Abstract Background Revascularization and reconstruction of the blood-spinal cord barrier (BSCB) following spinal cord injury (SCI) play crucial roles in supplying essential nutrients and fostering a supportive microenvironment for neural network reconstruction. Thus, facilitating vascular regeneration and maintaining BSCB integrity are key therapeutic targets for functional recovery post-SCI. Methods Ischemia-induced pathological alterations in spinal cord microvascular endothelial cells were modeled in vitro using oxygen–glucose deprivation/reperfusion (OGD/R)-exposed bEnd.3 cells to assess whether QCT protects endothelial cells and enhances their angiogenic capacity. Subsequently, motor function, histopathological morphology, vascular density, and BSCB integrity were evaluated in rats with SCI to examine the therapeutic efficacy of QCT. A network pharmacology approach was employed to predict the potential pharmacological mechanisms of QCT in the treatment of SCI, followed by experimental validation. Results QCT enhanced survival, tube formation, and migration of bEnd.3 cells following OGD/R exposure in vitro. In the rat SCI model, QCT demonstrated beneficial effects on vascular regeneration and BSCB integrity, contributing to improved functional recovery. The PI3K/Akt signaling pathway was investigated to elucidate the underlying molecular mechanisms. Conclusions These findings suggest that QCT can promote the regeneration of blood vessels in the injured spinal cord and protect the structure of the BSCB by activating the PI3K/Akt signaling pathway, thereby enhancing the neurological function of rats following SCI. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-025-06973-7. Keywords: Quercetin, Spinal cord injury, Angiogenesis, Endothelial cells, Blood-spinal cord barrier, PI3K/Akt pathway Background Spinal cord injury (SCI) is a traumatic disorder affecting the central nervous system, leading to deficits in sensory, motor, and autonomic functions below the site of damage [[46]1]. Given the exceptionally high disability rate, associated complications, limited therapeutic interventions, and considerable healthcare costs, SCI imposes a substantial disease burden and remains a major public health concern. The prevalence of traumatic SCI (TSCI) is 26.48 cases per million individuals, with an annual increase of 1.5%. Based on the severity, the cost of lifetime care for patients with SCI can range from $1 to $4 million, placing a significant strain on healthcare systems globally [[47]2, [48]3]. Despite numerous advances in SCI research and treatment, effective therapeutic options remain limited. Following SCI, acute mechanical trauma causes extensive damage to the local vascular network, affecting capillaries and small veins, which consequently leads to hemorrhage, thrombosis, and vasospasm, all of which disrupt microcirculation in the spinal cord [[49]4]. As axonal regeneration and clearance of harmful substances rely on a functional vascular system, ischemia significantly hinders neurological recovery [[50]5]. Conversely, the formation of new blood vessels at the injury site supplies essential nutrients that facilitate spinal cord repair. Therefore, promoting posttraumatic vascular remodeling, enhancing local microcirculation, and increasing tissue perfusion are critical for neuronal tissue survival. Additionally, hypoxia induced by injury contributes to endothelial dysfunction, compromising the integrity of the blood-spinal cord barrier (BSCB) [[51]6]. Increased BSCB permeability is a key pathophysiological characteristic of SCI. BSCB regulates the exchange of oxygen, nutrients, and metabolites between the spinal cord and the bloodstream while simultaneously serving as a protective barrier that prevents toxic substances and cells from entering the spinal cord, thereby maintaining its internal homeostasis. Following BSCB disruption, inflammatory cells, including neutrophils and macrophages, infiltrate the spinal cord tissue and release inflammatory mediators that worsen secondary injuries [[52]7]. Thus, strategies to protect BSCB integrity are equally vital in SCI treatment. Herbal medicine is widely used in traditional Chinese and Korean medicine to treat central nervous system-related diseases [[53]8–[54]10]. Quercetin (QCT), a widely studied bioactive compound found in numerous medicinal plants, exhibits potent anti-inflammatory, antioxidant, and anti-cancer properties [[55]11, [56]12]. QCT can reduce apoptosis induced by hypoxia-reoxygenation damage in human brain microvascular endothelial cells [[57]13], and its derivatives can enhance capillary density in ischemic muscles in a murine hind limb ischemia model [[58]14]. Moreover, QCT inhibits ferroptosis and alleviates the severity of brain injury by stimulating the Nrf2/HO-1 signaling pathway during cerebral ischemic events [[59]15]. Based on these observations, we hypothesized that QCT promotes neovascularization and expedites the restoration of BSCB integrity following SCI. To test this hypothesis, we systematically examined the changes in vascular endothelial cells and spinal cord tissue following QCT treatment in vivo and in vitro. Additionally, through network pharmacological screening, we identified and validated the potential molecular pathways involved in the QCT-mediated modulation of vascular tissue. Methods Cell culture and treatment The bEnd.3 cell line, derived from mouse brain tissue, was obtained from ProCell (Wuhan, China). These cells were cultured in high-glucose DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Solarbio, China). The cultures were kept at 37 °C in a humidified incubator with 5% CO[2]. To model in vitro ischemia–reperfusion injury following SCI, oxygen–glucose deprivation/reperfusion (OGD/R) injury was induced as previously described [[60]16]. Briefly, bEnd.3 cells were initially cultured in glucose-free DMEM (Gibco, USA) under hypoxic conditions (37 °C, 95% N2, 5% CO2, ThermoFisher, USA) for 6 h. Following this, the medium was replaced with standard high-glucose DMEM, and the cells were returned to normoxic conditions (37 °C, 21% O2, 5% CO2, ThermoFisher, USA) for an additional 6 h. Control samples consisted of bEnd.3 cells cultured under standard conditions. To assess the role of QCT on injury induced by OGD/R, bEnd.3 cells were treated with varying concentrations of QCT (Purity ≥ 95%, Sigma–Aldrich, USA) at the onset of OGD. To effectively inhibit PI3K signaling, cells were administered 10 μM of the PI3K inhibitor LY294002 (MCE, USA). As a solvent to facilitate drug solubilization, dimethyl sulfoxide (DMSO, Sigma–Aldrich, USA) was utilized, with its final concentration maintained below 0.1% to prevent any interference with the experimental outcomes. Cell viability assay Cell viability was assessed utilizing a Cell Counting Kit-8 (CCK-8, Beyotime, China) as per the instructions provided by the manufacturer. Briefly, the cells were plated in 96-well plates at a density of 10^4 cells per well. Following the respective treatments, 10 μl of CCK-8 detection solution was introduced to each well and incubated at 37 °C for 1 h. The absorbance was quantified at a wavelength of 450 nm. Each group was comprised of five replicates. TdT-mediated dUTP nick end labeling (TUNEL) staining The cell apoptosis rate was evaluated utilizing a TUNEL apoptosis detection kit (ApexBio, USA), following the guidelines provided by the manufacturer. The nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI). A total of six images were randomly captured, and analyzed using a fluorescence microscope to quantify the apoptotic cells. Cell counting and intensity quantification were performed using ImageJ software. EdU incorporation assay To assess whether QCT promoted cell proliferation, an EdU assay was performed. Briefly, bEnd.3 cells were seeded at a density of 5 × 10^3 cells per well in 24-well plates. Treatments were applied once the cells reached approximately 80% confluence. The endothelial cells were stained following the manufacturer's protocol (RiboBio, China), and nuclei were counterstained with Hoechst 33,342. Fluorescent images were captured, and the percentage of EdU-positive cells was quantified using ImageJ software. Each experiment was repeated with six replicates. Scratch migration assay As previously described [[61]17], the horizontal migration capability of cells was assessed through a scratch wound healing assay. Briefly, bEnd.3 cells were cultured in 6-well plates until they achieved 90% confluence. A cross-shaped scratch was then created in the cell monolayer with a P-200 pipette tip. Detached cell debris was subsequently removed with phosphate-buffered saline (PBS), and fresh medium was added. Wound closure was imaged using a microscope at 0 and 12 h under different treatments. The scratch width was measured in three random fields using the ImageJ software. Transwell migration assay The vertical migration ability of bEnd.3 cells was evaluated using a Transwell assay, as previously described [[62]18]. In short, bEnd.3 cells (2 × 10^4 cells/ wells) were introduced into the upper chamber of a Transwell featuring an 8.0 μm pore size (Corning, USA), while the lower chamber was filled with 600 μL of DMEM supplemented with 10% FBS and QCT. After a 12-h incubation period, the cells were stained with 0.1% crystal violet solution (LEAGENE, China). Subsequently, non-migrated cells were removed, and images were captured for analysis. The quantification of migrated cells was conducted using ImageJ software. Each experiment was repeated six times. Tube formation assay Tube formation assays were conducted to model endothelial cell tube formation in vitro [[63]19]. Briefly, the matrix gel (Matrigel, Corning, USA) was unfrozen at 4 °C, and 50 μL were dispensed into each well of a 96-well plate. The plate was then incubated at 37 °C for 30 min to facilitate polymerization. The bEnd.3 cells (4 × 10^4 per well) were seeded onto the Matrigel-coated wells, followed by incubation with the respective treatments at 37 °C for 12 h. The formation of capillary-like structures was imaged using a microscope, and the number of meshes and junctions was calculated utilizing ImageJ software. Immunofluorescence staining For immunofluorescence staining, tissue sections or cultured cells were first fixed in 4% paraformaldehyde (PFA; LEAGENE, China) for 15 min at room temperature, followed by permeabilization with 0.1% Triton X-100 (Sigma–Aldrich, USA) in PBS for 10 min. After three washes with PBS, the samples were blocked with 5% bovine serum albumin (BSA, Solarbio, China) in PBS for 1 h. Primary antibodies were then applied and incubated overnight at 4 °C in a humidified chamber. Following incubation with primary antibodies, the sections or cells were washed three times with PBS and incubated with fluorescence-conjugated secondary antibodies for 1 h at room temperature, protected from light. After secondary antibody incubation, the samples were washed with PBS and mounted with a DAPI-containing mounting medium to counterstain the nuclei. The localization and expression of the target proteins were assessed using confocal microscopy (Olympus GmbH, Hamburg, Germany); the brand names, article numbers, host species, and dilution ratios of the specific antibodies used are listed in Table [64]1. Table 1. Primary antibody information Antibody Source Catalog number Host species Application Dilution ratio Anti-CD31 abcam ab9498 Mouse IF 1:200 Anti-ZO1 abcam ab221547 Rabbit IF 1:200 Anti-Ki67 abcam ab15580 Rabbit IF 1:200 Anti-PI3K Cell Signaling Technology 4257 Rabbit WB 1:1000 Anti-p-PI3K Cell Signaling Technology 4228 Rabbit WB 1:1000 Anti-Akt Cell Signaling Technology 4685 Rabbit WB 1:1000 Anti-p-Akt Cell Signaling Technology 4060 Rabbit WB 1:2000 Anti-Claudin5 abcam ab131259 Rabbit WB 1:1000 Anti-Occludin abcam ab216327 Rabbit WB 1:1000 Anti-CD31 abcam ab9498 Mouse WB 1:1000 Anti-GAPDH abcam ab9484 Mouse WB 1:5000 Anti-rabbit lgG, HRP-linked Antibody Proteintech SA00001-2 Goat WB 1:5000 Anti-mouse lgG, HRP-linked Antibody Proteintech SA00001-1-A Goat WB 1:5000 [65]Open in a new tab Animal usage In this study, a cohort of 50 adult female Sprague–Dawley (SD) rats, aged 6–8 weeks and weighing between 250 and 300 g, was utilized. All the animals were acquired from the Guangdong Medical Experimental Animal Center. The rats were maintained in a environment under specific pathogen-free conditions, subjected to a 12-h light/dark cycle, and were given unrestricted access to food and water. Rat spinal cord injury models, treatment, and motor function scores In this study, we used a rat model to replicate SCI using a clamping technique [[66]20]. Prior to the procedure, the rats were acclimatized to a standard laboratory environment under quiet conditions for 3 days. To reduce the risk of postoperative gastrointestinal complications and mortality from infections associated with SCI, a preoperative fasting period of 24 h and an additional 8 h of alcohol abstinence were implemented. The rats were subjected to anesthesia using isoflurane, administered at a concentration of 2% for induction and 1.5% for maintenance, and a dorsal incision was made to expose the thoracic vertebrae T9 to T11. Precise laminectomy at T10 exposed the spinal cord, which was compressed using an arterial clip (Sugita, Japan) with 30 g of force for 1 min to simulate compressive trauma similar to that in human SCI. Following the injury, the incision site was irrigated with antibiotic-loaded saline and sutured. The rats were randomly allocated into five distinct groups (n = 10 per group): sham operation group, SCI group, low-dose QCT treatment group, high-dose QCT treatment group, and blocker group. After surgery, the rats in the QCT treatment groups were administered intraperitoneal injections of QCT (Purity ≥ 95%, Sigma–Aldrich, USA) dissolved in saline at dosages of 20 and 50 mg/kg/day over a period of 28 consecutive days, based on the concentrations demonstrated to be effective in previous studies. The blocker group received the PI3K/Akt signaling pathway inhibitor LY294002 (1.5 mg/kg/day) (MCE, USA) in combination with 50 mg/kg/day QCT, administered intraperitoneally immediately after the SCI model was established for 28 consecutive days. The SCI group was administered an equivalent volume of saline as that provided to the control group. The sham-operated group underwent a laminectomy without spinal cord compression. Postoperatively, the rats were administered analgesics and antibiotics to prevent infections and were monitored for urinary and bowel function. Behavioral analysis Motor function was assessed using the Basso (BBB) scale at baseline (prior to surgical intervention) and on postoperative days 1, 3, 7, 14, 21, and 28 [[67]21]. Two investigators independently conducted the behavioral assessments and remained blinded to the group assignments. The BBB evaluates motor function in three areas: joint movement, gait, coordination, and fine motor skills. The assessment scale extends from 0, representing total paralysis, to 21, which indicates normal motor function. The scoring system is classified into three categories: low scores (0–7) signify severe motor impairment or a complete lack of hind limb movement, medium scores (8–13) suggest inconsistent movement, and high scores (14–21) denote synchronized movement of both forelimbs and hind limbs. Neuroelectrophysiology Motor evoked potentials (MEPs) were recorded to evaluate the recovery of neuroelectric signals following SCI [[68]22]. The rats were anesthetized using isoflurane and positioned in a prone orientation. Stimulating electrodes were placed on the motor cortex, whereas recording electrodes were inserted into the hind limb muscle tissue. The grounding electrode was positioned caudally to minimize electrical noise. The stimulation parameters were optimized to achieve a clear and reproducible motor response without causing tissue damage. The responses were amplified and recorded using an electrophysiological recording system (Taimeng Software, Chengdu, China). MEPs were then analyzed to assess changes in amplitude, which reflect the functional integrity of the motor pathway. Histological analysis The spinal cord tissue was subjected to fixation in 4% paraformaldehyde (LEAGENE, China) for 24 h to facilitate histological examination. Following the fixation process, the tissues were dehydrated using a series of graded alcohol solutions, eliminated in xylene, and embedded in paraffin. Sections were prepared at a thickness of 10 μm utilizing a microtome and subsequently mounted onto slides for further analysis. Prior to the application of hematoxylin and eosin (H&E) staining, paraffin was removed from the tissue sections using xylene, followed by rehydration with a descending series of alcohols in water. Hematoxylin was used to impart a blue coloration to the nuclei, after which the sections were rinsed with water. The sections were subsequently counterstained with eosin, which resulted in pink coloration of the cytoplasm and various tissue structures. (The HE staining kit purchased from Solarbio, Beijing, China) After staining, the slides were encapsulated in a neutral resin. The stained sections were analyzed under a light microscope to evaluate the tissue architecture. Assessment of BSCB permeability The BSCB permeability was assessed using the Evans blue (EB; LEAGENE, China) dye extravasation method, as described previously [[69]23]. On day 7 post-SCI, the rats were administered 2% EB dye via the tail vein, 2 h before euthanasia. After anesthesia, rats were subjected to transcardial perfusion with PBS. Spinal cord tissue was collected and incubated in N,N-dimethylformamide (Sigma-Aldrich, Germany) for 72 h. Following this incubation period, the samples underwent centrifugation at 15,000 rpm for 1 h. The supernatant was collected, and the fluorescence intensity was measured using a spectrophotometer, with excitation and emission wavelengths set at 610 nm and 680 nm, respectively. The concentration of EB dye per gram of spinal cord tissue was determined using a standard curve. Western blot assay Spinal cord and cell samples were subjected to homogenization in RIPA buffer (Beyotime, China) containing 1% protease inhibitor and 1% phosphatase inhibitor (Roche, Switzerland), followed by a 10-min lysis period. The lysates were spun at 12,000 rpm for 10 min at 4 °C. The resulting supernatant was then collected, and the protein content was assessed using the BCA method. A reducing sample buffer (Beyotimehour, China) was incorporated, and the samples were heated to 100 °C for 5 min. Equal protein amounts were applied to 8–12% SDS-PAGE gels for separation, and then transferred onto specific membranes (Millipore, USA). Following transfer, the membranes were blocked with 5% BSA (Solarbio, China) solution for 1 h and incubated overnight at 4 °C with primary antibodies. The brand names, article numbers, host species, and dilution ratios of the primary antibodies are listed in Table [70]1. Following three washes with TBST, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies, tailored for either mouse or rabbit. Finally, the membranes were exposed to ECL reagent (Bio-Rad, USA) and imaged using the ChemiDoc™ XRS+ imaging system (Bio-Rad, USA). Network pharmacology analysis Using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analytical Platform, the Swiss Target Prediction Database, the PharmMapper database, and UniProt for ID mapping, we searched for QCT-related targets by entering “ingredient”, “chemical name”, or “compound” as “quercetin.” Gene targets associated with spinal cord injury were identified using the GeneCards, OMIM, and TTD databases, with search attributes restricted to “H. sapiens”. Predicted targets from the Swiss database had a probability score > 0, while targets from PharmMapper had a fit score of ≥ 0.7. Finally, QCT-related and SCI-related gene targets were cross-referenced using Venny 2.1 and Evenn software to identify potential active targets of QCT in treating SCI. In the process of constructing the protein–protein interaction (PPI) network, topological data were obtained from the STRING database, focusing on targets that are likely to be active. Interactions with a confidence score > 0.7 were selected for network analysis and screened according to centrality. A visual network representing the interactions between QCT and SCI-associated common targets was constructed. PPI data were integrated into the Cytoscape version 3.10.1 software to identify hub targets, and CytoHubba plug-ins were used to calculate the degree of interaction between nodes. Every node within the PPI network was assigned a degree value, with higher values indicating closer proximity to the network core. The core targets identified in the database underwent analyses for enrichment in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. An enrichment analysis was conducted utilizing the “functional annotation” feature, with the identification code designated as “OFFICIAL_GENE_SYMBOL” and restricting the species to “Homo sapiens.” A P-value threshold of less than 0.05 was set to determine statistical significance. Molecular docking Molecular docking was employed to explore the binding interactions between the identified key components and core proteins. The component structure files in the mol2 format were retrieved from the TCMSP database, while the three-dimensional structures of the key components were acquired from the PubChem database. These structures were imported into AutoDock 1.5.7, which included the removal of water molecules, hydrogen addition, and charge assignment. The grid parameters were set using the autogrid plug-in, the center grid box was defined, and the map file was calculated. The AutoDock plug-in was used to configure the search and docking parameters for the molecular docking calculations. The resulting binding conformations were imported into Discovery Studio and PyMOL 2.6 to visually analyze the docking results. Molecular dynamics simulation Molecular dynamics simulations of the protein–ligand complex were conducted using GROMACS on a Linux server to evaluate the stability of the PIK3R1-QCT complex, with PIK3R1 alone as the control. The Amber99sb force field was used to define the force field parameters for the protein atoms, whereas the GAFF generic force field was applied to the small-molecule ligands. The SPC water model was selected, and the solvent and ion pairs were introduced to equilibrate the system. Chloride and sodium ions were introduced in place of water molecules to maintain electrical neutrality within the system. The system underwent energy minimization, followed by NVT and NPT equilibration. Molecular dynamics (MD) simulations were carried out for 100 ns at 300 K and atmospheric pressure. Upon completion of the simulations, critical parameters, such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (RG), and solvent-accessible surface area (SASA), were computed to evaluate the stability of the complex. Statistical analysis Statistical analyses were conducted utilizing SPSS version 20.0, which was developed in Chicago, New York, USA. The data are presented as mean ± standard error of the mean. Unpaired Student’s t-tests were employed to evaluate statistical significance in comparisons between two groups. For comparisons involving three or more groups, one-way ANOVA was performed, followed by least significant difference (LSD) post-hoc analysis, assuming equal variances, or Dunnett’s T3 method when the assumption of equal variances was not met, to identify significant differences among groups. Additionally, two-way ANOVA with Bonferroni post hoc tests was utilized to evaluate BBB scores. A significance level of P < 0.05 was established. Results Quercetin enhances the proliferation and attenuates the apoptosis of endothelial cells in vitro following OGD/R-induced injury As depicted in the flowchart (Fig. [71]1A) of the in vitro experiments, bEnd.3 cells were treated with or without QCT and subjected to 6 h of oxygen–glucose deprivation (OGD) to simulate ischemia, followed by 6 h of reoxygenation. After 12 h, the endothelial cells were analyzed using various assays, including TUNEL staining, EdU assay, western blotting, and immunofluorescence, to evaluate cell apoptosis, proliferation, and protein expression. Furthermore, functional assays, such as proliferation, scratch migration, Transwell migration, and tube formation, were conducted to assess cell viability, migration, and angiogenic potential. Cell proliferation constitutes a critical component of angiogenesis, which is vital for the processes of tissue repair and regeneration [[72]24]. To determine the optimal dose of QCT for mitigating OGD/R injury in endothelial cells, we employed the CCK-8 assay to evaluate the proliferative effects of QCT across a concentration range of 0.1–20 μM (Fig. [73]S1A). The results demonstrated that QCT at concentrations of 0.5–10 μM significantly enhanced the viability of OGD/R-injured bEnd.3 cells, with 10 μM being the most effective concentration. Therefore, for subsequent experiments, we established low- and high-dose treatment groups with concentrations of 5 and 10 μM, respectively. To further assess the temporal effects of QCT on cell viability, we analyzed endothelial cell proliferation at different time points (0–24 h) using a CCK-8 assay. As shown in Supplementary Fig. [74]1B, no cytotoxic effects were observed at the 10 μM concentration over this time frame, indicating that QCT maintains cell viability during prolonged exposure. TUNEL staining was performed to assess the extent of apoptosis in endothelial cells among various experimental groups. As illustrated in Fig. [75]1B and D, a significant increase in the number of TUNEL-positive endothelial cells was observed in the OGD/R group, indicating increased apoptosis, whereas QCT treatment markedly reversed this effect. Additionally, as presented in Fig. [76]1C and E, revealed that QCT-treated OGD/R-bEnd.3 cells exhibited a notably stronger proliferative capacity than the untreated OGD/R group. Collectively, these findings suggest that QCT confers protective effects on endothelial cells following OGD/R injury by promoting cell proliferation and reducing apoptosis, thereby supporting its potential role in angiogenesis and vascular repair. Fig. 1. [77]Fig. 1 [78]Open in a new tab QCT promotes endothelial cell proliferation and reduces apoptosis following OGD/R-induced injury. A Schematic diagram of the in vitro experimental protocol. bEnd.3 cells were subjected to OGD/R followed by treatment with QCT at various concentrations. After 12 h, multiple assays, including TUNEL staining, EdU assay, and others, were performed to assess cell proliferation, apoptosis, and other functional properties. B TUNEL staining showing apoptosis in bEnd.3 cells following treatment with OGD/R and QCT. Scale bar: 50 μm. C Representative EdU incorporation assay. Scale bar: 50 μm. D Quantification of TUNEL-positive cells after drug treatment. n = 6. ****p < 0.0001, versus the control group; ##p < 0.01 and ###p < 0.001, versus the OGD/R group. E Quantification of EdU-positive cells following drug treatment. n = 6. ****p < 0.0001, versus the control group; ##p < 0.01 and ###p < 0.001, versus the OGD/R group Quercetin enhances the angiogenic activity of bEnd.3 cells following OGD/R-induced injury Angiogenesis is a critical process in SCI recovery as it facilitates tissue repair by restoring blood supply and promoting neurovascular regeneration [[79]25]. To assess the potential of QCT to promote angiogenesis in SCI, we conducted an in vitro analysis of its effects on endothelial cells. Wound healing and Transwell migration assays were performed to assess the migratory ability of the cells. Our findings demonstrated that QCT significantly accelerated wound closure and increased the number of migrating endothelial cells in comparison to those in the OGD/R group (Fig. [80]2A–E). These results suggest that QCT enhances the migratory capacity of endothelial cells, a key feature of angiogenesis. Moreover, as depicted in Fig. [81]2C, bEnd.3 cells following OGD/R injury and treated with various concentrations of QCT exhibited an increased number of capillary-like structures. Quantitative analysis demonstrated that both the number of meshes and junctions in the QCT-treated groups were significantly higher than those in the OGD group (Fig. [82]2F, G). In summary, these results indicate that QCT not only accelerates endothelial cell migration but also enhances tube formation, both of which are vital steps in angiogenesis. This suggests QCT as a promising therapeutic agent for enhancing angiogenesis and facilitating recovery after SCI. Fig. 2. [83]Fig. 2 [84]Open in a new tab QCT promotes angiogenesis by enhancing endothelial cell migration and tube formation following OGD/R. A Representative images from the wound healing assay at 0 and 12 h. Scale bar: 500 μm. B Representative images of the Transwell migration assay in different groups. Scale bar: 100 μm. C Representative images of the tube formation assay in different groups. Scale bar: 100 μm. D Quantification of migration rate from the wound healing assay. n = 6. ****p < 0.0001, versus the control group; ##p < 0.01 and ###p < 0.001, versus the OGD/R group. E Quantification of migration rate from the Transwell assay. n = 6. ***p < 0.001, versus the control group; ##p < 0.01 and ###p < 0.001, versus the OGD/R group. F and G Quantification of the number of meshes (F) and junctions (G) from the tube formation assay. n = 6. ***p < 0.001, versus the control group; ##p < 0.01 and ####p < 0.0001, versus the OGD/R group Quercetin enhances the recovery of tight junctions following endothelial cell injury The integrity of the spinal cord microvasculature is crucial for preserving BSCB function, and tight-junction proteins play a fundamental role in safeguarding this barrier [[85]26]. Hypoxic injury compromises the expression of these essential proteins in endothelial cells, leading to structural disruption and increased BSCB permeability [[86]27]. Given the critical role of tight junctions in maintaining BSCB integrity, examining the effects of QCT on intercellular junctions offers important insights into its protective mechanisms. As shown in Fig. [87]3A, B, QCT significantly upregulated the expression of the tight junction protein ZO-1 in endothelial cells subjected to OGD/R-induced injury. Similarly, WB results showed that QCT significantly upregulated the expression of two other tight junction proteins, Claudin-5 and Occludin, in endothelial cells with OGD/R-induced injury (Fig. [88]3C–E). These results suggest that QCT aids in the restoration of endothelial barrier function, thereby reinforcing vascular integrity and preventing further damage. This in vitro evidence underscores QCT's potential to enhance the structure and function of the BSCB, making it a promising candidate for therapeutic strategies aimed at improving recovery outcomes following SCI. Fig. 3. [89]Fig. 3 [90]Open in a new tab QCT restores ZO-1 expression in endothelial cells following OGD/R-induced injury. A Representative immunofluorescence staining of the tight junction protein ZO-1 (red) in endothelial cells in different groups. Nuclei are counterstained with DAPI (blue). OGD/R significantly reduces ZO-1 expression, while QCT treatment notably enhances ZO-1 levels in a dose-dependent manner following injury. Scale bar: 25 μm. B Quantification of the relative immunofluorescence intensity of ZO-1 in each group (n = 6). ***p < 0.001, versus the control group; ##p < 0.01 and ###p < 0.001, versus the OGD/R group. C Representative western blot images showing Occludin and Claudin-5 in each group. D Quantification of Occludin relative expression as percentages of control. n = 3. ***p < 0.001 versus the control group; #p < 0.05 and ####p < 0.0001 versus the OGD/R group. E Quantification of Claudin-5 relative expression as percentages of control. n = 3. ***p < 0.001 versus the control group; ##p < 0.01 versus the OGD/R group Quercetin promotes functional recovery and reduces the extent of injury following SCI We explored the effects of QCT on functional recovery, vascular regeneration, and BSCB integrity in vivo by administering intraperitoneal QCT injections following SCI. Based on prior literature and preclinical studies, the therapeutic concentration gradients of QCT were set to 20 and 50 mg/kg/day, for animal models [[91]28, [92]29]. Relevant experiments were conducted at specific time intervals to assess the outcomes (Fig. [93]4A). To further assess the impact of QCT on tissue repair and functional recovery post-SCI, we performed behavioral tests and histological analyses. The BBB scores revealed that QCT significantly enhanced hind limb locomotor function over time compared with the control group (Fig. [94]4B). Furthermore, electrophysiological tests indicated that QCT-treated rats exhibited a markedly higher MEP amplitude 28 days post-SCI, suggesting improved neural conductivity (Fig. [95]4C, D). Moreover, histological examination via H&E staining revealed a marked reduction in the injured area in rats treated with QCT (Fig. [96]4E, F), indicating enhanced tissue preservation. Collectively, these findings suggest that QCT significantly accelerates tissue repair, improves motor function, and mitigates damage following SCI, highlighting its therapeutic potential in spinal cord regeneration. Fig. 4. [97]Fig. 4 [98]Open in a new tab QCT promotes functional recovery and tissue repair following SCI. A Schematic of the experimental timeline in SCI rats treated with QCT. The treatments and assessments included BBB scores, electrophysiology, histological analysis, and BSCB integrity evaluation at different time points. B BBB scores of rats in each group at 28 days post-injury. n = 6. ***p < 0.001, versus the SCI group; ##p < 0.01 and ###p < 0.001, versus the untreated SCI group. C Representative waveform of MEPs at 28 days post-injury. D Quantification of MEPs amplitudes. n = 6. ****p < 0.0001, versus the sham group; #p < 0.05 and ##p < 0.01, versus the untreated SCI group. E H&E staining of spinal cord sections from each group at 28 days post-SCI. Scale bar: 500 μm. F Quantitative analysis of the lesion area. n = 6 per group. ###p < 0.001 and ####p < 0.0001, versus the untreated SCI group Quercetin promotes vascular regeneration and protects the structure of BSCB following SCI Primary mechanical trauma caused by SCI disrupts local capillaries and the BSCB, leading to ischemic changes in spinal cord tissue [[99]30]. Thus, promoting angiogenesis and maintaining BSCB integrity are key strategies for SCI intervention [[100]31]. Immunofluorescence analysis revealed that QCT treatment resulted in a notable reduction the proportion of TUNEL-positive cells at the site of injury 7 days post-SCI compared to the SCI group (Fig. [101]5A, B), indicating that it can significantly reduce apoptosis in spinal cord tissue after SCI. Complementary Evans blue extravasation assays revealed pronounced dye permeability in SCI cohorts compared to Sham group (Fig. [102]5C, D), quantitatively confirming BSCB disintegration. Notably, QCT administration significantly mitigated this vascular leakage phenomenon, suggesting potent barrier restorative properties through modulation of endothelial permeability. Molecular investigations further elucidated that QCT treatment effectively counterregulated the injury-induced downregulation of critical tight junction proteins—Occludin and Claudin-5 (Fig. [103]5E–H). This molecular evidence substantiates the mechanistic basis for QCT-mediated BSCB stabilization via endothelial junctional complex reinforcement. Concomitant analysis of CD31 expression patterns demonstrated significant vascular network disruption post-SCI, which was substantially ameliorated by QCT intervention (Fig. [104]5E, F). Notably, dual immunofluorescence mapping of KI67 (proliferation marker) and CD31 revealed enhanced co-localization indices in SCI group versus sham group (F[105]5g. [106]5I, [107]5), indicating endogenous compensatory neovascularization attempts in response to ischemic pathophysiology. QCT treatment potentiated this reparative angiogenesis, as evidenced by amplified KI67+/CD31+ cellular populations, suggesting pharmacologically enhanced angiogenesis capacity. Together, these findings suggest that QCT contributes to functional recovery after SCI, and the mechanism may involve vascular regeneration and stabilization of BSCB integrity to restore local blood flow. Fig. 5. [108]Fig. 5 [109]Open in a new tab QCT accelerates vascular regeneration and maintains BSCB integrity following SCI. A Representative TUNEL staining images in each group. Scale bar: 50 μm. B Quantification of TUNEL-positive cells as shown in A. n = 6. #p < 0.05 and ##p < 0.01, versus the SCI group. C Representative images of EB leakage at 7 days post-SCI in each group. Scale bar: 2.5 mm. D Quantitative analysis of EB content in the spinal cord tissue. n = 3. **p < 0.01, versus the SCI group. E Representative western blot images showing CD31, Occludin and Claudin-5 in each group. F–H Quantification of CD31, Occludin and Claudin-5 relative expression as percentages of control. n = 3. ***p < 0.001 and ****p < 0.0001 versus the control group; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 versus the SCI group. I Representative immunofluorescence images showing Ki67 (red) and CD31 (green) expression in the spinal cord at 7 days post-SCI. Scale bar: 50 μm. J Quantification of Ki67^+/CD31^+ double-positive cells in different groups. n = 6. ***p < 0.001, versus the sham group; ##p < 0.01 and ###p < 0.001, versus the SCI group Network pharmacology analysis of quercetin in SCI To explore the pharmacological mechanisms of QCT in the treatment of SCI, predicted molecular targets were analyzed. QCT-related targets were screened using the TCMSP, PharmMapper, and SwissTarget databases. After eliminating duplicates, 293 QCT-associated targets were identified. Targets related to SCI and potential therapeutic targets were retrieved from the GeneCards, OMIM, and TTD databases, yielding 1,005 targets after screening, aggregation, and removal of duplicates. A total of 76 common targets were identified, which could serve as potential therapeutic candidates for QCT treatment of SCI (Fig. [110]6A). Next, the Cytoscape software was employed to develop a drug-target network that linked the active components of QCT to potential therapeutic targets relevant to SCI treatment (Fig. [111]6B). To elucidate the relationships among these potential target genes, a PPI network was constructed for the 76 potential therapeutic targets (Fig. [112]6C). The centrality of nodes within the network indicates the relative importance of each target. Based on the degree value, closeness centrality, and betweenness centrality, the top 10 key targets were identified as HSP90AA1, EGFR, AKT1, SRC, MMP9, PIK3R1, ESR1, ALB, MAPK8, and CASP3. These targets likely play essential roles in the therapeutic effects of QCT on SCI. GO and KEGG pathway enrichment analyses were conducted to examine the possible biological functions and associated pathways of these therapeutic targets. To elucidate the biological activities and mechanisms of QCT in the treatment of SCI, the DAVID database was employed to conduct GO and KEGG pathway enrichment analyses for the 76 identified targets, and the resulting data were subsequently visualized. (Fig. [113]6D). In GO analysis (Fig. [114]6E), biological process (BP) enrichment indicated that QCT negatively regulated apoptosis and positively influenced cell migration following SCI, consistent with our previous in vitro and in vivo experimental findings. Additionally, BP analysis suggested that QCT promoted protein autophosphorylation and signal transduction. Previous studies have demonstrated that the PI3K/Akt signaling pathway plays a crucial role in initiating various fundamental cellular responses involved in angiogenesis, such as cell survival, migration, and tube formation [[115]32–[116]34]. Based on the KEGG pathway enrichment results, we hypothesized that QCT facilitates SCI repair by promoting neovascularization and maintaining BSCB integrity via the PI3K/Akt signaling pathway. Fig. 6. [117]Fig. 6 [118]Open in a new tab Identification and enrichment analysis of QCT regulatory targets in SCI. A Venn diagram of the targets of QCT for the treatment of SCI. The diagram displays 293 drug-related targets on the left, 1005 disease-related targets on the right, and 76 lapping targets in the center. B The interaction network of the overlapping targets. C PPI network of overlapped targets between QCT and SCI. The size of the nodes indicates their degree values, with the core target located in the inner circle. D KEGG pathway enrichment analysis of overlapping candidate targets involved in QCT-mediated SCI treatment. E Top 10 GO functional categories in molecular function, cellular component, and biological process Quercetin exerts pro-angiogenic effects and protects BSCB structure through a PI3K/Akt-dependent mechanism We evaluated the role of the PI3K/Akt signaling pathway in treating SCI by examining the expression levels of PI3K, p-PI3K, Akt, and p-Akt proteins in QCT-treated endothelial cells (Fig. [119]7A–C) and rat spinal cord tissue (Fig. [120]7M–O). To further validate the involvement of QCT in spinal cord injury recovery, the PI3K/Akt pathway was inhibited using the PI3K inhibitor LY294002. Western blot analysis showed a significant decrease in the ratios of p-PI3K/PI3K and p-Akt/Akt after injury compared to the control group. In contrast, QCT treatment significantly increased the ratios of p-PI3K/PI3K and p-Akt/Akt, an effect that was markedly reduced when LY294002 was applied. These findings collectively indicate that QCT activates the PI3K/Akt signaling pathway in both OGD/R-injured endothelial cells and spinal cord-injured rats, corroborating the results from network pharmacological analysis. Next, we examined the angiogenesis-related ability of endothelial cells in the PI3K pathway inhibition group and found that following the administration of LY294002, which inhibits the PI3K signaling pathway, the migration (Fig. [121]7D, E, H, I) and tube formation (Fig. [122]7F, J, K) abilities of endothelial cells were significantly diminished compared to those treated with the same dose of QCT. The immunofluorescence results indicated a marked reduction in the expression of the tight junction protein ZO-1 (Fig. [123]7G, L). In addition, EB dye penetration in the spinal cord tissues of the QCT-treated combined blocker rats was markedly higher than that in the same-dose-treated group, indicating increased permeability of the BSCB (Fig. [124]7P, R). In addition, immunofluorescence showed a significant reduction of Ki67^+/CD31^+- positive areas in the QCT-treated combined blocker group, suggesting that the ability of angiogenesis in the injured localities of SCI rats was significantly reduced after inhibition of the PI3K signaling pathway. (Fig. [125]7Q, S). These findings suggest that LY294002 inhibits the stimulation of the PI3K/Akt signaling pathway by QCT, thereby affecting the ability of QCT to enhance angiogenesis and decrease BSCB permeability. Collectively, these findings indicated that QCT enhanced angiogenesis and decreased BSCB permeability following SCI by stimulating the PI3K/Akt signaling pathway. Fig. 7. [126]Fig. 7 [127]Open in a new tab Quercetin enhances angiogenesis and protects BSCB structure through activation of the PI3K/Akt signaling pathway. A Representative western blot images showing PI3K, p-PI3K, Akt, and p-Akt expression in each group. B and C Quantification of p-PI3K/PI3K (B) and p-Akt/Akt (C) as percentages of control. n = 6. ***p < 0.001 and ****p < 0.0001 versus the control group; #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 versus the OGD/R group. D Representative images of wound healing assay in each group. Scale bar: 500 μm. E Representative images of Transwell test in each group. Scale bar: 100 μm. H Quantification of migration rate as shown in (D). n = 6. ***p < 0.001 versus the OGD/R+QCT group. I Quantification of vertical migration rate in percentage of control as shown in (E). n = 6. ***p < 0.001 versus the OGD/R+QCT group. F Representative tube formation images for each group. Scale bar: 100 μm. J–K Quantitative analysis of the number of meshes (J) and junctions (K) in tube formation. n = 6. ***p < 0.001 and ****p < 0.0001 versus OGD/R+QCT group. G Representative immunofluorescence images showing ZO-1 expression (red) in each group. Scale bar: 25 μm. L Quantification of ZO-1 relative density in each group. n = 6. ****p < 0.0001 versus the OGD/R+QCT group. M Representative western blot images of PI3K, p-PI3K, Akt, and p-Akt expression in spinal cord tissues post-SCI with QCT and LY294002 treatments. N–O Quantification of p-PI3K/PI3K (N) and p-Akt/Akt (O) in SCI model. n = 6. ****p < 0.0001 versus the Sham group; ##p < 0.01, ###p < 0.001, and ####p < 0.0001 versus the SCI group. P Representative images of EB staining showing blood-spinal cord barrier permeability in each group. Scale bar: 2.5 mm. Q Representative immunofluorescence images of CD31 (green) and Ki67 (red) expression in each group. Scale bar: 50 μm. R Quantification of EB content in spinal cord tissues. n = 3. **p < 0.01 versus the SCI+QCT-H group. S Quantification of Ki67^+/CD31^+ double-positive cells in each group. n = 6. ****p < 0.0001 versus the SCI+QCT-H group Molecular docking and dynamics simulation reveal the stable binding of quercetin with key target proteins To gain deeper insight into how QCT interacts with its target proteins, Fig. [128]8A, B illustrates the secondary and tertiary structures of QCT, respectively. The secondary structure highlights the arrangement of the QCT chemical bonds, whereas the tertiary structure reveals three-dimensional folding. This 3D conformation is crucial for understanding how QCT fits within the binding sites of potential protein targets, thereby facilitating interaction and binding affinity. Molecular docking was used to evaluate the binding interactions between small-molecule drugs and their potential targets after conducting structural analysis. A negative binding energy indicates successful ligand-receptor binding, with lower values reflecting stronger binding affinity. As shown in Fig. [129]8C–L, the molecular docking analysis revealed the interactions between QCT and the top 10 core proteins. The results revealed that EGFR, HSP90AA1, AKT1, SRC, MMP9, ESR1, ALB, PIK3R1, MAPK8, and IGF1 exhibited favorable binding affinities for QCT. The binding energies of QCT to these target proteins are summarized in Table [130]2, all of which are below − 5 kcal/mol, signifying robust binding interactions. The binding affinity of the PIK3R1-QCT complex, which demonstrated strong docking results, was further validated through molecular dynamics simulations. Fig. 8. [131]Fig. 8 [132]Open in a new tab Molecular docking and molecular dynamics simulations. A and B Two-dimensional and three-dimensional structures of QCT. B–L Visual analysis of molecular docking. M RMSD, N RMSF, O RG, and F SASA analysis plots for QCT and PI3KR1 Table 2. Binding energies of quercetin and each key target Key target PDBID Binding energy(kcal/mol) EGFR 3POZ − 7.1 HSP90AA1 3O0I − 6.16 Akt1 1UNQ − 5.09 SCR 1FMK − 6.64 MMP9 4XCT − 8.18 ESR1 1XP6 − 5.29 ALB 1BJ5 − 6.03 PI3KR1 6D81 − 5.36 MAPK8 4AWI − 5.32 IGF1 1IMX − 5.08 [133]Open in a new tab The stability of the simulated system was evaluated through RMSD analysis. As shown in Fig. [134]8M, the PIK3R1-QCT complex stabilized at approximately 17 ns with a fluctuation range of only 0.05 nm throughout the simulation, indicating a significant level of stability and equilibrium in the binding mode. RMSF analysis revealed reduced flexibility of the PIK3R1-QCT complex compared with that of the PIK3R1 protein alone (Fig. [135]8N). Apart from the terminal regions, the RMSF values for the remaining sequence segments of the complex were relatively low, suggesting that the amino acid residues were stable. The overall structural flexibility was minimal, with fluctuations within 0.1 nm, indicating that the interactions within the binding pocket remained stable. RG was employed to evaluate the compactness of the protein structure during simulation (Fig. [136]8O). The RG values remain relatively stable, reflecting a good degree of compactness. SASA analysis revealed the exposure of the protein–ligand complex to the solvent during the simulation (Fig. [137]8P). The two curves remained within close proximity and were stable throughout the simulation, indicating that QCT-PIK3R1 binding maintained good stability with consistent solvent interactions during the simulation process. Discussion This study investigated the therapeutic efficacy of QCT in enhancing angiogenesis and preserving BSCB in a rat model of SCI, elucidating the molecular mechanisms through which QCT exerts its effects via the activation of the PI3K/Akt signaling pathway. The findings demonstrated that QCT enhanced the survival, migration, and tube formation capacity of bEnd.3 endothelial cells after OGD/R injury in vitro and facilitated the recovery of motor function, stimulated vascular regeneration, and maintained the integrity of the BSCB in vivo. Primary mechanical damage from SCI leads to rupture of the vascular system in the spinal cord tissue, causing an imbalance between hemorrhage and ischemia. This imbalance results in pathological changes, including inflammatory infiltration and neural tissue edema [[138]35]. A crucial aspect of post-SCI recovery is the re-establishment of functional microvasculature to support tissue repair and mitigate secondary injury [[139]36]. Angiogenesis plays a pivotal role in this process, ensuring an adequate supply of oxygen and nutrients to the injured area, which is necessary for neural tissue regeneration and alleviation of ischemia-induced damage. This study confirmed the angiogenic properties of QCT in SCI models both in vitro and in vivo. These findings align with earlier research that has illustrated the proangiogenic effects of QCT in various pathological contexts [[140]13]. QCT stimulates the PI3K/Akt signaling pathway in human umbilical vein endothelial cells (HUVECs), thereby enhancing endothelial cell migration and tube formation [[141]37]. The BSCB, similar to the blood–brain barrier, is a specialized barrier system that protects the spinal cord by preventing macromolecules and harmful substances from entering the spinal cord parenchyma and maintaining the balance of nutrients and metabolic substances required by the spinal cord tissues [[142]38]. The microvasculature of the spinal cord is a key component. Endothelial cells within the spinal microvasculature form the BSCB through tight junctions that restrict nonselective substance transport, allowing only specific molecules to cross [[143]39]. Following SCI, the integrity of the BSCB is compromised, leading to increased permeability, which allows inflammatory cells and harmful substances to infiltrate the spinal cord parenchyma, exacerbating the secondary injury [[144]40]. BSCB permeability increases in two phases following SCI: an initial peak shortly after injury and a second peak 3–7 days later [[145]41]. Owing to the short duration of treatment in the early stages of SCI, no significant recovery in motor and neurological functions was observed. Therefore, we assessed the BSCB structure at the second permeability peak 7 days post-injury, using an EB leakage assay and immunofluorescence staining. QCT treatment preserved the BSCB integrity by reducing its permeability and preventing the infiltration of inflammatory cells and harmful substances. Additionally, QCT inhibits cytokines that promote inflammation, namely interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α), which are known to disrupt endothelial tight junctions and increase vascular permeability [[146]42]. Our results corroborate these reports, as QCT treatment not only reduced BSCB permeability but also attenuated inflammation at the injury site [[147]43]. Future research should further explore the relationship between BSCB integrity and the local spinal cord microenvironment, as well as the connection between BSCB structure and inflammatory infiltration following nerve injury. One of the major highlights of this study was the systematic elucidation of the multi-target and multi-pathway mechanisms of QCT in treating SCI through the application of network pharmacology. Compared to previous work by Shen et al. [[148]44], this study identifies potential targets associated with SCI by integrating a broader and more accurate range of databases (TCMSP, Swiss Target Prediction, PharmMapper, GeneCards, OMIM, TTD) for predicting QCT targets and performing intersection-based screening. This multidimensional network pharmacology approach offers a more comprehensive validation of targets than traditional investigations that rely on a single database, thereby enhancing the reliability of the results. A total of 76 overlapping targets were identified, and the drug-target network and PPI analysis revealed key targets, including HSP90AA1, EGFR, and AKT1, which play critical roles in regulating apoptosis, inflammation, and tissue repair. GO analysis indicated that QCT exerts its effects mainly by inhibiting apoptosis, promoting cell migration, and modulating biological processes, such as signal transduction and phosphorylation. Notably, the PI3K/Akt signaling is the central pathway for SCI repair and regeneration and a key mediator of QCT’s action [[149]45]. Molecular docking and dynamic simulations further validated the strong binding affinity of QCT for its key targets, particularly PIK3R1. These simulations demonstrated the stability of the QCT-PIK3R1 complex, highlighting the strong interactions between QCT and its molecular targets. This suggests that QCT’s efficacy in treating SCI is mediated through stable high-affinity interactions with multiple key proteins, thereby supporting its role in regulating critical cellular pathways involved in SCI repair. These findings broaden our insight into the therapeutic potential of QCT and emphasize the value of network pharmacology in exploring the mechanisms of multitarget drugs, such as QCT, in treating complex diseases such as SCI. The significance of the PI3K/Akt signaling pathway in the process of angiogenesis is well-documented. Stimulation of this pathway upregulates vascular endothelial growth factor (VEGF) and other angiogenic factors, thereby fostering endothelial cell proliferation, migration, and survival [[150]32, [151]46]. Using network pharmacology, a reliable bioinformatics tool, we identified the PI3K/Akt pathway as a key signaling pathway activated by QCT in the process of SCI repair. Our study demonstrates that QCT activates the PI3K/Akt pathway in both endothelial cells and spinal cord tissues. This contrasts with previous work indicating that QCT inhibits the PI3K/Akt pathway in SCI [[152]44]. One possible explanation for this discrepancy is the difference in experimental time points. Previous studies primarily focused on the acute phase of injury, during which PI3K/Akt activation is largely associated with inflammatory cascades and cytoprotective mechanisms. At this critical stage, QCT may exert its therapeutic effects by attenuating pathological hyperproliferation, excessive inflammation, and fibrotic scarring through selective inhibition of the PI3K/Akt signaling axis. Supplementary Fig. [153]2 demonstrates the direct experimental evidence supporting this interpretation. At the acute phase (3 days post-SCI), QCT significantly suppresses PI3K/Akt hyperactivation, thereby reducing inflammatory responses and aberrant proliferation, consistent with previous reports on PI3/Akt inhibition. In our study, however, protein pathway analysis was performed four weeks after QCT treatment, a later stage in which PI3K/Akt activation plays a critical role in neuronal survival and tissue repair. PI3K/Akt signaling facilitates repair during the chronic phase, while it may suppress unwanted cell proliferation and inflammation during the acute phase. Thus, the time-dependent dual role of PI3K/Akt signaling is likely a key factor in explaining the contrasting effects observed in our study. Furthermore, treatment with the PI3K inhibitor LY294002 confirmed that inhibition of this pathway diminished the protective effects of QCT, resulting in reduced endothelial cell migration, impaired tube formation, and compromised BSCB integrity. These findings highlight the crucial role of PI3K/Akt signaling in QCT-induced vascular regeneration, underscoring its significance in the therapeutic mechanisms of QCT. Moreover, these findings align with earlier studies that have shown the role of PI3K/Akt signaling in promoting angiogenesis after ischemic events [[154]47]. For instance, activating the PI3K/Akt pathway in an ischemic stroke model increased angiogenesis and improved recovery outcomes [[155]48]. The involvement of the PI3K/Akt pathway in the maintenance of BSCB integrity highlights its therapeutic potential. Activation of this pathway has been shown to upregulate tight junction proteins, such as claudin-5 and occludin, essential for preserving BSCB integrity [[156]49]. Enhancing PI3K/Akt signaling, QCT may stabilize tight junctions and prevent BSCB disruption and secondary injury. We used PI3K/Akt pathway-specific inhibitors to validate their function in QCT-mediated vascular repair. Further supporting the involvement of this pathway in SCI recovery, inhibition of PI3K/Akt signaling was found to diminish the protective effects of QCT on endothelial cells and BSCB integrity. These findings highlight the potential of modulating the PI3K/Akt signaling pathway as an effective strategy for enhancing vascular regeneration after SCI. The therapeutic implications of these findings are substantial, particularly given the lack of effective treatments for SCI. The ability of QCT to promote angiogenesis while simultaneously protecting the BSCB offers a novel therapeutic strategy for SCI by effectively targeting both the vascular and neural components of the injury. The overall mechanism by which QCT promotes angiogenesis and protects BSCB integrity following SCI is summarized in Fig. [157]9. Fig. 9. [158]Fig. 9 [159]Open in a new tab Mechanisms by which QCT promotes angiogenesis and reduces BSCB permeability following SCI Although this study provides robust evidence for QCT’s angiogenic and barrier-protective efficacy, its restriction to female rats warrants caution when extrapolating outcomes across sexes. Researchers should conduct future investigations to systematically evaluate sex as a biological variable, particularly given the established neuromodulatory role of estrogen in SCI. Comparative analyses across species will further enhance translational relevance. This study demonstrates QCT’s efficacy in promoting angiogenesis and BSCB integrity via PI3K/Akt signaling. However, we acknowledge that inclusion of a methylprednisolone-treated control group would provide direct benchmarking against current clinical standards. Prior experimental data [[160]50] indicate that QCT and methylprednisolone exert comparable neuroprotective and antioxidant effects in acute SCI models, with no significant difference in reducing oxidative stress or histopathological damage. These outcome equivalencies directed our focus on QCT’s relatively unexplored role in vascular repair. Future studies directly comparing QCT with methylprednisolone will be instrumental for clinical translation. Although our findings validate QCT's therapeutic potential, its clinical translation is limited by intrinsic pharmacokinetic challenges, including poor aqueous solubility and restricted systemic bioavailability. To overcome these limitations, advanced nano delivery strategies offer promising solutions. Lipid-based systems encapsulate hydrophobic QCT within amphiphilic matrices to enhance dissolution and prolong circulation. Polymeric nanoparticles, such as chitosan-based or PLGA vectors, improve intestinal absorption and reduce first-pass metabolism via mucoadhesive and controlled-release properties [[161]51]. Cyclodextrin inclusion complexes and metal–organic frameworks utilize host–guest interactions to stabilize QCT against enzymatic degradation while enabling pH-responsive release at target sites [[162]52]. To further improve QCT's bioavailability, we aim to develop next-generation nanocarriers engineered for intelligent, spatiotemporal control. Innovations in ligand-targeted delivery and stimuli-responsive platforms will enhance therapeutic precision while minimizing systemic exposure. Future work will focus on multifunctional nanocomposites capable of co-delivering synergistic agents via biodegradable vectors, ultimately bridging the mechanistic promise of QCT toward clinical realization in neural repair. Conclusion This study demonstrated that QCT enhances angiogenesis and preserves BSCB structure in a rat model of SCI through the activation of the PI3K/Akt signaling pathway. These findings indicated that QCT has the potential to serve as a promising therapeutic agent for SCI, providing advantages in neuroprotection and angiogenesis. Supplementary Information Below is the link to the electronic supplementary material. [163]Supplementary Material 1.^ (1.7MB, docx) Acknowledgements