Animals

To mitigate potential confounding effects of estrogen-mediated neuroprotection and cyclical hormonal variations in female rodents, this study exclusively employed male C57BL/6 mice (body weight: 20–30 g; Certification No. SCXK 2019–0004) obtained from Hangzhou Ziyuan Laboratory Animal Co., Ltd. (Zhejiang, China). All experimental protocols received ethical approvals (2024-N(A)-134; LLSC20242502) from the Institutional Animal Care Committees of the First Affiliated Hospital of USTC and Anhui Medical University. Animals were maintained under controlled environmental conditions (humidity: 55 ± 5%; 12:12 h light–dark cycle) with ad libitum access to food and water. Following a 7 day acclimatization period, eighteen mice were randomly allocated into three experimental cohorts (n = 6/group): 1) Sham-operated controls, 2) Spinal cord injury (SCI) model, and 3) SCI + Xanthoxylin therapeutic intervention.

Drug Administration

All drugs were dissolved in 2% DMSO saline and administered via tail vein injection. Mice in the Xanthoxylin group were injected with 0.1 ml Xanthoxylin (10 μM, MCE, Cat# HY-N1063) daily post-SCI. Both the control group and the injury model group received daily injections of 100 μL PBS for control purposes. Xanthoxylin was administered immediately after spinal cord injury modeling and then every two days thereafter.

SCI Animal Model

We first anesthetized the mice using isoflurane at a concentration of 3%, and then maintained the anesthesia with isoflurane at a concentration of 1%. During the procedure of establishing the spinal cord injury model, the mice were placed on a warming blanket. For the surgical modeling, we selected the T10-11 segment of the mice. We first incised the lamina to expose the dura mater of the spinal cord, and then caused a spinal cord injury by dropping a weight of approximately 5 g from a height of 3 cm.After injury, the fascia, muscles, and skin were sutured in layers using non-absorbable sutures (He et al., 2024; Zhong et al., 2019). Sham-operated mice underwent the same surgical procedures as the injury group. To alleviate postoperative pain in the mice and enhance the welfare of the experimental animals, we administered a subcutaneous injection of 0.5 mg/kg Carprofen before surgery and then injected the same dose three times over the 48 h post-surgery to relieve pain. All mice received manual bladder expression three times daily to assist with urination.

Spinal Cord Function Assessment—Basso Mouse Scale (BMS) Score

The overall behavioral scoring of mice lasted for four weeks, during which we selected different time points to evaluate the motor ability and recovery of mice from different groups after modeling. We mainly observed the gait, limb movement, and limb coordination of the mice. We used the Basso Mouse Scale (BMS) for systematic assessment, with a total score ranging from 0 to 9 (where 0 indicates complete loss of motor function and 9 indicates normal motor function). We conducted motor assessments at 1, 3, 7, 21, and 28 days after modeling.

Locomotor Gait Analysis

To evaluate the neuromotor function of mice, we conducted a gait analysis experiment. We applied blue ink to the left hind limb and red ink to the right hind limb of the mice. The mice were then placed on a clean sheet of white paper, and they were encouraged to walk in a straight line, leaving footprints on the paper. Based on the footprints left on the paper, we calculated the stride length and the step width. All experimental assessments were performed by personnel who were blinded to the grouping of the mice （Zeng et al., 2018; Yao et al., 2021）.

Inclined Plane Test

Mice were placed on an inclined plane, covered with rubber pads having 3 mm spacing between them. The longitudinal axis of the mouse’s body was aligned parallel to the inclined plane, with the head facing upward. The angle of the inclined plane was gradually increased by 5° until the mouse could no longer maintain its position for 5 s. We recorded the maximum inclined board angle that each mouse could maintain. All experimental assessments were performed by personnel who were blinded to the grouping of the mice. Each mouse was tested three times, and the average of these measurements was used as the final value.

Cell Culture

The PC12 neuronal cell line (CL-0481) and BV2 microglial cells (CL-0493) were acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Cells were maintained in high-glucose DMEM ( Ketu Biotech, Hefei) containing 10% heat-inactivated fetal bovine serum (FBS; Corning, NY, USA) and an antibiotic–antimycotic cocktail (100 U/mL penicillin, 100 μg/mL streptomycin; Ketu Biotech). All cultures were grown under standard conditions in a humidified 5% CO₂ atmosphere at 37 ℃.

Co-culture Experiment

PC12 cells were pretreated with Xanthoxylin (10 μM) for 24 h. Subsequently, the conditioned supernatant was collected and co-cultured with BV2 cells in 6-well plates. After 24 h of co-culture, the BV2 cells were harvested for subsequent experiments.

Oxygen–Glucose Deprivation (OGD) Experiment

We first cultured the BV2 cells in glucose-free DMEM medium. Then, we created a hypoxic environment by filling a sealed chamber with 95% nitrogen and 5% carbon dioxide and placed the glucose-free cultured BV2 cells into the chamber for 4 h of treatment.

Cell Viability Assay

To determine the optimal experimental concentration of Xanthoxylin, PC12 cells were plated in 96-well plates and exposed to a concentration gradient spanning 0 μM to 160 μM (0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM). Following 24 h treatment, cellular viability was assessed by adding 10 μL of CCK-8 reagent (Beyotime, Shanghai) to each well. After 30-min incubation at 37 ℃, optical density measurements were performed at 450 nm using a full-spectrum microplate reader.

Western Blotting

Protein lysates were prepared from BV2 microglial cultures and spinal cord specimens across experimental cohorts. Following denaturation, samples underwent electrophoretic separation via 12% SDS-PAGE (Bio-Rad) and were subsequently electrophoretically transferred to PVDF membranes (0.45 µm, Millipore) using semi-dry transfer apparatus. Membranes were subjected to blocking with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at ambient temperature (25 ℃). Primary antibody incubation was performed overnight at 4 ℃ with gentle agitation using the following reagents: iNOS (1:1000, Proteintech, Wuhan, China, Cat# 189851AP, RRID: AB_2782960), CD206 (1:1000, Proteintech, Wuhan, China, Cat# 187041AP, RRID: AB_10597232), TNF-α (1:1000, Proteintech, Wuhan, China, Cat# 175901AP, RRID: AB_2271853), GAPDH (1:50,000, Proteintech, Wuhan, China, Cat# 60,004–1-IG, RRID: AB_2107436), P65 (1:1000, Proteintech, Wuhan, China, Cat# 10,745–1-AP), p-P65 (1:2000, Proteintech, Wuhan, China, Cat# 82,335–1-RR), IκBα (1:1000, Proteintech, Wuhan, China, Cat# 18,220–1-AP), and p-IκBα (1:2000, Proteintech, Wuhan, China, Cat# 68,999–1-IG). The next day, the membrane was incubated with secondary antibody, Goat Anti-Rabbit IgG (1:50,000, Proteintech), at 25 ℃ for 2 h. We used enhanced chemiluminescence (ECL) to visualize the proteins on the membranes and analyzed the images and results using ImageJ software. All experiments were performed in triplicate.

RT-qPCR

Total RNA isolation from cellular and murine spinal cord tissue samples across experimental groups was performed using Trizol reagent. First-strand cDNA synthesis was carried out with the HiScript IORT SuperMix for qPCR (Vazyme Biotech, China) following the manufacturer’s protocol. Quantitative real-time PCR (qPCR) amplification was conducted using AceOg SYBR Green Master Mix (Roche, Switzerland) on a thermal cycler system. Gene expression levels were normalized to the housekeeping gene GAPDH, with relative quantification calculated through the comparative threshold cycle (2^(-ΔΔCt)) method. Target-specific primer pairs for CD206, TNF-α, iNOS, and GAPDH are detailed in Table 1.

Table 1 | Primer sequencesImmunofluorescence Staining

BV2 microglial cells were cultured on glass coverslips within 24-well plates. Following PBS rinsing to remove residual medium, cellular fixation was achieved by ice-cold 4% paraformaldehyde treatment for 20 min. Fixed specimens were permeabilized with 0.5% Triton X-100. For spinal cord histological sections, standard procedures included xylene-mediated deparaffinization, graded ethanol rehydration, and antigen retrieval using EDTA buffer (pH 8.0) under heated conditions. All samples (cells and tissues) underwent blocking with 10% BSA prior to overnight incubation at 4 ℃ with primary antibodies. Secondary antibody conjugation was performed at ambient temperature (25 ℃) for 45 ± 15 min. Nuclear counterstaining employed DAPI (Servicebio, China). Mounted slides were imaged via fluorescence microscopy (Olympus IX83) for quantitative assessment of iNOS (M1 marker) and CD206 (M2 marker) expression.

Histological Analysis

Histochemical analyses were conducted on spinal cord specimens harvested from SCI model mice, employing H&E (Solarbio, Beijing; Cat# G1120) and Nissl staining (Cat# G1434) following vendor-prescribed protocols. Tissue processing encompassed sequential ethanol dehydration series (70%, 95%, 100%), xylene-based clearing (10 min immersion), and neutral balsam-mediated slide mounting. Digitized whole-slide scanning was executed using the Pannoramic DESK P-MIDI P250 platform (3DHISTECH, Hungary), enabling systematic evaluation of cytoarchitectural alterations at lesion sites.

Transcriptome Sequencing and Bioinformatics Analyses

We performed transcriptomic profiling on spinal cord tissues from both spinal cord injury (SCI) and SCI with Xanthoxylin treatment groups, with three mice examined per group at 14 days post-injury. After euthanizing the animals by cervical dislocation, we carefully dissected a 1 cm spinal cord segment encompassing the lesion site (extending 0.5 cm rostral and caudal to the injury epicenter). Total RNA was immediately extracted from the fresh tissue samples, followed by mRNA enrichment using oligo(dT)-attached magnetic beads. For library construction, we first generated single-stranded circular cDNA through PCR amplification, then employed phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) to produce DNA nanoballs via isothermal amplification.

High-throughput sequencing was carried out on the DNBSEQ-T7 ultra-high-throughput platform. Comparative transcriptomic profiling between experimental groups was executed via DESeq2 (v1.26.0) in R, implementing Benjamini–Hochberg false discovery rate (FDR) correction for multiple hypothesis testing. Differentially expressed genes (DEGs) were defined by stringent thresholds (FDR-adjusted p < 0.05, |log2 fold change|> 1). Functional annotation of DEGs was conducted through Gene Ontology (GO) enrichment analysis using the GOseq package, employing the Wallenius non-central hypergeometric algorithm to account for gene length bias. KEGG pathway enrichment was assessed through KOBAS 3.0 with Fisher’s exact test, and results were sorted by ascending q-value to prioritize biological significance. The complete RNA-seq dataset has been deposited in NCBI’s Sequence Read Archive (Accession: PRJNA1148461), ensuring public accessibility.

Flow Cytometry

Apoptosis was analyzed by flow cytometry using the ™488/PI double-staining apoptosis detection kit (Abbkine, China). Data acquisition and analysis were performed on a Beckman Coulter CytoFLEX flow cytometry system.

Enzyme-Linked Immunosorbent Assay (ELISA)

Spinal cord tissues underwent three cycles of rapid freezing in liquid nitrogen followed by thawing at room temperature to achieve complete cellular lysis. The lysates were then spun at 10,000 × g for 10 min under refrigerated conditions (4 ℃), after which the clarified supernatant was aliquoted and cryopreserved at − 80 ℃ for subsequent biochemical analyses. Quantitative detection of inflammatory cytokines was performed using species-specific ELISA kits: TNF-α (CSB-E09315h, CUSABIO) and IL-10 (CSB-E04594M-IS, CUSABIO), following optimized protocols. Optical density measurements at 550 nm were acquired using the Synergy™ HT multi-mode microplate reader (BioTek Instruments, Vermont, USA), with all assays conducted in technical triplicates.

TdT-Mediated dUTP Nick-End Labeling (TUNEL)

We performed TUNEL apoptosis assays using a commercial kit (Beyotime Biotechnology, Nantong, Jiangsu, China) following the manufacturer’s protocol. Apoptotic nuclei were visualized under confocal microscopy, exhibiting red fluorescence, while normal nuclei were counterstained blue with DAPI. Quantitative analysis of cell survival rates was conducted using ImageJ software (NIH, USA).

Small RNA Interference

The siRNA constructs were designed through bioinformatic prediction algorithms and synthesized commercially by General Biol (Anhui, China). For cellular transfection, a lipid-based delivery system was employed: Lipofectamine 3000 transfection reagent (Invitrogen, USA) was reconstituted in 125 μL of Opti-MEM reduced-serum medium (5 μL reagent per preparation) to generate Solution A. Concurrently, siRNA oligos (5 μL) were diluted in an equal volume of Opti-MEM (125 μL) supplemented with 10 μL of P3000 transfection enhancer, constituting Solution B. The two solutions were homogenously mixed at a 1:1 volumetric ratio and maintained at standard culture conditions (37 ℃) for 15 min to promote nanoparticle self-assembly. These siRNA-lipid complexes were then administered to BV2 microglial cells pre-cultured in 6-well plates. Post-transfection silencing efficacy was validated 48 h later via quantitative Western blot analysis of target protein downregulation.

Statistical Analysis

Quantitative data are expressed as mean ± SD and were statistically evaluated using SPSS 25.0 (IBM, Armonk, NY). Analytical approaches were guided by experimental design parameters: independent samples t-tests for dual-group comparisons, one-way ANOVA for single-factor multi-group analyses, and two-way ANOVA for factorial designs. Following significant omnibus ANOVA results (p < 0.05), Bonferroni-adjusted pairwise comparisons were implemented to delineate intergroup variations. Statistical significance threshold was maintained at α = 0.05 throughout all analyses.