 Promotes Peripheral Nerve Injury Repair by Inhibiting the Ferroptosis‒Inflammation Axis via the ERK/Nrf2 Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 12:29:11","doi":"10.21203/rs.3.rs-7846278/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-05T13:22:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T07:20:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-16T09:31:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66694600136280526533139605348294163168","date":"2025-10-16T00:07:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255638577820597651914160607791829365250","date":"2025-10-15T18:44:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-15T16:46:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T00:52:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-14T00:50:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation","date":"2025-10-13T08:07:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b24afc03-cd08-40b1-b4c5-fdb6c9fcf597","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:01:28+00:00","versionOfRecord":{"articleIdentity":"rs-7846278","link":"https://doi.org/10.1007/s10753-026-02453-2","journal":{"identity":"inflammation","isVorOnly":false,"title":"Inflammation"},"publishedOn":"2026-03-12 15:58:13","publishedOnDateReadable":"March 12th, 2026"},"versionCreatedAt":"2025-10-29 12:29:11","video":"","vorDoi":"10.1007/s10753-026-02453-2","vorDoiUrl":"https://doi.org/10.1007/s10753-026-02453-2","workflowStages":[]},"version":"v1","identity":"rs-7846278","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7846278","identity":"rs-7846278","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}