VJJ was purchased from Hebei Renxin Pharmaceutical Co., LTD. The extraction process was as follows: the crude powder of VJJ was subjected to 70% ethanol extraction, with an initial extraction ratio of 8:1 for 24 h, followed by a subsequent 6:1 extraction for 12 h. The resultant extracts were amalgamated and then concentrated under reduced pressure to remove any residual alcoholic odor, yielding an ethanolic extract. For purification, the ethanolic extract was homogeneously dispersed in water through ultrasonication and subsequently loaded onto a D101 macroporous resin column, characterized by a specific adsorption capacity of 72 mg/g, an adsorption flow rate of 1 BV/h, and an elution flow rate of 2 BV/h. Elution was performed sequentially with water at 6BV, followed by 60% ethanol at 4BV, and finally 95% ethanol at 4BV. The 95% ethanol fraction was collected, concentrated into a paste under reduced pressure, and subsequently dried in a vacuum at 40°C. The quantification of total iridoids in the extract, as determined by ultraviolet spectrophotometry, was established at 83.25%.

Forty-eight healthy male Sprague Dawley rats, weighing between 250 to 290 grams, were acquired from SiPeiFu (Beijing) Biotechnology Co. Ltd. (Animal Certificate No.: SCXK (Beijing) 2019–0010). The housing conditions were meticulously maintained, ensuring cleanliness and adequate ventilation, with a stable room temperature of 22 ± 2°C, relative air humidity maintained at 60%, a 12-h light–dark cycle, and ad libitum access to food. The conduct of the animal study received ethical approval from the Ethics Committee of the General Hospital of the Western Theater Command of the Chinese People’s Liberation Army.

Anesthesia induction in the rats was achieved through intraperitoneal injection of 5% pentobarbital sodium, administered at a dosage of 30 mg/kg. Subsequent to the T10 laminectomy, the spinal cord was clamped at the corresponding level for 10 s using 70 g medical aneurysm clips. Following the closure of the surgical incision, penicillin was administered via intramuscular injection at a dosage of 200,000 U/day for a period of 3 days. Additionally, manual bladder expression was conducted thrice daily to facilitate urinary excretion until normal voiding was reestablished. In the sham group, the procedure was limited to laminectomy, ensuring the spinal cord was left intact.

Forty-eight rats were randomly assigned into four groups using the random number table method: Sham, SCI, IRFV, and IRFV+LY294002. The IRFV extract, weighing 0.5 g, was dissolved via ultrasonic agitation in a 0.5% carboxymethyl cellulose sodium (CMC-Na) solution, which was subsequently brought to a final volume of 500 mL. The IRFV group received the IRFV solution orally at a dose of 10 mg/(kg·d). For the IRFV+LY294002 group, in addition to the same oral dose of IRFV, an intraperitoneal injection of LY294002 was administered at 1 mg/(kg·d). The Sham and SCI groups were administered an equivalent volume of CMC-Na solution daily via gavage. The duration of this intervention extended over a period of 28 days.

For the assessment of hind limb motor function in rats, the Basso, Beattie, and Bresnahan scoring (BBB score) (Basso et al., 1995) was employed. Two independent experimenters, well-versed in the scoring criteria and blinded to the animal groupings, conducted the evaluations. Each limb was scored separately over a duration of 5 min, and the average of the two evaluators’ scores was recorded for each assessment.

Spinal cord tissues were fixed in a 10% neutral buffered formalin solution for 24 h. Following fixation, the tissues underwent standard dehydration and embedding procedures, after which 4 μm serial sections were prepared for Luxol Fast Blue (LFB) and Nissl staining. For LFB staining, sections were deparaffinized to 95% ethanol, followed by immersion in the fast blue staining solution (Solarbio, Beijing, China) overnight at room temperature, subsequently rinsed in 95% ethanol to remove excess dye, followed by distilled water and then differentiated in LFB Differentiation solution for 15 s, and further in 70% ethanol for 30 s until the demarcation between gray and white matter was distinct, followed by a distilled water rinse, counterstaining with Eosin for 2 min, and then rapid washing and routine dehydration, clarification, and sealing of the sections. In the Nissl staining process, following routine dewaxing, sections were immersed in toluidine blue staining solution (Solarbio, Beijing, China), maintained at 50–60°C for 20–40 min, Upon completion of the staining, the tissue sections were gently rinsed with distilled water, swiftly differentiated using 95% ethanol, and subsequently underwent standard dehydration, clarification, and sealing procedures.

Treatment Highly differentiated PC12 cells were purchased from Wuhan Pricella Life Science and Technology Co. PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, United States) and maintained in an incubator at 37°C and 5% CO₂. In the study, the cells were allocated into four experimental groups: Control, H₂O₂, H₂O₂ + IRFV, and H₂O₂ + IRFV+LY294002. Before commencing the experiment, PC12 cells were seeded in 96-well plates at a concentration of 2 × 10⁴ cells/mL. The control group was maintained in DMEM without additional treatments. The H₂O₂ group received 400 μmol/L H₂O₂ (dissolved in DMEM) solution for 2 h. The H₂O₂ + IRFV group was exposed to 400 μmol/L H₂O₂ solution for 2 h, followed by treatment with 10 μg/mL IRFV solution (dissolved in DMEM) for 24 h. The H₂O₂ + IRFV+LY294002 group underwent 400 μmol/L H₂O₂ exposure for 2 h, succeeded by a combined treatment (dissolved in DMEM) of 10 μg/mL IRFV and 10 μmol/L LY294002 for 24 h. Each group consisted of three replicate wells to ensure experimental validity.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. A range of H2O2 concentrations (100–800 μmol/L) and IRFV concentrations (0.1–20 μg/mL) were applied to cells in 96-well plates. The Cell Counting Kit-8 reagent (MedChemExpress, New Jersey, United States) was introduced to the 96-well plates and the plates were incubated for 1 h in a cell incubator. Subsequently, the optical density at 450 nm was determined using a spectrophotometer (ThermoFisher Scientific, United States). Each experiment was replicated three times for consistency. The formula for calculating cell viability was as follows: Cell viability (%) = [(Absorbance in drug-treated wells - Absorbance in blank wells) / (Absorbance in control wells - Absorbance in blank wells)] × 100%.

Spinal cord tissues were fixed in a 10% neutral buffered formalin solution for 24 h. Following fixation, tissues underwent standard dehydration and embedding processes, were serially sectioned at 4 μm thickness, and the sections were subjected to antigen retrieval in EDTA solution (Solarbio, Beijing, China) using a microwave. PC12 cells were fixed using 4% paraformaldehyde for a duration of 20 min. Tissue and cell samples were permeabilized in Triton X-100 (Solarbio, Beijing, China) for 20 min, blocked with 3% BSA for 1 h at room temperature, and incubated overnight at 4°C with anti-neurofilament 200 (NF200) (1:200, 2,836, Cell Signalling Technology, Beverly, MA, United States) and growth-associated protein 43 (GAP43) (1:200, 8,945, Cell Signalling Technology, Beverly, MA, United States) primary antibodies. Subsequently, the samples were incubated with rabbit (1:200, Cat No: SA00013-1, Proteintech, Wuhan, China) or mouse (1:200, Cat No: SA00013-1, Proteintech, Wuhan, China) secondary antibodies conjugated with fluorochrome for 1 h, followed by re-staining of cell nuclei with DAPI for 1 h and sealing. Imaging was performed using a Nikon ECLIPSE CI-L microscope, and fluorescence intensity was quantitatively analyzed with Image J software.

The total protein from spinal cord tissues and cell samples was extracted using a protein extraction kit (Solarbio, Beijing, China), following the provided instructions, and the protein concentration was quantified using the BCA method. After gel preparation, the samples were loaded in equal amounts of total protein. The proteins was separated by 10% SDS-PAGE (80 V, 2 h, then 130 V, 1 h) and transferred to a PVDF membrane. The membrane was blocked with 5% milk for 1 h, followed by overnight incubation at 4°C with primary antibodies against NF200 (1:1000, 2,836, Cell Signalling Technology, Beverly, MA, United States), GAP43 (1:1000, 8,945, Cell Signalling Technology, Beverly, MA, United States), PI3K (1:1000, 4,292, Cell Signalling Technology, Beverly, MA, United States), p-PI3K (1:500, AF3242, Affinity, China), Akt (1:1000,bs-6951R, Bioss, Beijing, China), p-Akt (1:1000,bsm-60645R, Bioss, Beijing, China), and GAPDH (1:8000, 2,118, Cell Signalling Technology, Beverly, MA, United States). This was succeeded by incubation with HRP-conjugated secondary antibodies (1:10000, 7,074, 7,076, Cell Signalling Technology, Beverly, MA, United States) for 1 h at room temperature. Lastly, the membranes were visualized using ECL reagents and the Azure Biosystems NIR Fluorescence Imaging system, followed by quantitative grayscale analysis using Image J software.

Statistical analysis was performed using SPSS software version 25.0 (SPSS, Chicago, IL, United States). Comparative analysis among multiple groups was conducted using one-way ANOVA, while the differences between two groups were evaluated through unpaired Student’s t-test. A p-value of less than 0.05 was deemed to indicate statistical significance.

IRFV was administered via gastric gavage at a consistent dosage of 10 mg/kg daily for 28 days following SCI. Postoperatively, motor function was evaluated on days 0, 7, 14, 21, and 28 post-SCI using the BBB score (Figure 1A). The results indicate that on the first day post-operation, the Sham group had a BBB score of 21, while all other groups scored 0, suggesting the model was successfully established. The results also indicated that no significant difference in BBB scores between the SCI and IRFV groups from day 0 to 7. However, from day 14 to 28 post-operation, IRFV treatment significantly enhanced BBB scores, consistently outperforming the SCI group (Figure 1B). Histological analysis of spinal cord tissues using Nissl staining revealed a marked reduction in Nissl bodies in the SCI group compared to the sham group. Conversely, IRFV treatment notably increased the number of Nissl bodies compared to the SCI group (Figure 1C). Myelin loss in tissue was assessed using LFB staining, demonstrating that IRFV treatment significantly mitigated myelin loss compared to the SCI group (Figure 1D). In conclusion, these findings indicate that IRFV plays a pivotal role in facilitating tissue repair and remyelination, thus enhancing functional recovery post-SCI in vivo.

NF200, a crucial neurofilament predominantly located in the cytoplasm of neurons and axons, serves as an indirect indicator of the extent of axonal damage and regeneration following SCI (Hu X. et al., 2023; Hu Z. et al., 2023). GAP43 is an axonal membrane protein that plays a role in axonal development, regeneration and plasticity (Liu et al., 2020). Immunofluorescence co-localization and western blotting echniques were utilized to assess the expression of axon-related proteins NF200 and GAP43 in various groups of rats, four weeks post-SCI. Immunofluorescence co-localization results revealed a significant reduction in the expression of NF200 (p < 0.05) and GAP43 (p < 0.01) in the SCI group compared to the Sham group, while IRFV treatment markedly enhanced the expression levels of NF200 (p < 0.05) and GAP43 (p < 0.01) (Figures 2A–C). This result was further corroborated by Western blotting. Moreover, western blotting analysis demonstrated that IRFV treatment elevated the protein expression of p-Akt (p < 0.05), suggesting a potential link between IRFV’s axon regeneration-promoting effect in SCI and Akt phosphorylation (Figures 2D–G).

The CCK-8 assay was employed to determine the optimal intervention concentration of H₂O₂ and IRFV. Results indicated that the viability of PC12 cells was approximately 50% following 400 μmol/L H₂O₂ treatment for 2 h (Figure 3A). Consequently, 400 μmol/L was chosen as the H₂O₂ treatment concentration. Following 2 h of H₂O₂ treatment, PC12 cells treated with 10 μg/mL IRFV for 24 h exhibited the most significant increase in viability compared to other concentrations of IRFV administration (Figure 3B), thus 10 μg/mL was selected as the administration concentration in this study.

Optical microscopy revealed that the morphology of PC12 cells in the Control group was pike-shaped, featuring large cytosol, thickened and elongated cytosolic branches and trunks, strong refractive index, extended pseudopods, and a high cell count. Compared with the Control cells, the synapses of the PC12 cells in the H₂O₂ group and the H₂O₂ + IRFV+LY294002 group were observed to be shorter or the pseudopods absent. Additionally, a portion of these cells crumpled, rounded, and detached; the intercellular gap widened, and there was a noticeable reduction in cell count. In the H₂O₂ + IRFV group, there was a notable extension of pseudopods, reduction in cell gap, increase in cell number, and a clear improvement in the growth status of the cells (Figure 3C). These findings imply that IRFV contributes to the enhancement of PC12 cell growth, and that this effect can be reversed by the inhibition of PI3K/Akt through LY294002. The CCK-8 assay results demonstrated a significant reduction in the viability of the PC12 cells in the H₂O₂ group compared to the Control group (p < 0.001). Conversely, cell viablity in the H₂O₂ + IRFV group was significantly elevated compared with that of H₂O₂ group (p < 0.01), while the H₂O₂ + IRFV+LY294002 group exhibited a marked decrease in cell viability compared to the H₂O₂ + IRFV group (p < 0.01) (Figure 3D). This suggests that IRFV improves the cell viability, an effect that can be reversed by the inhibition of PI3K/Akt via LY294002.

Immunofluorescence and western blotting were employed\d to assess axon-related proteins NF200 and GAP43 in PC12 cells. Immunofluorescence results revealed a significant decrease in the protein expression of NF200 and GAP43 in the H₂O₂ group compared to the Control group (p < 0.05). The protein expression levels of NF200 and GAP43 in the H₂O₂ + IRFV group were significantly elevated compared to those in the H₂O₂ group (p < 0.05). Protein expression levels of NF200 (p < 0.01) and GAP43 (p < 0.05) in the H₂O₂ + IRFV+LY294002 were significantly decreased in comparison to the H₂O₂ + IRFV group (Figures 4A–C), which was further verified by western blotting results (Figures 4D–F). These findings indicate that IRFV facilitates axon regeneration following PC12 cell injury, and that this effect can be reversed by inhibiting the PI3K/Akt signaling pathway through the application of LY294002.

Western blotting was utilized to detect the expression of PI3K/Akt pathway proteins, aiming to elucidate the potential mechanism underlying the promotion of axonal regeneration. The expression levels of PI3K/Akt protein were significantly upregulated in IRFV-treated, H₂O₂-pre-stimulated PC12 cells (p < 0.05), while a notable reduction was observed in the expression of PI3K/Akt protein following the application of LY294002 (p < 0.05), indicating effective inhibition of the PI3K/Akt signaling pathway (Figures 5A–C). These results imply that IRFV may promote axonal regeneration in PC12 cells by activating the PI3K/Akt signaling pathway.

Rats with SCI treated with IRFV were administered LY294002 via intraperitoneal injection. Western blot analysis of PI3K/Akt signaling pathway proteins in the rat spinal cord revealed that post-IRFV treatment, the expression of these proteins was significantly upregulated (p < 0.05), indicating activation of PI3K/Akt signaling pathway. After the intraperitoneal injection of LY294002, a significant decrease in the expression of PI3K/Akt signaling pathway proteins was observed in the rat spinal cord, suggesting effective inhibition of the PI3K/Akt pathway (Figures 6A,C–D). Immunofluorescence and Western blot analyses demonstrated that IRFV treatment significantly elevated the protein expression of NF200 and GAP43 in the spinal cord of the SCI rats, whereas LY294002 administration via intraperitoneal injection markedly reduced their expression (Figures 6A–D). Nissl and LFB staining results indicated that intraperitoneal administration of LY294002 reversed IRFV’s effects on increasing the number of Nissl bodies (p < 0.001) and ameliorating demyelination (p < 0.001) (Figures 6E,F). Behavioral assessments also revealed that intraperitoneal administration of LY294002 led to a significant reduction in motor function compared to IRFV-treated rats (Figure 6G). These findings demonstrate that the inhibition of the PI3K/Akt signaling pathway reverses IRFV’s effect on promoting axonal regeneration in SCI rats, further suggesting that IRFV promotes axonal regeneration by activating the PI3K/Akt pathway.