Nesfatin-1 (NF-1) is derived from its precursor protein, nucleobindin 2, through the specific cleavage by prohormone convertases at certain sites [13], which is a peptide with multiple roles, including regulating fat metabolism, food intake, sleep, mental activity, gastrointestinal motility, and glucose metabolism [14,15,16,17]. In neurological disorders, NF-1 can counteract the damage and apoptosis of dopaminergic neurons induced by MPP(+)/MPTP through the activation of the C-Raf-ERK1/2 dependent anti-apoptotic signaling pathway. This offers a new potential target for treating neurodegenerative diseases such as Parkinson's disease [18]. Additionally, NF-1 inhibits the excessive production of reactive oxygen species (ROS) within cells following high glucose exposure, thereby reducing cell apoptosis. This makes NF-1 a potential therapeutic candidate for treating diabetic neuroinflammatory lesions [19]. NF-1 has recently been reported to upregulate the expression of TJ proteins such as ZO-1 [20]. In this study, the protective effects of NF-1 against Aβ1-42-induced senescence in BECs and disruption of the BBB were investigated to provide a potential therapeutic strategy for AD.
For in vitro experiments, bEnd.3 cells were divided into four groups (control, Aβ1-42, Aβ1-42 + 30 nM NF-1, Aβ1-42 + 60 nM NF-1) with n = 6 wells per group for each assay (e.g., SA-β-gal staining, FITC-dextran, TEER, real-time PCR, Western blotting). Each well represented an independent biological replicate, seeded from a single stock (iCell, China). All in vitro experiments were independently repeated three times. For in vivo experiments, male and female Tg APPswe/PSEN1dE9 and WT mice (Jackson Laboratories, USA), aged 6-8 months, were divided into four groups: WT (n = 8), Tg (n = 8), Tg + 10 μg/kg NF-1 (n = 8), and Tg + 20 μg/kg NF-1 (n = 8). Each group included 4 males and 4 females to reflect the clinical prevalence of AD in both sexes and comply with NIH guidelines on sex as a biological variable [6]. The 6-8-month age range was chosen to capture significant amyloid plaque burden and BBB dysfunction, which are robust in Tg mice at this stage [8, 12]. Each mouse represented an independent biological replicate, with tissues harvested from the frontal cortex. In vivo experiments were performed once, as preliminary studies confirmed consistent BBB permeability changes in Tg mice [18]. Sample sizes were chosen based on prior studies detecting significant differences in cellular senescence and BBB permeability [10, 18]. No samples or animals were excluded, and all cells with ≥80% confluence and mice with confirmed Tg APPswe/PSEN1dE9 genotype were included, as per pre-established criteria. Mice were randomly allocated to groups using a random number generator, and investigators were blinded to group allocation during outcome assessments (e.g., SA-β-gal staining, FITC-dextran, TEER, immunostaining) to minimize bias.
bEnd.3 cells were purchased from iCell (China) and cultured in complete endothelial culture medium (ECM) under conditions of 37 °C and 5% CO. bEnd.3 cells were authenticated by STR profiling within the last 6 months and tested negative for mycoplasma contamination using a PCR-based assay. Each well represented an independent biological replicate, seeded at 3 × 10⁵ cells per well for permeability assays or 2 × 10⁵ cells for telomerase activity (n = 6 per group). Experiments were repeated three times. To overexpress VEGF-R1, bEnd.3 cells were transduced with an adenovirus supplemented with the overexpression vector of VEGF-R1 (Ad-VEGF-R1) for 48 h. NF-1 concentrations of 30 and 60 nM were selected for in vitro experiments based on prior studies demonstrating effective modulation of cellular responses in neuronal and endothelial cells without toxicity [18, 20]. The 14-day treatment duration was chosen to reflect chronic Aβ-induced senescence, consistent with established models [10].
Wild-type (WT) mice and Tg (APPswe/PSEN1dE9) AD model mice were purchased from Jackson Laboratories (USA). Each mouse represented an independent biological replicate (n = 8 per group). Each group included 4 males and 4 females to reflect the clinical prevalence of AD in both sexes and comply with NIH guidelines on sex as a biological variable [6]. Mice were divided into four groups: WT, Tg, Tg + 10 μg/kg NF-1, and Tg + 20 μg/kg NF-1. In the Tg + 10 μg/kg NF-1 and Tg + 20 μg/kg NF-1 groups, Tg mice were intraperitoneally injected with 10 μg/kg/day and 20 μg/kg/day NF-1 for 3 months. In the WT and Tg groups, WT and Tg mice were intraperitoneally injected with the same volume of normal saline for 3 months. All animal experiments complied with the ARRIVE guidelines and were carried out following the National Institutes of Health guide for the care and use of Laboratory animals. Animal experiments utilized NF-1 doses of 10 and 20 μg/kg/day, previously shown to be neuroprotective in mouse models [18]. The 3-month treatment duration was selected to correspond with the chronic AD pathology timeline in Tg APPswe/PSEN1dE9 mice. Animal experiments were approved by the Ethics Committee of the Seventh Affiliated Hospital of Sun Yat-sen University.
SA-β-gal staining detects β-galactosidase activity at pH 6.0, a widely used marker of cellular senescence reflecting lysosomal changes in senescent cells. bEnd.3 cells (n = 6 wells per group) were fixed with 2 mL of 4% paraformaldehyde for 15 min. Subsequently, 1 mL of freshly prepared SA-β-Gal staining working solution was added. After sealing and incubating at 37 °C in a CO-free incubator for 16 h in the dark, images were captured using a fluorescence microscope (Zeiss, Germany). Experiments were repeated three times.
Telomerase activity and hTERT expression indicate telomere maintenance capacity, which is reduced in senescent cells, while TERF2 protects telomere ends, and its dysregulation is associated with senescence [21]. A total of 2 × 10 bEnd.3 cells (n = 6 wells per group) were counted and collected, then resuspended in 200 μl of CHAPS lysis buffer (Takara, Japan) on ice for 30 min. Cells were centrifuged at 4 °C at 16,000 × g for 30 min. The supernatant was aliquoted and stored at -80 °C. Telomerase activity detection followed Voglauer's qPCR method: a 20 μl reaction system included 1 μl of cell lysate (equivalent to 1 × 10 cells), 0.5U Hotstar DNA polymerase (Takara, Japan), 200 nM TS primer (5'-AATCCGTCGAGAACAGTT-3'), 100 nM Cxa primer (5'-GTGTAACCCTAACCCTAACCC-3'), and 0.4×SYBR-Green I. The reaction conditions were as follows: incubation at room temperature for 30 min, denaturation at 95 °C for 5 min, followed by qPCR with 95 °C for 15 s and 50 °C for 60 s for 45 cycles. PCR was performed using the GeneAmp 9700 system (Thermo Fisher Scientific, USA). Experiments were repeated three times.
Brain tissues from the frontal cortex of each mouse (n = 8 per group) were used for RNA and protein analyses, as this region is significantly affected in AD pathology. RNA was extracted from bEnd.3 cells (n = 6 wells per group) and brain tissues. Each sample was run in triplicate (technical replicates), with n = 6 wells per group for cells and n = 8 mice per group for tissues. The concentration and purity of the extracted RNA were measured and recorded using a nucleic acid detector (KAIAO, China), ensuring that the sample's OD260/OD280 ratio was within the range of 1.8 to 2.0. Subsequently, first-strand cDNA was synthesized using the MonScript RT III All-in-One Mix with dsDNase (Monad Biotech, China). The PCR amplification mixture consisted of 1 μL each of first-strand cDNA, forward primer, and reverse primer, 10 μL of MonAmp 2× Taq Mix Pro (+Dye) (Monad Biotech, China), and 7 μL of ddHO. The reaction protocol was as follows: initial denaturation at 94 °C for 3 min, followed by 25 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 15 s, and a final extension at 72 °C for 5 min. Experiments were repeated three times.
bEnd.3 cells (n = 6 wells per group) were lysed using RIPA cell lysis solution and PMSF solution (100 mM) at a ratio of 100:1. Further lysis was performed with an ultrasonic cell disruptor. The lysate was then centrifuged at 4 °C at 12,000 r·min-1 for 30 min, and the protein concentration was determined using the BCA method. The supernatant was taken, and an appropriate volume of protein loading buffer (diluted 4 times) was added. The mixture was denatured by heating in a 100 °C water bath for 10 min and then stored at -80 °C. SDS-PAGE and transfer were conducted, followed by a 1-h blocking with 5% non-fat milk. The diluted primary antibodies against p53, p21, VEGF-R1, VEGF-R2, ZO-1, claudin-5 (CST, USA, 1:1000), hTERT, TERF2 (Abcam, USA, 1:1000) and β-actin (CST, USA, 1:4000) were introduced, and the mixture was incubated for 12 h at 4 °C. After extensive washing with 1 × TBST, the diluted secondary antibody (CST, USA, 1:2000) was added, and the mixture was incubated for 2 h. Following further extensive washing with 1 × TBST, chemiluminescence was performed. Protein expression levels were detected using Image J software, with β-actin as an internal reference. Experiments were repeated three times.
bEnd.3 cells (n = 6 wells per group) were plated at a density of 3×10 per well in a 12-well Transwell culture plate. When cells reached approximately 80% confluence, 1 mg/ml of FITC-Dextran was added to the upper chamber in a light-protected manner. The plate was then incubated in a cell culture incubator for 6 h. Subsequently, 200 μl of the culture medium from the lower chamber was aspirated, and the OD value at 492 nm was measured to construct a standard curve for FITC-Dextran. Based on this standard curve, the concentration of FITC-Dextran in the lower chamber of each well was calculated. Experiments were repeated three times.
TEER measures the electrical resistance across the endothelial monolayer, reflecting the integrity and permeability of the BBB, with lower values indicating barrier dysfunction. bEnd.3 cells (n = 6 wells per group) were seeded in a 12-well Transwell culture plate. After completing the drug treatment, the resistance of each well was measured using an electrical resistance meter (Sigma, Germany). During the measurement, the longer end of the electrode from the resistance meter was in contact with the bottom of the culture plate, while the shorter end was positioned near the bottom of the chamber. Three different locations within each chamber were selected for measurement, and the average value was taken as the actual TEER measured value. The resistance values were expressed in Ω·cm². The actual TEER was calculated by subtracting the blank value from the measured value and then multiplying by 1.12 cm². Experiments were repeated three times.
Two hours before tissue collection, mice (n = 8 per group) in each experimental group were injected via the tail vein with 2% Evans Blue (EB) staining solution (4 mL/kg). After 2 h of systemic circulation, brains were harvested following cardiac perfusion and placed in a brain-slicing mold to prepare 2 mm continuous coronal brain sections. The areas of EB staining in the brains of mice were documented through photography, and their absorbance was measured using a microplate reader (MD, USA) to establish a standard curve. Based on the standard curve, the content of EB within the brain tissue of each experimental group (ng/mg protein) was calculated. Experiments were independently repeated twice, with results averaged to ensure reproducibility.
Paraffin sections were prepared from the frontal cortex of mouse brains (n = 8 per group). After deparaffinization, antigen retrieval, and blocking of endogenous peroxidase, the sections were incubated for 20 min. An appropriate number of primary antibodies against VEGF-R1, ZO-1, and claudin 5 (1:100, CST, USA) was then applied and allowed to incubate at 4 °C overnight. On the following day, after washing off the primary antibodies, secondary antibodies (TRITC conjugated secondary antibody was used for VEGF-R1 staining, 1:200, CST, USA) were applied and incubated for 1 h. For ZO-1 and claudin 5 staining, DAB chromogen solution was then added. The reaction was terminated when the cells were observed to turn brown-yellow under the microscope (Zeiss, Germany). The sections were then dehydrated, cleared, and mounted. The average optical density values were analyzed using Image J software. Experiments were independently repeated twice, with results averaged to ensure reproducibility.
Data are expressed as mean ± standard deviation (SD), with error bars in all figures representing SD. A two-way ANOVA (sex × treatment) was used to assess potential sex differences in outcomes, with no significant sex interactions detected (P > 0.05). For sample sizes (n = 6 for in vitro experiments, n = 8 for in vivo experiments), one-way analysis of variance (ANOVA) with Tukey post hoc tests was used for multiple group comparisons. Data were tested for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene's test to confirm ANOVA assumptions.
All assays used sample sizes n ≥ 5 (n = 6 for in vitro, n = 8 for in vivo), therefore descriptive statistics (mean ± SD) were used to summarize the data. Tests were two-sided, and no adjustments for multiple comparisons were applied, as each figure tested a single primary hypothesis. P-values are reported in figure legends, with significance set at P < 0.05. Statistical analyses were performed using GraphPad Prism 8.0.2.