The Combination of Sodium Tanshinone IIA Sulfonate and Mesenchymal Stem Cell-derived Exosomes Against Spinal Cord Injury via VEGF Pathway

Abstract

Spinal cord injury (SCI), a prevalent cause of severe disability, primarily stems from trauma, degenerative pathologies, or underlying diseases. Spinal cord injury presents significant therapeutic challenges. This study investigated the potential synergistic effect of combining Sodium Tanshinone IIA Sulfonate (STS) and bone mesenchymal stem cell-derived exosomes (BMSC-Exos) for SCI treatment, focusing on the vascular endothelial growth factor (VEGF) pathway.The STS-Exo combination significantly protected Mouse Brain Microvascular Endothelial Cells (bEnd.3) against H₂O₂-induced cytotoxicity, as shown by CCK-8. This synergistic treatment robustly enhanced angiogenic capacity measured through tube formation analysis and accelerated endothelial cell migration in scratch wound healing assays, outperforming individual monotherapies. In SCI rats, combined therapy promoted functional recovery, evidenced by elevated BBB locomotor scores and footprint analysis. Histopathological assessment via HE staining revealed attenuated tissue damage, while Evans Blue extravasation tests confirmed restoration of blood-spinal cord barrier(BSCB) integrity. Furthermore, immunofluorescence quantification showed marked reduction in glial scar formation. Mechanistically, WB and IF analyses confirmed that the STS-Exo combination potently activated the VEGF signaling pathway. This combination strategy represents a promising therapeutic approach for SCI.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12035-025-05422-2.

Background

Spinal cord injury (SCI) is a leading cause of severe disability, often resulting from trauma, disease, or degenerative conditions. SCI not only causes irreversible damage to the spinal cord structure, but also triggers a series of complex pathophysiological processes, including inflammation, cell apoptosis, ischemia, and disruption of the BSCB [1–3]. BSCB plays a crucial role in maintaining the stability of the spinal cord microenvironment, and its dysfunction increases neural damage and reduces regenerative potential [4, 5]. Therefore, promoting BSCB repair is essential for nerve regeneration and functional recovery after SCI.

In recent years, bone marrow mesenchymal stem cells (BMSCs) and their derived exosomes (BMSC-Exos) have gained attention for their promising regenerative and reparative properties [6–8]. BMSC-Exos are rich in various bioactive molecules, such as miRNA, proteins, and lipids, which can regulate inflammation, promote cell survival, and enhance regeneration through multiple mechanisms, showing potential for SCI treatment treatment [9, 10] However, a significant challenge remains in the efficient delivery of exosomes to specific tissue sites to reduce therapeutic efficacy. Adapting BMSC-Exos to the damaged microenvironment may be the key to improving SCI treatment.

The sodium tanshinone IIA sulfonate (STS), a water-soluble compound from the traditional Chinese medicinal herb Salvia miltiorrhiza, has drawn attention for its anti-inflammatory, antioxidant, and neuroprotective effects [11–13]. Studies have shown that STS reduces inflammation and oxidative stress after SCI, promotes nerve growth factor release, and contributes to nerve regeneration [14, 15]. Notably, STS shows potential for BSCB repair by affecting endothelial cell function and enhancing angiogenesis [16, 17]. The VEGF pathway is plays an important role in angiogenesis, endothelial cell proliferation, and survival, and plays a critical regulatory role in nerve regeneration [18–20]. Studies suggested that activating the VEGF pathway improves SCI repair, and STS enhances VEGF and VEGFR2 protein expression in brain tissues after ischemia/reperfusion. However, it remains unclear whether STS can improve the microenvironment after SCI to enhance the function of exosomes by activating VEGF pathway?

In this study, we aimed to systematically evaluate the effects of STS combined with BMSC-Exos on BSCB permeability, inflammation, nerve regeneration, and motor function, and explore the potential role of the VEGF pathway in these processes. Our research will provide new research ideas and directions for the treatment of spinal cord injuries.

Materials and Methods

Materials

SD Rat-derived Bone marrow mesenchymal stem cells (BMSCs), mouse brain microvascular endothelial cell line (bEnd.3), DMEM basic medium (Bios-128–0001), and fetal bovine serum (FBS) (UBS297) were obtained from Guangzhou Xinyuan Technology Co., Ltd. Exosome-depleted FBS was sourced from ViVaCell (cat. C38010050, Shanghai, China). The Total Exosome Isolation Kit (from cell culture media) was obtained from Invitrogen™ (cat. 4,478,359, USA). Matrigel was purchased from BD (CA, USA). Additionally, α-MEM medium (cat. 8,123,668) and Trypsin–EDTA (cat. 25,300,054) were obtained from Gibco. STS (purity > 99% using HPLC) was purchased from Chengdu Herbpurify Co. Ltd. (Chengdu, China). The full-wavelength microplate spectrophotometer was acquired from Thermo Fisher Scientific Co., Ltd.

Isolation of Exosomes

BMSCs were cultured in α-MEM medium containing 10% exosome-depleted FBS. Exosome extraction was performed using an extracellular vesicle isolation reagent kit [21]. First, cell culture media were collected and centrifuged at 2000 × g for 30 min to remove cells and debris. The cell-free supernatant was transferred to a new tube, and 0.5 times the volume of total exosome isolation reagent was added. The mixture was thoroughly vortexed or pipetted up and down, then incubated overnight at 2–8℃ After incubation, the sample was centrifuged at 10,000 × g for 1 h at 2–8℃. The supernatant was discarded, and the particle-containing pellet was resuspended in PBS and stored at −80℃.

Characterization of Exosomes

The particle size and concentration of exosomes were examined using ZetaView PMX 110 (Meerbusch, Germany). Exosome morphology was observed and photographed under a Hitachi TEM (Tokyo, Japan). Before assessment of exosomal marker proteins CD63 (1:200,25,682–1-AP, Proteintech, USA), and TSG101(1:2000, ET1701-59, HUABIO, China) through Western blot (WB) assay [22],a BCA protein assay kit (P0010, Beyotime, China) was used to quantify the protein concentration and control the amount of exosomes loaded to 10 μg.

Cell Culture and Treatment

The bEnd.3 cells were cultured in DMEM (NaHCO3 1.5 g/L) basic medium (1 ×) containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. The bEnd.3 cells were placed in a 37℃ humidified cell incubator with 5% CO₂.

For cell treatment experiments, the safe concentrations of STS on bEnd.3 cells were first determined using a CCK-8 assay. Cells were seeded in a 96-well plate and treated with different concentrations of STS (1, 3, and 10 μM) and H₂O₂ (100, 200, 300, 400, and 500 μM). Cells were treated with H₂O₂ (300 μM) for 12, 24, and 48 h. After treatment, the supernatant was aspirated, and each well was washed twice with 1 × PBS. Complete culture medium (100 µL) and CCK-8 reagent (10 µL) were then added to each well, and cells were incubated at 37℃ for 2 h. Absorbance was measured at 450 nm using a full-wavelength microplate spectrophotometer. Based on cell viability, we determined the concentration of 300 µM H₂O₂ with a 24-h treatment duration and an STS concentration of 3 µM. Additionally, CCK-8 assays were conducted to detect the protective effects of Exo, STS, and Exo combined with STS on endothelial cell activity. All experiments were performed with five replicates.

Tube Formation

The bEnd.3 cells were seeded onto matrigel to assess tube formation [23]. A 50 μL aliquot of Matrigel was added to the center of each well in a 96-well plate and allowed to solidify at 37 °C for 30 min. Cells were then treated with H₂O₂ and incubated. After 24 h of treatment with STS, Exo, or STS combined with Exo, 1.5 × 10⁴ cells per well were plated for tube formation. The cells were incubated at 37 °C with 5% CO₂ for the designated period. Tube formation was captured using an inverted microscope (200 ×), and ImageJ software was used to quantify the number of junctions and total tube length.

Migration Analysis

The migration of bEnd.3 cells was assessed using Transwell insert chamber (BD, USA) with an 8 μm polycarbonate filter membrane [24]. After exposure to H₂O₂ and the cells were incubated with STS, Exo, or STS combined with Exo for 24 h. The cell density was adjusted to 5 × 10⁵ cells/mL in EGM-2 supplemented with 1% FBS. For the Transwell assay, 200 μL of the cell suspension was added to the upper chamber, while EGM-2 with 5% FBS was placed in the lower chamber. After 24 h of incubation, migrated cells were fixed in formaldehyde for 30 min and stained with crystal violet for 4 h. Non-migrated cells were removed from the upper chamber with a cotton swab. Images were captured using an optical microscope (100 ×), and the number of migrating cells was counted and averaged across five random fields.

Scratch Wound Healing Assay

The bEnd.3 cells were seeded into a 6-well plate at a density of 5 × 10⁵ cells/well. After exposure to H₂O₂ and 24-h incubation with STS, Exo, and STS combined with Exo, a uniform scratch was made in the cell layer using a sterile 200 μL pipette tip. The cells were then washed with PBS to remove debris and floating cells. Wound closure was further washed by PBS and then imaged at the same location at 0, 12, and 24 h using an inverted microscope [25].

Animals

SD male rats aged 8–10 weeks and weighing 180–200 g were obtained from the Guangdong Medical Experimental Animal Center. They were housed in a temperature-controlled environment with access to standard rodent chow and water. All procedures involving animals were approved by the Ethics Committee of Guangzhou University of Chinese Medicine and conducted in accordance with the guidelines of the Chinese National Institutes of Health (Certificate No. 20220803007, Guangzhou, China).

SCI Model and Treatment

Rats were anesthetized pentobarbital sodium, and aseptic procedures were used to incise the skin on the back and expose the spine. The T9-T10 segment was selected for laminectomy to fully expose the spinal cord. Using a SCI percussion device (RWD, CA, USA), an 8 g rod and 3 mm was dropped from a height of 6 cm onto the dorsal surface of the spinal cord to cause standardized SCI. Immediate post-operative assessment revealed localized edema and vascular congestion at the injury epicenter. Upon recovery from anesthesia, animals exhibited complete hindlimb motor paralysis, urinary retention, and loss of tail elevation reflex with characteristic hindlimb dragging. The Sham surgery group only exposed the spinal cord without inducing injury. After surgery, the muscles and skin were sutured in layers, and appropriate antibiotics were administered to prevent infection. After the establishment of the model, rats were given bladder massages once a day to assist with urination [26].

The experiment was divided into five groups in total, namely the Sham group, the SCI group, the STS group, the Exo group and the STS + Exo group. The STS group received an immediate postoperative intravenous injection of 200 μL PBS via the tail vein, followed by daily oral administration of 10 mg/kg STS (dissolved in normal saline) by gavage. In the Exo group, 200 μg of Exo (dissolved in 200 μL PBS) was administered via tail vein injection immediately after surgery, and an equivalent volume of normal saline was given by daily gavage. The STS + Exo group received both intragastric administration of STS and tail vein injection of Exo simultaneously, with dosages identical to those used in the respective STS and Exo groups. The Sham group and the SCI group were administered 200 μL PBS via tail vein injection immediately post-surgery and received daily gavage with the same volume of normal saline. All treatments were continued for a total duration of 7 or 21 days.

Functional Behavior Evaluation

To evaluate hind limb motor function, we used the Basso Beattie Bresnahan (BBB) scale [27]. Before the experiment, rats were allowed to move freely in an open area for 5 min. Two independent, well-trained experimenters observed the hind limb motor function of the rats, including joint movement, gait, coordination, and tail balance, and scored them on a scale ranging from 0 (complete paralysis) to 21 (normal movement). Each group of rats (n = 8) was evaluated before surgery and on days 1, 3, 7, 14, and 21 post-injury. After each evaluation, scores were recorded and analyzed to monitor motor function recovery trends. A footprint test was also conducted by immersing the hind limbs of each rat in black dye, encouraging the rats to walk along a narrow pathway, and recording their footprints. The footprints were scanned, and digital images were used to analyze their gait.

Evans Blue Assay

Evans blue dye extravasation was used to assess BSCB permeability [28]. Briefly, seven days post-spinal cord injury, rats received intraperitoneally injected of 200 μL of 2% Evans Blue dye (in sterile saline), 3 h post-injection, and performed cardiac perfusion using PBS and 4% paraformaldehyde (PFA). The spinal cords were removed, and photos were taken to observe the dye-stained spinal cord. Cross-sectional Sects. (30 μm) were cut using an ultra-thin semi-automatic micro knife (Leica RM2255, Germany) and observed under a microscope to measure fluorescence intensity.

HE and Nissl Staining

Fixed tissues were dehydrated, embedded, and sectioned. For HE staining, sections were deparaffinized, stained with hematoxylin for nuclei, differentiated, stained with eosin for cytoplasm, dehydrated, and sealed. For Nissl staining, after dewaxing and hydration, neuronal Nissl bodies were stained with toluidine blue), washed, dehydrated, and sealed [29]. Images were captured under a microscope.

Western Blot

The fresh 5 mm spinal cord segments centered on the injury epicenter was collected and homogenized, or proteins were extracted from cells after intervention. Protein concentration was determined using the bicinchoninic acid (BCA) assay (20 μg per lane). Equal amounts of protein samples were separated using SDS-PAGE, based on molecular weight, and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat dry milk for 1 h to prevent non-specific binding. Specific primary antibodies, including anti-β-catenin (1:1000, Abclonal, A20221), anti-occludin (1:1000, Abclonal, A2601), anti-VEGFA (1:1000, Abclonal, A12303), anti-VEGFR2 (1:1000, Affinity, AF6281), anti-MAP2 (1:1000, Boster, BM1243), anti-GFAP (1:1000, Boster, PB9082), anti-Arg1 (1:1000, Abclonal, A1847), anti-NOS2(1:1000, Boster, BA0362), and anti-GAPDH (1:1000, Cell Signaling Technology, 2118), were added and incubated overnight at 4 °C. HRP-conjugated rabbit/mouse secondary antibodies (1:1000, Abcam, ab205718, ab6728) were then applied. Protein bands were visualized using chemiluminescence. ImageJ software was used to calculate the grayscale values of the bands and then the resulting data were normalized for statistical analysis.

Immunofluorescent on Cells and Sections

For each experimental group, five rats spinal cords were taken and fixed in 4% PFA at room temperature for one week, after which the intact spinal cords were peeled off and later paraffin-embedded. The lesion site of each wax block was used as the starting site for sectioning, and ten consecutive 5 μm sections were made from each block. The sections were then numbered sequentially. During staining, sections with the same serial number in all blocks were selected for simultaneous immunofluorescence staining. Paraffin-embedded tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed to expose epitopes. For in vitro experiments, cells were fixed on coverslips with 4% PFA for 2 h post-intervention. The subsequent steps for cell coverslips were the same as for tissue sections: blocking with PBS containing 10% goat serum and 1% Triton X-100 to prevent non-specific antibody binding. Primary antibodies, including anti-occludin (1:200, Abclonal, A2601), anti-β-catenin (1:200, Abclonal, A20221), anti-GFAP (1:600, Boster, PB9082), anti-MAP2 (1:200, Boster, BM1243), anti-Iba1 (1:200,Huabio, RT1316), anti-Arg1 (1:200, Abclonal, A1847), anti-NOS2 (1:200,Boster, BA0362), anti-VEGFA (1:200, Abclonal, A12303) and anti-VEGFR2 (1:200, Affinity, AF6281) were applied and incubated overnight at 4 °C. Alexa Fluor®-conjugated secondary antibodies (1:300,ab150113, ab6939) were used to detect the primary antibodies. Finally, nuclei were stained with DAPI, and an anti-fade mounting medium was used before observation under a fluorescence microscope. Finally, the average fluorescence intensity within the region of interest was quantified using ImageJ software.

Statistical Analysis

Statistical analyses were performed using SPSS 29.0 and GraphPad Prism 9.0 software. Image quantification was conducted using ImageJ. Experimental measurements were derived from at least five independent biological replicates. Data conforming to a normal distribution are expressed as mean ± standard deviation (SD), while non-normally distributed data are presented as median and interquartile range (IQR). For comparisons among multiple independent groups, one-way analysis of variance (ANOVA) was applied to normally distributed data, followed by appropriate post-hoc tests for multiple comparisons. The Kruskal–Wallis H test was used for non-normally distributed data. A p-value < 0.05 was considered statistically significant.

Results

Identification of Exosomes Extracted from BMSC Culture Medium

After isolating the exosomes from the BMSCs culture medium, the results were consistent with expectations, confirming the presence and characteristics of the exosomes. Morphological features (Fig. 1A-C): Transmission electron microscopy revealed that the exosomes had a typical cup-shaped or disc-shaped morphology, consistent with characteristic exosome structures. Particle size distribution: Nanoparticle tracking analysis (NTA) showed that the particle size of the extracted exosomes was mainly concentrated between 60–150 nm, which conforms to the standard size range of exosomes. Characteristic protein expression (Fig. 1D): Western blot showed the expression of CD63, a marker protein for exosomes, further confirming that the extract is an extracellular vesicle. Moreover, the presence of intracellular proteins such as TSG101 supports the exosomal origin. In summary, these identification results indicated that the exosomes extracted from BMSCs culture medium possess the expected characteristics, with good purity and specificity, laying a solid foundation for subsequent research.

Fig. 1.

Identification of BMSCs-Exo and enhancement of bEnd.3 cells vitality after H₂O₂ damage by STS combined with Exo. A The morphology of extracellular vesicles was observed by transmission electron microscopy. B-C Size distribution of extracted extracellular vesicles. D Western blot showed that the expression of exosome marker proteins CD63 and TSG101 was detected in extracellular vesicles. E The structure of STS. F The bEnd.3 cells were treated with different concentrations (1, 3, and 10 µM) of STS for 24 h. G The bEnd.3 cells were treated with 300 µM H₂O₂ for 0, 12, 24, and 48 h. Cell viability was assayed by cck-8. H Treatment of bEnd.3 cells with different concentrations (100, 200, 300, 400, and 500 µM) of H₂O₂ for 24 h. I Effect of different concentrations of STS (1, 3, and 10 µM) on cell viability in H₂O₂-treated bEnd.3 cells. J 300 µM H₂O₂ was selected to induce cellular oxidative stress, and cell survival was detected using 3 µM STS and 10 µg/ml exosomes in grouped interventions on bEnd.3 cells. Data are presented as the mean ± SD of five independent experiments. ^∗P < 0.05

Protective Effects of STS and Exo on bEnd.3 Cells Exposed to H₂O₂

The CCK8 assay results indicated that STS (1, 3, and 10 µM) had no adverse effect on the viability of bEnd.3 cells (Fig. 1F). The survival rate of bEnd.3 cells under H₂O₂ exposure was evaluated at concentrations ranging from 100 to 500 µM (Fig. 1G). Exposure to 300 µM H₂O₂ for 24 h resulted in approximately 50–60% cell death (Fig. 1H). To investigate the protective effect of STS on H₂O₂-induced bEnd.3 death, we conducted a CCK8 assay. The results demonstrated that 1 µM STS did not significantly affect the cell survival, whereas 3 µM and 10 µM STS significantly improved cell viability (p < 0.05), with no significant difference between the effects of 3 µM and 10 µM (Fig. 1I). For subsequent experiments, 3 µM STS was selected as the optimal concentration. Both STS and Exo increased the survival rate of bEnd.3 cells following H₂O₂-induced damage (p < 0.05), with the combination of STS and Exo showing the most pronounced protective effect (Fig. 1J).

STS and Exo Promote bEnd.3 Cells Tube Formation after H₂O₂ Exposure

We observed the promotive effect of STS and Exo on bEnd.3 cells angiogenesis after H₂O₂ exposure. Specifically, H₂O₂ treatment significantly weakened the tube-forming ability of bEnd.3 cells, while the addition of STS and Exo significantly improved this ability (p < 0.05). Notably, the combination of STS and Exo had the best (Fig. 2A). Combined treatment promotd intercellular interactions and tubular structure formation, and demonstrated a synergistic effect in cell protection and tube formation.

Fig. 2.

STS and Exo promoted tube formation and migration in bEnd.3 cells after H₂O₂ exposure. A Tube-forming experiments to observe blood vessel formation, bar graph showing total tube length counts. B Scratch wound healing assay showing effects of STS and Exo on migration at 0, 12, and 24 h and bar graphs of wound closure. C Analysis of the effect of Exo and STS on the migration after H₂O₂ treatment and quantification of the mobility by Transwell experiments. Data are presented as the mean ± SD of five independent experiments. ^∗P < 0.05

STS and Exo Enhance Migration of bEnd.3 Cells Exposed to H₂O₂

In our study, we evaluated the effects of STS and Exo on the migration ability of bEnd.3 cells after H₂O₂ exposure using a cell scratch assay and Transwell migration assay. As shown in Fig. 2B, the cell scratch assay results indicated that H₂O₂ treatment significantly reduced the migration ability of bEnd.3 cells (p < 0.05). However, after treatment with STS and Exo, the cell migration ability was significantly improved, and the scratch healing rate increased significantly (p < 0.05). Further Transwell migration experiments confirmed this finding (Fig. 2C), showing that both STS and Exo effectively promoted bEnd.3 cells migration, with the combined treatment of STS and Exo having the most pronounced effect after 12 and 24 h.

STS and Exo Protect bEnd.3 Cells from H₂O₂ Induced Reduction of β-catenin and Occludin

The expression of adhesion junction (AJ) and tight junction (TJ) proteins in endothelial cells plays a crucial role in maintaining and enhancing endothelial barrier function.We evaluated the effects of STS and Exo on the protein expression of AJ and TJ markers in bEnd.3 cells after H₂O₂ exposure, using immunofluorescence and Western blot. The results of immunofluorescence experiments showed that H₂O₂ exposure significantly reduced the expression of β-catenin (AJ marker) and Occludin (TJ marker). However, treatment with STS or Exo significantly restored the expression of these proteins (p < 0.05) (Fig. 3A). Western blot analysis further confirmed that both STS and Exo treatments effectively increased the expression levels of β-catenin and Occludin proteins (p < 0.05). when STS was used in combination with Exo, the expression levels of AJ and TJ proteins reached optimal levels, significantly higher than with either treatment alone (p < 0.05) (Fig. 3B).This study suggests that STS and Exo have a synergistic effect in maintaining the core proteins of endothelial cell connections, especially in H₂O₂ induced cell damage responses. Combined therapy lays the foundation for restoring the structural integrity and function of BSCB.

Fig. 3.

STS and Exo prevent the loss of TJ and AJ proteins in H₂O₂-treated bEnd.3 cells. A Representative immunofluorescence images showing in situ expression of β-catenin and Occludin. B Westen blot showing β-catenin and Occludin protein levels in bEnd.3 cells after 24 h of H₂O₂ treatment. C Quantification of the relative protein levels. D Quantitative analysis of the mean fluorescence intensities. Data are presented as the mean ± SD of five independent experiments. ^∗P < 0.05

STS and Exo Promote VEGF Protein Expression in bEnd.3 Cells After H₂O₂ Exposure

In this study, immunofluorescence and Western blot were used to investigate the effects of STS and Exo on the expression of VEGF pathway proteins in bEnd.3 cells exposed to H₂O₂. As shown in Fig. 4A, immunofluorescence staining showed that H₂O₂ exposure significantly reduced the expression of key VEGF pathway proteins, such as VEGFR2 and VEGFA, indicating inhibition of the VEGF signaling pathway. However, treatment with STS or Exo led to a marked restoration of these protein expression levels. Western blot analysis further confirmed these observations, showing that both STS and Exo significantly upregulated the expression of VEGF pathway-related proteins, including VEGFR2 and VEGFA (p < 0.05). Notably, the combined treatment of STS and Exo resulted in the highest levels of VEGF signaling protein expression, indicating a synergistic effect (Fig. 4B). These findings suggested that the protective effects of STS and Exo on bEnd.3 cells may be closely associated with the activation of the VEGF signaling pathway.

Fig. 4.

STS and Exo exert protective effects on bEnd.3 cells after H₂O₂ exposure through the VEGF signaling pathway. A Representative immunofluorescence images showing in situ expression of VEGFR2 and VEGFA. B Westen blot showing VEGFR2 and VEGFA protein levels in bEnd.3 cells after 24 h of H₂O₂ treatment. C Quantification of the relative protein levels. D Quantitative analysis of the mean fluorescence intensities. Data are presented as the mean ± SD of five independent experiments. ^∗P < 0.05

STS and Exo Improve Motor Function and Reduce Tissue Damage after SCI

To determine the effects of STS and Exo treatments on functional recovery in SCI rats, We observed and photographed the recovery of the hind limbs of rats and conducted footprint tests and BBB scores. After 21 days of spinal cord injury, the SCI model group showed dragging and crawling on the back of the foot without effective weight-bearing capacity. The STS group and Exo group were able to achieve stable alternating gait, but had slightly insufficient fine motor skills; The combination therapy group showed good coordinated gait in the hind limbs, manifested as stable foot landing, flexible ankle joint movement, and toe grasping and pushing, as shown in Fig. 5A.The BBB scores indicated that both STS and Exo treatments significantly improved motor function compared to the control group. Notably, the combined treatment of STS and Exo resulted in the highest BBB scores, demonstrating a superior effect on functional recovery (Fig. 5B). Footprint tests showed that rats in the SCI group had inconsistent behavior of extensive dragging, while rats treated with STS and Exo showed relatively consistent hind limb footprints on the 21 st day post-injury. The gait improvement was most significant in the STS and Exo combined treatment group (Fig. 5C).

Fig. 5.

STS and Exo alleviate tissue damage and neuronal loss after SCI. A Behavioral observation images showing Characteristics of hind limb movement in different groups. B BBB motor scores of different groups. C Footprint analysis of different groups on day 21 after SCI. D Representative images of HE staining of longitudinal sections on day 21 after SCI. E Percentage of damaged areas based on relative quantification of lesion areas in different groups. F Survival of neurons in different groups based on relative quantification of the lesion area. G Representative images of Nissl staining of longitudinal sections on day 21 after SCI. Data are presented as the mean ± SD, n = 5 per group. ^∗P < 0.05

Histological analysis revealed that STS and Exo treatments effectively reduced pathological changes on the 21 st day after SCI. HE staining showed the histological structure of the injured spinal cord. Compared to the sham group, the SCI-injured site in the control group showed significant malformation and cavity formation, while the treatment groups showed clearer structure and reduced necrosis (p < 0.05) (Fig. 5D, E). Nissl staining revealed normal neurons in the sham group. In contrast, only a few neurons with normal Nissl substance were present at the injury site in the SCI group. However, a significant increase in the number of surviving neurons was observed following STS and Exo treatment (p < 0.05) (Fig. 5F, G). Overall, these results suggest that the combination of STS and Exo provides enhanced therapeutic benefits, promoting functional recovery and mitigating pathological changes after SCI in rats. This synergistic effect underscores the potential of combined therapies for improving outcomes in spinal cord injury.

STS and Exo Maintain the Integrity of BSCB after SCI by Preventing the Loss of Connecting Proteins

In this study, Evans Blue dye extravasation was used to assess the permeability of the BSCB, while Western blot and immunofluorescence analyses evaluated the expression of AJ and TJ proteins in a rat model of SCI. Evans Blue fluorescence in the injured spinal cord was higher than in the sham group, while a significant reduction in fluorescence intensity was observed in rats treated with STS or Exo, compared to the SCI group, indicating a protective effect on BSCB integrity (at 7 days post-injury) (p < 0.05). The combined treatment of STS and Exo resulted in the lowest levels of EB dye leakage, suggesting the most substantial improvement in BSCB function (Fig. 6A-D).

Fig. 6.

STS and Exo inhibit BSCB disruption after SCI. A Representative images of Evans blue staining of the spinal cord for each group. B Quantification of Evans blue in spinal cord of each group (μg/g). C Representative confocal image of a transverse section of the spinal cord showing Evans blue extravasation. D Quantification of Evans blue fluorescence intensity. E Representative immunoblot images showing Occludin and β-catenin protein expression levels. F-G Quantification of relative Occludin and β-catenin protein levels. Data are presented as the mean ± SD, n = 5 per group. ^∗P < 0.05

Western blot results indicated a significant reduction in β-catenin and Occludin proteins following injury compared to the sham group. However, STS and Exo treatments upregulated the expression of β-catenin and Occludin proteins in the SCI group, which are critical for maintaining BSCB integrity (p < 0.05). Notably, the combination of STS and Exo significantly increased the levels of these proteins (p < 0.05) (Fig. 6E-F).

STS and Exo Reduce Tissue and Neuronal Damage after SCI

We examined the effects of STS and Exo on glial scar formation and axonal regeneration, both of which are key factors in SCI repair. GFAP immunofluorescence staining showed that astrocytes in the damaged area were activated and migrated to the periphery of the lesion, forming a barrier structure. Significant glial scars were observed in the untreated SCI group, with their edges tightly enveloped by activated astrocytes; After treatment with STS and Exo, the area of glial scars significantly decreased, and the scar boundaries formed by activated astrocytes became blurred, indicating a decrease in astrocyte reactivity. To assess axonal regeneration, MAP2 immunostaining was used to measure the distance from the lesion center to the nearest neuron. In the STS and Exo treatment groups, this distance was significantly shortened compared to the SCI group, suggesting enhanced neuronal recovery and axonal regeneration (p < 0.05) (Fig. 7A-C). Western blot results further supported these findings, showing a decrease in GFAP expression and an increase in MAP2 expression in the STS and Exo-treated groups compared to the SCI group (p < 0.05) (Fig. 7D-F). These results indicated that the combined treatment of STS and Exo not only reduced glial scar formation, but also promoted axonal regeneration, facilitating improved spinal cord repair.

Fig. 7.

STS and Exo reduce the tissue and neuronal damage after SCI. A Co-immunofluorescence images showing GFAP (red) and MAP2 (green) 21 days after SCI. B Longest damage area in the SCI regions for each group. C Total area of cavities and scars in the SCI region for each group. D-F Western blot analysis and quantification of GFAP and MAP2 expression. Data are presented as the mean ± SD, n = 5 per group. ^∗P < 0.05

STS and Exo Reduce Inflammatory Reaction after SCI

We explored the role of the inflammatory response in SCI and assessed the effects of STS and Exo on modulating this response. Double immunofluorescence staining for Arg1 +/Iba1 + and NOS2 +/Iba1 + was performed to evaluate the polarization state of microglia/macrophages after SCI. The results demonstrated a significant increase in Arg1-positive cells and a decrease in NOS2-positive cells in the spinal cord tissue of STS and Exo-treated rats compared to the SCI group (p < 0.05), indicating a shift toward an anti-inflammatory. The combined treatment of STS and Exo produced the most pronounced effect, with the highest Arg1 +/Iba1 + and lowest NOS2 +/Iba1 + expression (Fig. 8A-D).

Fig. 8.

STS and Exo treatment promotes the expression of Arg1 in microglia. A-B Immunofluorescence staining of Iba1(green)/Arg1(red) and Iba1(green)/NOS2(red) at the site of spinal cord lesion 21 days after SCI. C-D Quantification the number of Arg⁺/Iba1⁺ and NOS2⁺/Iba1⁺ cells number in spinal cord. E–G Western blot analysis and quantification of NOS2 and Arg expression. Data are presented as the mean ± SD, n = 5 per group. ^∗P < 0.05

Western blot analysis further confirmed these findings. Compared to the sham group, the expression of inflammatory markers such as Arg1 and NOS2 was significantly increased in the SCI group, indicating a sustained inflammatory response after SCI (p < 0.05). However, after treatment with STS and Exo, there was an increase in Arg1 expression and a decrease in NOS2 expression, with the combined treatment showing the most pronounced effect (Fig. 8E-G). These results suggested that STS and Exo, especially when used together, effectively modulated the inflammatory response after SCI, promoting a more favorable environment for spinal cord repair.

STS and Exo Promote VEGF Pathway Protein Expression after SCI

Studies has shown that the VEGF pathway is crucial in SCI repair [30, 31]. It plays an important role in improving SCI recovery by promoting angiogenesis, regulating inflammation, supporting neuronal survival and regeneration, and other mechanisms. Immunofluorescence results showed that the expression of VEGFA was significantly reduced after SCI, while VEGFA expression increased after STS and Exo treatments (p < 0.05), with the combination treatment group showing the most pronounced effect (Fig. 9A, B). Western blot also showed consistent results, indicating that STS and Exo treatments significantly activate the VEGF pathway after SCI (p < 0.05) (Fig. 9C, D). These findings suggested that the protective effects of STS and Exo may be closely associated with the activation of the VEGF signaling pathway after SCI.

Fig. 9.

STS and Exo exerts microcirculation protective and neuroprotective effects after SCI through the VEGF signaling pathway. A-B Immunofluorescence staining and mean fluorescence intensity of VEGFA at the spinal cord lesion site after 21 days of SCI. C-D Western blot analysis and quantification of VEGFA expression. Data are presented as the mean ± SD, n = 5 per group. ^∗P < 0.05

Discussion

The initial mechanical damage to BSCB after SCI can trigger a series of secondary inflammations, vascular dissolution, and biochemical reactions, thereby exacerbating neuronal dysfunction [32]. Repairing the BSCB may be a breakthrough in SCI treatment strategies. In our study, we explored the therapeutic potential of combining STS and Exo for SCI, with a specific focus on their effects on the VEGF signaling pathway. Our results provide novel insights into how these agents may work to enhance recovery in both in vitro and in vivo.

Consistent with previous studies, our findings demonstrated that treating endothelial cells with H₂O₂ significantly reduces cell viability, tube formation, and migration capabilities. Additionally, there is a significant decrease in expression levels of AJ and TJ proteins [16, 33, 34]. Our findings showed that both STS and Exo interventions can protect bEnd.3 cells from H₂O₂ induced damage, especially when used in combination, as demonstrated by increased cell survival. Additionally, these treatments significantly improved endothelial cell migration, tube formation, and upregulated the expression of VEGFA and its receptor VEGFR2. These findings suggested that STS and Exo may promote angiogenesis and vascular repair by activating the VEGF signaling pathway, which is critical for improving blood supply and nutrient delivery to injured tissue. Maintaining endothelial function and promoting neovascularization are key to establishing a supportive environment for neuronal repair following SCI [35, 36].

To translate these in vitro findings, we employed a rat SCI model to assess functional and histological recovery. Strikingly, STS and Exo co-administration significantly accelerated motor function restoration. Given the pivotal role of BSCB integrity in SCI pathology, we next evaluated Evans Blue extravasation. As anticipated [37], SCI increased BSCB permeability, whereas STS and Exo treatment—particularly in combination—markedly restored barrier integrity. This repair likely shields the injury microenvironment from secondary damage, creating favorable conditions for neural recovery [38, 39].

Beyond vascular protection, glial scar modulation is equally critical for regeneration.The formation of glial scars following SCI offers limited benefits; however, extensive and persistent glial scars create a dense barrier that hinders neuronal axon regeneration. Thus, identifying treatments that limit glial scarring while protecting neurons is essential for SCI recovery. Among key markers, GFAP is specific to astrocytes, while MAP2 labels mature neurons. In consist with previous studies,we found that SCI significantly upregulated GFAP expression and reduced MAP2 expression. In SCI rats, activated astrocytes proliferated around the lesion site, forming a fence-like structure with overlapping projections encircling the injury. However, treatment with STS and Exo led to a significant decrease in GFAP expression and a less pronounced boundary of activated astrocytes, along with increased MAP2 expression. The combination therapy had the most significant effect, suggesting that STS and Exo treatment reduces glial scar formation, thereby fostering a favorable microenvironment for neuronal axon regeneration.

Concurrently,inflammation is a key component of the secondary injury following SCI, and its regulation is essential for promoting tissue repair [40, 41]. Consistent with previous study [42], we found that inflammatory responses continued to increase after SCI, with NOS2 and Arg1 expressions significantly higher in the SCI group compared to the Sham group. Our results showed that STS and Exo treatments significantly reduced NOS2 expression while increasing Arg1 expression, indicating a shift in macrophage polarization toward an anti-inflammatory phenotype. This shift is associated with decreased tissue damage, improved clearance of cellular debris, and enhanced tissue repair. The anti-inflammatory effects of STS and Exo, particularly in combination, likely contribute to improved spinal cord healing and functional recovery. The modulation of the inflammatory environment is essential for reducing secondary injury and creating conditions favorable for regeneration.

One of the key findings in this study is the activation of the VEGF signaling pathway following treatment with STS and Exo. VEGF is a key regulator of both angiogenesis and neuroprotection, and its upregulation has been associated with improved outcomes in SCI models [43, 44]. Upon binding to its receptors, primarily VEGFR-1 and VEGFR-2, on endothelial cells, triggering a series of downstream signaling events. Specifically, activation of VEGFR-2 triggers dimerization and autophosphorylation, subsequently activating several intracellular pathways, including the PI3K/Akt pathway [45], which promotes cell survival, and the MAPK/ERK pathway, which promotes cell proliferation [46]. In both in vitro and in vivo experiments, we observed a significant upregulation of VEGFA and its receptor VEGFR2 in the treatment groups. This pathway activation likely plays a key role in the observed improvements in angiogenesis, neuroprotection, and functional recovery, making it as a promising therapeutic target for SCI.

There were some limitations in our current study.These tasks have not been fully elucidated:Firstly,The research confirms that STS can reduce inflammation and oxidative stress in SCI models. In addition to the inflammation studied in this article, oxidative stress regulation is a possible complementary mechanism of STS combined with Exo therapy for spinal cord injury. Future research should quantitatively evaluate biomarkers to describe the contribution of oxidative stress regulation to functional recovery.Secondly, The observed synergy underscores the therapeutic imperative of multi-targeted intervention for complex SCI pathophysiology and establishes combination regimens as superior to monotherapies. While robustly validating therapeutic efficacy,our upcoming research will focus on the functional crosstalk between STS and Exo in the treatment of spinal cord injury, as well as in-depth investigation of the role played by the VEGF pathway in this process.

Conclusion

Overall, our study provides convincing preclinical evidence that the STS and Exo combination therapy is a promising strategy for treating spinal cord injury (SCI). STS and Exo can protect endothelial cells from damage caused by H₂O₂ through the activated VEGF signaling pathway. At the same time, STS and Exo therapy can significantly repair the function of BSCB after injury and reduce inflammatory response. This dual target approach demonstrating significant functional recovery effects. As shown in Fig. 10. These results emphasize the promising therapeutic advantages of combining STS and Exo transplantation for the treatment of SCI. This study thus paves the way for innovative therapeutic strategies aimed at improving outcomes for SCI patients.

Fig. 10.

A schematic diagram

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

STS

Sodium tanshinone IIA sulfonate

SCI

Spinal cord injury

BSCB

Blood spinal cord barriers

VEGF

Vascular endothelial growth factor pathway

BMSC

Bone mesenchymal stem cell

EXO

Exosomes

DMEM

Dulbecco's Modified Eagle's Medium

FBS

Fetal Bovine Serum

TEM

Transmission Electron Microscopy

BBB

Basso Beattie Bresnahan

EB

Evans Blue

HE

Hematoxylin and Eosin

SDS-PAGE

Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

CD63

Cluster of Differentiation 63

TSG101

Tumor Susceptibility Gene 101

VEGFA

Vascular Endothelial Growth Factor A

VEGFR1/R2

Vascular Endothelial Growth Factor Receptor ½

MAP2Microtubule

Associated Protein 2

GFAP

Glial Fibrillary Acidic Protein

ARG1

Arginase 1

NOS2

Nitric Oxide Synthase 2

GAPDH

Glyceraldehyde-3-Phosphate Dehydrogenase

HRP

Horseradish Peroxidase

Iba-1

Ionized Calcium Binding Adapter Molecule 1

DAPI

4',6-Diamidino-2-Phenylindole

CCK8

Cell Counting Kit-8

TJ

Tight Junction

AJ

Adherens Junction

PI3K/AKT

Phosphoinositide 3-Kinase/Protein Kinase B

Author Contribution

Dan Luo and Lin Ma performed cell experiments and wrote the manuscript. Zhijian Pan and Ling Chen performed animal experiments. Zhifeng Hu and Chaolun Liang analyzed the data. Zenglu Wang and Zhifeng Xiao Processed pictures. Dingkun Lin and Da Guo supervised the work. Jinfeng Li edited the manuscript. Li Xing provided financial support and conceived and designed experiments.All authors have read and approved this manuscript.

Funding

This work was supported by Natural Science Foundation of Guangdong, China (No.2024A1515030293, No. 2022A1515010793,No. 2024A1515140021); Research Fund for Bajian/Qingmiao Talents of Guangdong Provincial Hospital of Chinese Medicine (No.BJ2022KY07, No.SZ2022QN05); University—Hospital Joint Fund Project of Guangzhou University of Chinese Medicine (No.GZYZS2024D03,NO.GZY2025GB0402);the Science and Technology Program of Guangzhou, China (No. 2023A03J0239);Natural Science Foundation of China (No. 82104894);The Science and Technology Program of Guangzhou, China (No. 2024A03J0105); the Research Project of Traditional Chinese Medicine Bureau of Guangdong Province (No. 20231138).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

All animal experiments were approved by the animal ethics committee of Guangzhou University of Chinese Medicine.

Consent for Publication

All authors have read and approved the content and agree to submit for consideration for publication in the journal.

Conflict of interest

The authors declare no competing interests.