Bergapten attenuates oxidative stress and neuroinflammation to promote the survival and neuronal differentiation of neural stem/progenitor cells after ischemic insult

Abstract

Background

Ischemic stroke remains a leading cause of death and disability worldwide. A significant challenge in recovery is the hostile microenvironment in the ischemic penumbra, characterized by excessive oxidative stress and neuroinflammation, which leads to massive neuronal death and impedes the regenerative potential of endogenous neural stem/progenitor cells (NSPCs). Bergapten, a natural coumarin derivative, has demonstrated antioxidant and anti-inflammatory properties in various disease models, suggesting its potential as a neuroprotective agent.

Objective

This study aimed to investigate whether bergapten could protect against oxidative stress and inflammation in a simulated ischemic stroke model, and subsequently promote the survival, proliferation, and neuronal differentiation of NSPCs.

Methods

We established in vitro models of oxidative stress using hydrogen peroxide (H₂O₂) treatment on primary mouse NSPCs and BV2 microglial cells. NSPCs were identified via immunofluorescence staining for Nestin, SRY-box transcription factor 2 (SOX2), and paired box protein 6 (PAX6). The effects of bergapten were evaluated by measuring: 1) intracellular reactive oxygen species (ROS) levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis in NSPCs; 2) intracellular ROS levels and M1/M2 polarization (inducible nitric oxide synthase (iNOS)/Arginase-1 (Arg-1)) in BV2 cells; and 3) neuronal or astroglial differentiation of NSPCs (neuronal class III β-tubulin (TUJ-1)) under stress conditions.

Results

H₂O₂ treatment induced significant oxidative stress and apoptosis in NSPCs, as evidenced by increased ROS levels and TUNEL-positive cells. It also triggered ROS production and a pro-inflammatory M1 phenotype (iNOS⁺) in BV2 microglia. Crucially, under H₂O₂-induced stress, NSPCs showed impaired differentiation into neurons (TUJ-1⁺ cells). Bergapten treatment effectively mitigated these detrimental effects: it reduced ROS levels and apoptosis in NSPCs, suppressed M1 polarization in BV2 cells, and, most importantly, rescued the capacity of NSPCs to differentiate into neurons.

Conclusions

Our findings demonstrate that bergapten possesses potent antioxidant and anti-inflammatory properties in neural cell models of ischemic injury. By ameliorating the hostile microenvironment, bergapten enhances NSPC survival and facilitates their differentiation into neurons. This study provides the first experimental evidence supporting bergapten as a promising therapeutic candidate to promote endogenous neurogenesis and functional repair after ischemic stroke.

Highlights

• •Bergapten enhances neural stem/progenitor cells survival via antioxidative/antiapoptotic effects.
• •Bergapten shifts microglia to anti-inflammatory M2 phenotype.
• •Bergapten restores impaired neuronal differentiation.
• •Bergapten protects endothelial barrier and tight junctions post-OGD/R.
• •Bergapten reduces vascular hyperpermeability, supports BBB integrity.

1. Introduction

Ischemic stroke, resulting from the occlusion of a cerebral artery, is a devastating neurological event and a primary cause of long-term adult disability [1,2]. The initial ischemic insult triggers a complex cascade of pathological events, including excitotoxicity, ionic imbalance, and energy failure. Following reperfusion, either spontaneously or through clinical intervention, a secondary wave of damage known as ischemia-reperfusion injury occurs, which is largely driven by excessive production of reactive oxygen species (ROS) and the activation of a robust neuroinflammatory response [3,4]. This hostile microenvironment in the peri-infarct region exacerbates neuronal apoptosis and necrosis, leading to irreversible brain damage.

Current therapeutic strategies, such as thrombolysis and thrombectomy, are critically limited by a narrow treatment time window and are ineffective at reversing established neuronal loss [5,6]. Consequently, research has shifted towards promoting brain repair and regeneration. The adult mammalian brain harbors endogenous neural stem/progenitor cells (NSPCs) primarily in the subventricular zone and the hippocampal dentate gyrus, which hold the potential for self-repair [[7], [8], [9]]. Following ischemic injury, these NSPCs are activated and undergo proliferation. However, their regenerative capacity is severely hampered by the post-stroke milieu [10,11]. The overwhelming oxidative stress damages cellular lipids, proteins, and DNA, directly harming NSPCs [12,13]. Concurrently, activated microglia, the resident immune cells of the brain, polarize towards a pro-inflammatory M1 phenotype, releasing cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), which further inhibit NSPC proliferation and skew their differentiation away from neurons towards astrocytes, contributing to glial scar formation [[14], [15], [16]]. Therefore, a therapeutic strategy that simultaneously mitigates oxidative stress and neuroinflammation could create a permissive environment for endogenous NSPCs to survive, proliferate, and differentiate into functional neurons, thereby promoting structural and functional recovery.

Bergapten (5-methoxypsoralen), a natural furocoumarin found in bergamot and other citrus fruits, has garnered attention for its diverse pharmacological activities. Studies have reported its potent antioxidant effects, such as reducing infarct size and lipid peroxidation in models of myocardial infarction [17,18]. Its anti-inflammatory properties are evidenced by its ability to inhibit the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and modulate macrophage polarization in models of acute lung injury and colitis [19,20]. Notably, bergapten exhibits high blood-brain barrier permeability, making it a suitable candidate for central nervous system disorders [21,22]. Despite these promising attributes, its direct effects on the neural components critical for stroke recovery—particularly on NSPCs and microglia in the context of ischemic injury—remain largely unexplored.

Based on this rationale, we hypothesized that bergapten could protect NSPCs and microglia from oxidative stress and inflammation, thereby promoting a neurogenic environment. In this study, we utilized hydrogen peroxide (H₂O₂) to simulate the oxidative stress component of ischemic injury in vitro. We first characterized the detrimental effects of H₂O₂ on NSPC apoptosis, microglial activation, and neurogenesis. We then investigated the therapeutic potential of bergapten in counteracting these effects, focusing on its roles in reducing ROS, inhibiting microglial M1 polarization, enhancing NSPC survival, and crucially, rescuing neuronal differentiation.

2. Materials and methods

2.1. Mouse NSPCs culture

All experimental procedures were approved by the Animal Welfare Committee of Affiliated Hospital of Hebei Engineering University (No20240930t0500115[512]). NSPCs were harvested from the telencephalic regions of embryonic C57BL/6 mice at E15. Following dissection, the telencephalic tissues were immediately placed in chilled phosphate-buffered saline (PBS). Tissue dissociation was carried out enzymatically using TrypLE (Gibco, USA) for 25 min at 37 °C, after which the reaction was halted by adding Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Gibco, USA). The resulting cell suspension was passed through 70-μm and 40-μm cell strainers to eliminate any residual tissue aggregates and then centrifuged at 500g for 5 min. The cell pellet was resuspended in 8 mL of proliferation medium and transferred to a T25 culture flask (Corning, USA). The proliferation medium contained DMEM/F12 supplemented with 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin–streptomycin, 2% B27, 6 g/L glucose, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, and 1.83 μg/mL heparin. After seven days in culture, abundant neurospheres had formed, indicating that the cultures were suitable for further experimental procedures.

2.2. TUNEL assay

Apoptotic cells in tissue sections were labeled by TUNEL staining using a commercial kit (Yisheng, China) as per the manufacturer's instructions, followed by immunofluorescence imaging with a Zeiss (USA) microscope.

2.3. Measurement of ROS

Intracellular ROS levels were assessed with the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe (Beyotime, China). According to the manufacturer's instructions, NSPCs were washed with PBS and then incubated for 1 h at 37 °C with 20 μM DCFH-DA. This probe is initially non-fluorescent but becomes fluorescent when oxidized by intracellular ROS to form 2′,7′-dichlorofluorescein (DCF), allowing ROS detection. After incubation, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured using a fluorescence microscope with an excitation wavelength of 488 nm to visualize ROS generation. For quantification, the mean fluorescence intensity was measured in triplicate experiments using ImageJ software to ensure statistical reliability.

2.4. Flow cytometry

BV2 microglia were seeded uniformly into 6-well plates and cultured until they reached 80–95% confluence. The cells in the experimental and control groups were treated accordingly, with a blank sample (without probe loading) included for background subtraction. Subsequently, the cells were washed once or twice with PBS and detached using trypsin. The resulting cell suspensions were transferred into pre-labeled 1.5 mL centrifuge tubes and centrifuged at 1200 revolutions per minute (rpm) for 3 min at room temperature. A DCFH-DA working solution was prepared immediately before use by diluting the stock solution in serum-free medium at a ratio of 3 μL:3 mL, with gentle inversion to ensure mixing; all procedures involving the probe were performed under light-protected conditions. Following centrifugation, the supernatant was discarded, and the cell pellets were resuspended in 1 mL of the DCFH-DA working solution (or 1 mL of serum-free medium for the blank sample). After 20 min of incubation at 37 °C in the dark, the cells were centrifuged again under the same conditions (1200 rpm, 3 min, room temperature). The supernatant was carefully removed, and the cells were washed twice with 1 mL of cold PBS, followed by centrifugation after each wash. Finally, the cells were resuspended in 500 μL of cold PBS, transferred to flow cytometry tubes, and analyzed using a flow cytometer.

2.5. Evaluation of microglia phenotype

BV2 cells were stimulated with 400 μM H₂O₂ and treated with H2O2bergapten. After 1 day, protein levels of iNOS (M1-polarized microglia) and ARG-1 (M2-polarized microglia) were assessed by immunofluorescence staining.

2.6. In vitro differentiation assay

To promote neuronal differentiation, neurospheres were cultured for 7 days in a growth factor-free differentiation medium. The medium, modified from the proliferation formula by omitting growth factors, contained DMEM/F12 supplemented with 1% non-essential amino acids, 1% sodium pyruvate, 1% penicillin–streptomycin, 1% GlutaMAX (Gibco, USA), 2% B27, and 6 g/L glucose, along with an addition of 50 μM N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Selleck, USA) to potentiate the process.

2.7. Immunocytochemistry

For immunofluorescence staining, cells were first fixed overnight at 4 °C with 4% paraformaldehyde (PFA). After three PBS rinses, they were permeabilized and blocked for 60 min with a solution of 0.2% Triton X-100 in 5% bovine serum albumin (BSA)/PBS. Subsequently, the cells were incubated with primary antibodies (in blocking solution) for 1 h at RT, followed by application of Alexa Fluor–conjugated donkey secondary antibodies (1:500, Invitrogen) for 30 min after PBS washes. Prior to imaging, nuclei were stained with Hoechst 33342. All images were obtained using a Zeiss Imager.Z2 fluorescence microscope (USA).

2.8. Endothelial tube formation assay

The angiogenic capacity of endothelial cells was evaluated using a tube formation assay in vitro. Briefly, 96-well plates were pre-coated with 50 μL of Matrigel (Corning, USA) and allowed to polymerize at 37 °C for 30 min. Subsequently, endothelial cells were seeded onto the Matrigel layer at a density of 2 × 10⁴ cells per well in the respective treatment media, which included the control, oxygen-glucose deprivation/reoxygenation (OGD/R), and OGD/R groups supplemented with varying concentrations of bergapten (20, 40, and 100 μM). The cells were then incubated at 37 °C in a 5% CO₂ atmosphere for 6-8 h. Tube formation was observed under an inverted phase-contrast microscope. Three key parameters—total branching points, total loops, and total branching length—were quantified from at least three random fields per well using ImageJ software (National Institutes of Health, USA) with the Angiogenesis Analyzer plugin.

2.9. Fluorescein Isothiocyanate (FITC)-Dextran endothelial permeability assay

The integrity of the endothelial barrier was assessed by measuring the paracellular flux of FITC-labeled dextran. Endothelial cells were seeded at a density of 2 × 10⁵ cells/mL onto the upper chamber of 24-well Transwell inserts (0.4 μm pore size; Corning, USA) and allowed to form a confluent monolayer. After subjecting the cells to the OGD/R protocol, the culture medium was replaced. Cells in the treatment groups received Bergapten (20, 40, or 100 μM) in the upper chamber medium. Then, 40 kDa FITC-dextran (1 mg/mL; Sigma-Aldrich, USA) was added to the upper chamber. The lower chamber was filled with PBS. The plate was protected from light and incubated at 37 °C for 1 h. Following incubation, 40 μL of solution was collected from the lower chamber. The fluorescence intensity of the samples was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm using a microplate reader. The FITC-dextran transport rate (%) was calculated as follows: (Fluorescence intensity of lower chamber sample - Fluorescence intensity of PBS negative control)/Fluorescence intensity of the upper chamber positive control × 100%.

2.10. Statistical analysis

In this study, all experimental data were derived from a minimum of three independent replicates and are reported as mean ± standard deviation (SD). Outliers were determined using the mean ± 2SD criterion and excluded from the final analysis. The significance of differences between two groups was assessed by two-tailed Student's t-tests, while differences among multiple groups were evaluated using one-way repeated-measures ANOVA supplemented with post-hoc testing. Statistical significance was set at P < 0.05, with the following asterisk conventions: *P < 0.05, **P < 0.01, and ***P < 0.001.

3. Results

3.1. Isolation, culture, and characterization of primary mouse NSPCs

Primary NSPCs were successfully isolated from the brains of C57BL/6 mice. Under proliferating conditions, these cells formed characteristic free-floating neurospheres. To confirm their identity as NSPCs, the neurospheres were dissociated and plated, then subjected to immunofluorescence staining for well-established NSPCs markers. The quantitative data suggested that all cells exhibited strong positive expression for Nestin (an intermediate filament protein of NSPCs), SOX2 (a transcription factor for maintaining stem cell pluripotency), and PAX6 (a key transcription factor in neural development) (Fig. 1). The high percentage of cells expressing these markers confirm the successful enrichment and culture of a pure population of NSPCs for subsequent experiments.

Fig. 1.

Phenotypic characterization of mouse NSPCs. a After 7 days in proliferation medium, isolated primary NSPCs formed neurospheres. Neurospheres were enzymatically dissociated into single cells and were positive for the NSPC markers Nestin, SOX2, and PAX6. Scale bar = 20 μm b Quantification of Nestin⁺, SOX2⁺, and PAX6⁺ cells.

3.2. Bergapten protects NSPCs from H₂O₂-Induced oxidative stress and apoptosis

To model oxidative stress encountered after ischemia, NSPCs were exposed to 400 μM H₂O₂. This treatment resulted in a significant increase in intracellular ROS levels (Fig. 2a) (69.23 ± 7.86) and directly led to increased cell death, demonstrated by a marked rise in the number of TUNEL-positive apoptotic cells (Fig. 2c) (45.87% ± 6.17%), compared to the control group (ROS: 31.28 ± 8.21, p < 0.001; TUNEL: 3.21 ± 1.79, p < 0.001). Treatment with bergapten, however, significantly attenuated these effects. The bergapten-treated groups showed a substantial reduction in H₂O₂-induced ROS production (Fig. 2a) and a corresponding decrease in the number of TUNEL-positive cells (Fig. 2c). The effect of adding 100 μM bergapten was the best, and the mean fluorescence intensity of ROS or TUNEL-positive apoptotic cells was 39.83 ± 5.64 and 15.81% ± 2.56%, which was significantly lower than that of 20 μM (ROS: 67.35 ± 5.98; TUNEL: 40.09% ± 6.13%) or 40 μM dose groups (ROS:51.79 ± 10.26; TUNEL: 25.95% ± 4.75%) as demonstrated in Fig. 2b or 2d. Compared with the H₂O₂-only group, both the 40 μM and 100 μM bergapten groups showed significantly reduced mean fluorescence intensity of ROS (both p < 0.01) and TUNEL-positive apoptotic cells (40 μM: p < 0.05 and 100 μM: p < 0.001). These results indicate that bergapten effectively alleviates oxidative stress and protects NSPCs from apoptosis under pathological conditions.

Fig. 2.

Intracellular ROS levels and apoptosis (by TUNEL assay) were measured in NSPCs after treatment with 400 μM H2O2 to induce oxidative stress. After 12 h culture, NSPCs treated with different concentrations of bergapten were examined for intracellular ROS (a) or apoptosis levels (c). Scale bar = 40 μm in (a) and scale bar = 200 μm in (c). Quantification of ROS fluorescence intensity (b) or TUNEL positive cells (d). *p < 0.05, **p < 0.01, ***p < 0.001, compared with H₂O₂ group.

3.3. Bergapten alleviates oxidative stress and modulates microglial polarization in BV2 cells

We next investigated the effect of bergapten on microglia, key mediators of neuroinflammation. Exposure of BV2 microglial cells to 400 μM H₂O₂ induced a significant burst of intracellular ROS levels (87.09 ± 6.02), compared to the control group (21.08 ± 3.87) (Fig. 3a and b), confirming that oxidative stress also directly activates immune cells. Bergapten addition markedly reduced H₂O₂-induced ROS levels in BV2 cells. In 100 μM bergapten group, mean fluorescence ROS level was 47.74 ± 3.44, significantly lower than that in 20 μM group (83.87 ± 2.37) or 40 μM group (62.37 ± 3.66), as showed in Fig. 3a and b. In particular, both the 40 μM and 100 μM bergapten groups showed significantly reduced mean fluorescence intensity of ROS levels compared with the H₂O₂-only group (40 μM: p < 0.01 and 100 μM: p < 0.001).

Fig. 3.

Intracellular ROS levels were measured in BV2 microglia after treatment with 400 μM H2O2 to induce oxidative stress. After 12 h culture, BV2 microglia treated with different concentrations of bergapten were examined for intracellular ROS by DCFH-DA fluorescent probe (a) or flow cytometry (c). Scale bar = 200 μm in (a). b Quantification of ROS fluorescence intensity (b) or ROS positive cells (d). *p < 0.05, **p < 0.01, ***p < 0.001, compared with H₂O₂ group.

Flow cytometry result showed a similar trend as exhibited in Fig. 3c and d. In control group, about 3.16% ± 3.16% BV2 microglia expressed ROS and H₂O₂ addition increased the number of ROS positive cells to 78.54% ± 24.39%. The number of ROS positive cells in 20 μM, 40 μM or 100 μM bergapten group was 68.05% ± 7.56%, 50.48% ± 7.55% or 31.95% ± 6.34% respectively. We also observed that the percentage of ROS-positive cells in the 100 μM bergapten group was significantly lower than that in the H₂O₂-only group (p < 0.05).

Furthermore, immunofluorescence staining revealed that H₂O₂ treatment promoted the polarization of BV2 cells towards a pro-inflammatory M1 phenotype, as indicated by a significant increase in the expression of iNOS (a classic M1 marker) (Fig. 4a and b). Conversely, the expression of Arg-1, a marker for the anti-inflammatory, pro-repair M2 phenotype, was decreased (Fig. 4a and c). 100 μM bergapten treatment effectively countered these changes. It significantly suppressed the shift towards the M1 state, shown by decreased iNOS expression (30.79 ± 2.92, p < 0.01) and a concurrent increase in Arg-1 expression (64.55 ± 2.99, p < 0.001) compared with the H₂O₂ group. Similarly, the 20 or 40 μM bergapten group also exhibited reduced iNOS expression and elevated Arg-1 expression compared with the H₂O₂ group. All three bergapten-treated groups reduced iNOS expression and increased Arg-1 expression to varying degrees, with the 100 μM group showing the most pronounced effect. This suggests that bergapten not only reduces oxidative stress in microglia but also drives them towards a more protective, anti-inflammatory phenotype.

Fig. 4.

Immunofluorescence staining of BV2 microglia for iNOS (proinflammatory M1 phenotype) or ARG-1 (anti-inflammatory M2 phenotype). Nuclei are stained with DAPI (blue). a After 12 h culture, BV2 microglia treated with different concentrations of bergapten were examined for iNOS or ARG-1. Scale bar = 200 μm. Quantification of the expression levels of iNOS(b) and ARG-1(c) in (a). **p < 0.01, ***p < 0.001, compared with H₂O₂ group.

3.4. Bergapten rescues neuronal differentiation potential of NSPCs impaired by oxidative stress

A critical step for functional recovery is the generation of new neurons from NSPCs. Under normal differentiation conditions, NSPCs efficiently differentiated into neurons, evidenced by robust expression of the neuronal marker TUJ-1. However, when subjected to H₂O₂-induced oxidative stress during the differentiation process, the neurogenic capacity of NSPCs was severely compromised, leading to a significant reduction in the number of TUJ-1⁺ neurons and increase in the number of glial fibrillary acidic protein (GFAP)⁺ astrocytes (Fig. 5a–c). Remarkably, co-treatment with bergapten largely prevented this deficit. The proportion of TUJ-1⁺ cells in the 100 μM bergapten-treated group (9.43% ± 0.2%, p < 0.05) was significantly higher than in the H₂O₂-only group (3.28% ± 2.02%) and was comparable to control levels (9.63% ± 1.11%) (Fig. 5a and b). As shown in Fig. 5c, H₂O₂ exposure significantly increased the ratio of GFAP positive astrocytes (19.01% ± 3.58%, p < 0.01) compared with control group. Bergapten treatment reversed this effect. Bergapten at concentrations of 40 μM (8.18% ± 2.09%, p < 0.01) and 100 μM (11.3% ± 3.03%, p < 0.05) respectively exhibited significant reduced GFAP-positive astrocytes compared with the H₂O₂ group. This key finding demonstrates that bergapten preserves the neurogenic potential of NSPCs even in a hostile, oxidative environment.

Fig. 5.

Bergapten promoted neuronal differentiation and inhibited astroglial differentiation of NSPCs in vitro with 400 μM H₂O₂ to induce oxidative stress. a After 7 days of differentiation, immature neurons were immunostained for TUJ-1 (green) and astrocytes were immunostained for GFAP (red). Scale bar = 100 μm. Quantification of TUJ-1⁺ immature neurons (b) and GFAP⁺ astrocytes (c). *p < 0.05, **p < 0.01, compared with H₂O₂ group.

3.5. Bergapten preserves tight junction protein expression and enhances angiogenic capacity in endothelial cells under OGD/R conditions

To evaluate the protective effect of bergapten on endothelial barrier integrity, we examined the expression of key tight junction proteins, Claudin-5 and zonula occludens-1 (ZO-1), using immunofluorescence staining. As illustrated in Fig. 6a–d, exposure to OGD/R resulted in a significant reduction in the mean fluorescence intensity of both Claudin-5 (67.32 ± 4.86) and ZO-1 (15.69 ± 6.26) compared with the control group (114.42 ± 13.05, p < 0.001; 37.91 ± 3.01, p < 0.001). Treatment with bergapten at concentrations of 20, 40, and 100 μM notably attenuated this loss in a dose-dependent manner. Specifically, bergapten at 100 μM restored Claudin-5 (102.38 ± 2.31) and ZO-1 (104.97 ± 6.17) expression to levels comparable to the control, indicating a strong protective effect on endothelial junctional complexes under ischemic-like injury.

Fig. 6.

Bergapten attenuates OGD/R-induced downregulation of tight junction proteins in endothelial cells. a Representative immunofluorescence images showing the expression and distribution of Claudin-5 (green) in different treatment groups. Scale bar = 40 μm. c Representative immunofluorescence images showing the expression and distribution of ZO-1 (green). Scale bar = 40 μm. Quantitative analysis of the mean fluorescence intensity of Claudin-5 (b) or ZO-1 (d). *p < 0.05, **p < 0.01, ***p < 0.001 vs. OGD/R group.

The pro-angiogenic effect of bergapten was assessed using a tube formation assay. Fig. 7a–d shows that OGD/R significantly impaired endothelial tube formation, as reflected by decreased total branching points, total loops, and total branching length (p < 0.05, p < 0.001). Bergapten treatment markedly reversed these deficits. In particular, at the highest concentration (100 μM), bergapten restored all three parameters to near-normal levels, suggesting a potent role in promoting vascular network recovery following OGD/R.

Fig. 7.

Bergapten restores endothelial tube formation and reduces permeability following OGD/R. a Representative bright-field images of endothelial tube formation under different treatment conditions. Scale bar = 100 μm. b-d Quantitative analysis of total branching points (b), total branching length (c), and total loops (d). *p < 0.05, ***p < 0.001 vs. OGD/R group.e-f Endothelial monolayer permeability was assessed by FITC-dextran flux assay. (e) shows the comparison under post-OGD/R conditions, while (f) includes a pre-OGD/R control. **p < 0.01, ***p < 0.001 vs. OGD/R group.

3.6. Bergapten mitigates OGD/R-induced endothelial hyperpermeability

To further investigate the functional impact of bergapten on endothelial barrier function, we performed a FITC-dextran transwell permeability assay. As shown in Fig. 7e and f, OGD/R induced a significant increase in FITC-dextran transport across the endothelial monolayer compared to the control group (p < 0.01). Bergapten treatment dose-dependently reduced this hyperpermeability, with significant protection observed at 40 μM and 100 μM. The 100 μM bergapten group nearly normalized permeability to baseline levels, underscoring its efficacy in maintaining endothelial barrier integrity under pathological conditions.

4. Discussion

The present study provides compelling evidence for the multifaceted therapeutic potential of bergapten in a simulated ischemic stroke environment. Our major findings are that bergapten: 1) mitigates oxidative stress-induced apoptosis and supports the survival of NSPCs; 2) reduces oxidative stress and promotes a switch from a pro-inflammatory M1 to an anti-inflammatory M2 phenotype in microglia; and 3) crucially, rescues the impaired neuronal differentiation of NSPCs under oxidative stress conditions.

The post-ischemic brain is characterized by a surge in ROS, which directly damages all neural cell types [23,24]. Our data showing H₂O₂-induced ROS production and apoptosis in NSPCs are consistent with this notion and highlight the vulnerability of the endogenous stem cell pool. The efficacy of bergapten in scavenging ROS and promoting NSPC survival aligns with its known antioxidant properties reported in cardiac and other models [25]. By preserving this regenerative cell population, bergapten lays the foundational step for any subsequent repair process.

Neuroinflammation, orchestrated largely by microglia, is another major impediment to recovery. Activated M1 microglia release cytotoxic cytokines that inhibit neurogenesis and promote glial scar formation [26,27]. Our observation that H₂O₂ alone can drive BV2 cells towards an M1 state (iNOS⁺) and away from an M2 state (Arg-1⁺) underscores the interplay between oxidative stress and inflammation. Bergapten's ability to suppress M1 markers while enhancing M2 markers demonstrates a potent anti-inflammatory and immunomodulatory effect. This is likely mediated through the inhibition of key signaling pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as suggested by previous studies [28,29]. Shifting the microglial balance towards a pro-repair phenotype likely creates a more favorable secretome for neural regeneration.

The most significant finding of this study is that bergapten directly facilitates neurogenesis by overcoming the differentiation block imposed by oxidative stress. The hostile microenvironment after stroke not only kills existing neurons but also prevents the birth of new ones. The failure of NSPCs to differentiate into neurons under H₂O₂ stress, as observed here, mirrors the in vivo situation where neurogenesis is often aborted or skewed towards gliogenesis [30,31]. Bergapten's success in restoring neuronal differentiation is probably not a single-mechanism effect but a consequence of its combined actions: reducing the overall oxidative burden on differentiating NSPCs and creating a more supportive milieu through the modulation of microglial activation. This dual action makes it a particularly attractive candidate, as it targets two core pathological pathways simultaneously.

Our findings indicate that bergapten significantly preserves the expression of critical tight junction proteins, Claudin-5 and ZO-1, thereby helping to maintain the structural integrity of the endothelial barrier. Furthermore, bergapten enhanced angiogenic capacity, as evidenced by the restoration of tube formation parameters. Functionally, these structural and pro-angiogenic benefits were coupled with a marked reduction in OGD/R-induced hyperpermeability. The collective results suggest that bergapten mitigates key aspects of blood-brain barrier dysfunction following ischemic-like injury by fortifying junctional complexes, promoting vascular repair, and reducing vascular leakage. These findings position bergapten as a promising therapeutic candidate for ischemic stroke, warranting further investigation into its underlying molecular mechanisms and in vivo efficacy.

There are some limitations to our study. Firstly, we used H₂O₂ to model oxidative stress, which, while effective and reproducible, does not fully recapitulate the complex metabolic and inflammatory cascade of ischemia-reperfusion. Future studies should employ the OGD/R model, as planned in our research proposal, to validate these findings in a more physiologically relevant context. Secondly, the current experiments were conducted in monocultures. Co-culture systems of NSPCs with microglia would be invaluable to dissect the cell-cell interactions and paracrine mechanisms underlying bergapten's effects. Finally, the specific molecular targets of bergapten in neural cells, such as its potential interaction with the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway or the toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/NF-κB inflammatory axis, remain to be elucidated.

Despite these limitations, our findings strongly position bergapten as a promising pro-regenerative agent for stroke therapy. Its natural origin, established safety profile, and ability to cross the blood-brain barrier further enhance its translational potential.

5. Conclusions

In conclusion, this study demonstrates that the natural compound bergapten effectively protects against key pathological processes in an in vitro model of ischemic injury. It alleviates oxidative stress and apoptosis in NSPCs, modulates microglia towards an anti-inflammatory phenotype, and, most importantly, preserves the critical ability of NSPCs to differentiate into neurons. These results provide a robust scientific rationale for the further development of bergapten as a novel therapeutic strategy to promote endogenous repair and recovery after ischemic stroke. Future work will focus on validating these effects in OGD/R and in vivo stroke models, and on deciphering the precise molecular mechanisms involved.

CRediT authorship contribution statement

Zelong Tang: Data curation, Formal analysis. Yannian Li: Supervision, Validation. Yan Gao: Writing – review & editing. Yiping Wu: Investigation, Project administration. Wei Tian: Funding acquisition, Visualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

ROS: reactive oxygen species, NSPCs: neural stem/progenitor cells, OGD/R: oxygen-glucose deprivation/reperfusion.

Data availability

The data that support the findings of this study are available within the article and its supplementary materials.