Kaempferol regulates apoptosis and migration of neural stem cells to attenuate cerebral infarction by O‐GlcNAcylation of β-catenin

2.1 Reagents

Kae (C₁₅H₁₀O₆; purity ≥97%), triphenyl-2,3,5-tetrazoliumchloride (TTC), methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate (MSAB) (Wnt/β-catenin pathway inhibitor; purity ≥95%), D-hanks, trypsin, crystal violet, paraformaldehyde, and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Media/Ham’s F-12 (DMEM/F12), B27, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were purchased from Gibco (Grand Island, NY, USA). Glucose-free DMEM/F12 was purchased from Procell (Wuhan, China). Cell counting kit-8 (CCK-8) was obtained from MedChemExpress (Monmouth Junction, NJ, USA). Annexin V PE/7-AAD apoptosis detection kit, TUNEL BrightGreen apoptosis detection kit, bicinchoninic acid (BCA) protein quantification kit, and electro-chemi-luminescence (ECL) detection kit were acquired from Vazyme (Nanjing, China). Protein A/G agarose beads were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

2.2 Animal experiment design

The animal study was approved by the Ethics Committee of The Second Hospital of Hebei Medical University (No. 2023-AE303). Male C57BL/6 mice (8–10 weeks old; Charles River, Beijing, China) were housed at 12/12 h light/dark, 20–22°C conditions with free water and food. The mice were randomly divided into three groups (five mice per group): sham, middle cerebral artery occlusion (MCAO), and MCAO + Kae groups. The mice in the MCAO group received MCAO surgery. The sham mice received the same procedure except for carotid artery occlusion. The mice in the MCAO + Kae group underwent a tail vein injection of 30 mg/kg Kae at 2 h after the MCAO procedure. The neurological function was analyzed when the mice recovered from anesthesia. All mice were sacrificed at 24 h following the MCAO process. The brain tissues were collected and washed in pre-cold normal saline to remove blood, slightly frozen in the refrigerator, and cut into five brain sections.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and has been approved by the Ethics Committee of The Second Hospital of Hebei Medical University (no. 2023-AE303).

2.3 MCAO model establishment

Mice were anesthetized using 1.5% isoflurane to alleviate pain. An opening was made in the middle of the mouse’s neck to expose the left common carotid artery and its branches. A silicon-coated 6–0 nylon monofilament was inserted into the common carotid artery to the opening of the middle cerebral artery to block the blood flow under a surgical microscope. A decrease in cerebral blood flow of more than 70% was observed with a Laser Doppler flowmeter (Perimed, Sweden), indicating success in MCAO. After 2 h, the monofilament was removed and the wound was closed to restore blood flow. A heating pad was used to maintain the mice’s body temperature at 37 ± 0.5°C during the whole process.

2.4 Neurological function analysis

The neurological status was scored using a 5-point grading method as previously described [19]: 0 point: the mouse was normal; 1 point: the contralateral front paw of the mouse could not fully extend when holding the tail; 2 points: the mouse turned around in a circle towards the ipsilateral side; 3 points: the mouse fell on the contralaterally; and 4 points: the mouse could not move autonomously.

2.5 TTC staining

The brain sections were incubated with 1% TTC reagent at 37°C for 0.5 h. After washing in PBS, the results were observed and photographed. The infarct region was shown in white. The total area of each brain section and its infarct size were analyzed using the Image-Pro Plus software. The infarct volume was calculated using the following formula: infarcted area/total brain area × 100%.

2.6 Isolation and culture of NSCs

NSCs were isolated from the cerebral cortex of fetal rats (E14). The cortex tissue was washed in D-hanks and cut into small pieces with ophthalmic scissors. The tissues were digested with 0.25% trypsin for 15 min at room temperature. After filtration with the filter (200 mesh), the cells were centrifugated at 1,000 rpm for 5 min and the supernatant was removed. The cells were maintained in DMEM/F-12 supplemented with 2% B-27, EGF (20 ng/mL), and bFGF (20 ng/mL) at 37°C with 5% CO₂. The cells were passaged every 6–7 days. The third-passage NSCs were used in this study.

2.7 Oxygen and glucose deprivation (OGD)/reoxygenation (R) cell model establishment

For the OGD process, the NSCs were incubated in glucose-free DMEM/F12 under low oxygen conditions (94% N₂, 1% O₂, and 5% CO₂) at 37°C for 1 h. Then, for the R process, the NSCs were incubated in the normal medium at 37°C with 95% atmosphere and 5% CO₂ for 24 h.

2.8 Cell treatment

To study the role of Kae, the NSCs were exposed to 30 µM Kae for 1 h before OGD/R treatment. MSAB is a selective inhibitor of Wnt/β-catenin signaling that can bind to β-catenin to promote its degradation [20]. Therefore, to inactive the Wnt/β-catenin pathway, the NSCs were pre-treated with 10 µM MSAB for 24 h before OGD/R treatment.

2.9 CCK-8

A CCK-8 kit was used to analyze cell viability. After cell treatment and OGD/R induction, the CCK-8 solution was added to the plates to incubate for 4 h. The absorbance was read at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

2.10 Transwell assay

Cell migration was determined by Transwell assay. The 24-well chambers (8 µm pore) were purchased from Corning (Corning, NY, USA). The cell suspension was added to the top chambers, while the culture medium was added to the bottom chambers. After 24 h, the cells on the upper surface of the filter were removed, and the migrated cells were stained with crystal violet. The stained NSCs were visualized under a light microscope (Olympus, Tokyo, Japan) at 5 random fields.

2.11 Flow cytometry

An Annexin V PE/7-AAD kit was used to analyze cell apoptosis. The NSCs (5 × 10⁵ cells) were suspended in 1× binding buffer to make the single-cell suspension. Annexin V-PE (5 µL) and 7-AAD (5 µL) were incubated with cells for 10 min. Apoptosis was assessed using a flow cytometer (Thermo Fisher Scientific) with 1 h of adding 400 µL 1× binding buffer.

2.12 TUNEL assay

Cell slides were fixed with 4% paraformaldehyde and incubated with 2 µg/mL proteinase K reagent. Then, the cells were incubated with 50 µL TdT buffer (10 µL 5× Equilibration Buffer, 5 µL BrightGreen Labeling Mix, 1 µL Recombinant TdT Enzyme, and 34 µL ddH₂O₂) at 37°C for 1 h. After washing with PBS, the cells were re-stained with 2 μg/mL DAPI for 5 min at room temperature. The signals were visualized under a fluorescence microscope (Olympus).

2.13 Western blot

The NSCs were lysed using radio immunoprecipitation assay buffer on ice. Protein concentration was detected using the BCA protein quantification kit. The proteins were run using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes. The membranes were incubated with specific primary antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. The bands were developed using the ECL detection kit.

The antibodies used were shown below: anti-O-GlcNAc (MA1-072, 1:1,000; Invitrogen, Carlsbad, CA, USA), anti-OGT (PA5-22071, 1:1,000; Invitrogen), anti-OGA (PA5-119277, 1:1,000; Invitrogen), anti-β-catenin (#9582, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-c-myc (#9402, 1:1,000; Cell Signaling Technology), and anti-cyclin D1 (#2922, 1:1,000; Cell Signaling Technology). The secondary antibodies including HRP-linked anti-mouse IgG (#7076, 1:3,000) and HRP-linked anti-rabbit IgG (#7074, 1:3,000) were purchased from Cell Signaling Technology.

2.14 Immunoprecipitation

The lysate of NSCs was incubated with protein A/G agarose beads at 4°C overnight. Then, the complex was incubated with anti-β-catenin or anti-IgG at 4°C overnight. After centrifugation to remove the beads, the β-catenin was detected using the Western blot.

2.15 Molecular docking

The 3D structure of Kae (ID: 5280863) was obtained from the PubChem database and saved in a mol2 format. The structure of human OGT (ID: 5NPS) was downloaded from the RCSB Protein Data Bank (RCSB PDB) database in the PDB format. The molecular docking was performed using the AutodockTools-1.5.6.

2.16 Bioinformatics

The O‐GlcNAcylation sites of β-catenin were predicted using the online tool (https://services.healthtech.dtu.dk/service.php?YinOYang-1.2).

2.17 Statistical analysis

All data in this study were analyzed using GraphPad Prism 8 software and are exhibited as mean ± standard deviation. Comparisons of difference were evaluated by Student’s t-test (two groups) and one-way analysis of variance (multiple groups). P < 0.05 was considered statistically significant.

3.1 Kae suppresses MCAO-induced infarction and neurological injury

First, the MCAO mice model was established and treated with Kae to study the effects of Kae on infarction. As shown in Figure 1b and c, the infarct volume was increased in MCAO mice than in the sham mice, suggesting that the model was successfully established. Kae significantly reduced MCAO-induced infarction volume. In addition, the neurological scores of mice in the MCAO group were higher than those in the sham group, while Kae decreased the scores induced by MCAO (Figure 1d). The results indicated that Kae attenuated the cerebral infarction and neurological injury induced by MCAO.

Figure 1

Kae suppresses MCAO-induced infarction and neurological injury. (a) The chemical structure of Kae. (b) The photograph of the brain tissues of the mice in the sham, MCAO, and MCAO + Kae group. The white is the infarct area, and the red is the normal area. (c) The infarct volume was quantified. (d) The neurological function was evaluated by neurological scores. ^## P < 0.01.

3.2 OGD/R inhibits cell migration and induces apoptosis

The OGD/R NSC injury model was established, and the biological functions were analyzed. Cell viability was inhibited by OGD/R treatment (Figure 2a). As compared with the control group, OGD/R suppressed cell migration (Figure 2b). Besides, according to the results of flow cytometry and TUNEL assay, ODG/R facilitated cell apoptosis, compared with the control cells (Figure 2c and d). The data suggested that the OGD/R cell model was successfully generated.

Figure 2

OGD/R inhibits cell migration and induces apoptosis. After the OGD/R NSCs model was established: (a) cell viability was analyzed by CCK-8; (b) cellular migration was assessed using Transwell assay; and apoptosis was evaluated by (c) flow cytometry and (d) TUNEL assay. ^## P < 0.01.

3.3 Kae suppresses OGD/R-induced NSC injury

To assess the function of Kae, we used Kae to treat OGD/R cells. We found that Kae promoted the cell viability of OGD/R cells (Figure 3a). Besides, the migration of OGD/R cells was facilitated by Kae treatment (Figure 3b). Inversely, cell apoptosis induced by OGD/R was abrogated by Kae (Figure 3c and d). To sum up, Kae promoted cell viability, and migration, and inhibited apoptosis of OGD/R cells, suggesting Kae attenuated NSC injury.

Figure 3

Kae suppresses OGD/R-induced NSC injury. The OGD/R cell model was established and treated with Kae: (a) CCK-8 was carried out to analyze cell viability; (b) transwell assay evaluated cell migration; and apoptosis was assessed using both (c) flow cytometry and (d) TUNEL assay. ^## P < 0.01.

3.4 Kae promotes O‐GlcNAcylation of β-catenin

To explore the underlying mechanism of Kae, we performed the molecular docking between Kae and OGT. Details of the visualization of molecular docking are shown in Figure 4a. Hydrogen bond interaction force promotes the binding of molecules to active sites. The results showed that forms hydrogen bonds with OGT at ALA-896 and THR-922 sites (Figure 4a). Then, we assessed O‐GlcNAcylation modification. The results showed that OGD/R suppressed total O‐GlcNAcylation levels, whereas Kae reversed the suppression of O‐GlcNAcylation levels (Figure 4b). OGD/R treatment decreased the protein levels of OGT and increased OGA protein levels, as well as reduced O‐GlcNAcylation levels of β-catenin, while Kae reversed the OGD/R effects (Figure 4b). Moreover, multiple O‐GlcNAcylation sites of β-catenin were predicted (Figure 4c). In addition, the protein levels of c-Myc and cyclin D1 were downregulated by OGD/R treatment, which was abrogated by Kae treatment (Figure 4d). The data indicated that Kae promoted O‐GlcNAcylation of β-catenin and thus activated the Wnt/β-catenin pathway in the OGD/R NSCs.

Figure 4

Kae promotes O‐GlcNAcylation of β-catenin. (a) The molecular docking between Kae and OGT. (b) The OGD/R cells exposed to Kae were lysed and the total O‐GlcNAc, OGT, OGA, and β-catenin levels were assessed by the Western blot. GAPDH was the endogenous control. (c) The O‐GlcNAcylation sites of β-catenin were predicted. (d) The protein levels of c-Myc and cyclin D1 were examined in OGD/R cells stimulated by Kae.

3.5 Kae inhibits NSC injury by activating the Wnt/β-catenin pathway

To confirm that Kae regulated the biological functions by the Wnt/β-catenin pathway, we used MSAB to suppress the activation of this pathway. MSAB counteracted the promotion of cell viability induced by Kae (Figure 5a). Kae promoted cell migration, while MSAB inhibited the cell migration of Kae-stimulated NSCs (Figure 5b). In addition, cell apoptosis suppressed by Kae was partly abrogated by MSAB (Figure 5c and d). Taken together, the inactivation of the Wnt/β-catenin pathway reversed the Kae-induced neuroprotective effect.

Figure 5

Kae inhibits NSC injury by activating the Wnt/β-catenin pathway. After the OGD/R NSCs treated with Kae and MSAB: (a) cell viability was determined using CCK-8; (b) transwell assay evaluated cellular migration; and (c) flow cytometry and (d) TUNEL assay were performed to evaluate apoptosis. ^## P < 0.01. ^# P < 0.05.