Hesperidin Improves Memory Function by Enhancing Neurogenesis in a Mouse Model of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is an irreversible neurodegenerative disease characterized by memory and cognitive impairments. Neurogenesis, which is related to memory and cognitive function, is reduced in the brains of patients with AD. Therefore, enhancing neurogenesis is a potential therapeutic strategy for neurodegenerative diseases, including AD. Hesperidin (HSP), a bioflavonoid found primarily in citrus plants, has anti-inflammatory, antioxidant, and neuroprotective effects. The objective of this study was to determine the effects of HSP on neurogenesis in neural stem cells (NSCs) isolated from the brain of mouse embryos and five familial AD (5xFAD) mice. In NSCs, HSP significantly increased the proliferation of NSCs by activating adenosine monophosphate (AMP)-activated protein kinase (AMPK)/cAMP-response element-binding protein (CREB) signaling, but did not affect NSC differentiation into neurons and astrocytes. HSP administration restored neurogenesis in the hippocampus of 5xFAD mice via AMPK/brain-derived neurotrophic factor/tropomyosin receptor kinase B/CREB signaling, thereby decreasing amyloid-beta accumulation and ameliorating memory dysfunction. Collectively, these preclinical findings suggest that HSP is a promising candidate for the prevention and treatment of AD.

Keywords: Alzheimer’s disease; hesperidin; neural stem cells; neurogenesis.

Figure 1

The effects of hesperidin on cell proliferation in neural stem cells isolated from mouse embryonic brain. (A) In vitro neurosphere culture and experiment scheme. Cells were pretreated with 10, 50, 100, and 200 µM hesperidin for 4 days. (B) Cell proliferation (%) was measured using a WST-1 assay. (C,D) Representative images showing proliferation and the number of neurospheres of at least 100 µm in the graph. (E) Neural stem cell-specific proliferation was confirmed by measuring the mRNA levels of DCX, nestin, and Sox2 by qRT-PCR. Statistical analysis included one-way analysis of variance and Tukey’s post hoc test. * p < 0.05 and *** p < 0.001 compared with the vehicle-treated control group. Data are shown as mean ± SEM (n = 3 per group).

Figure 2

The effects of hesperidin on AMPK/CREB signaling in neurospheres. To prove the neurogenic effects of HSP, we measured AMPK and CREB protein levels in vitro. Phosphorylated/total AMPK and CREB were analyzed by western blot. (A) Representative images of AMPK and CREB in neurosphere lysates. (B,C) Quantification of AMPK (B) and CREB (C) expression. Statistical analysis included Student’s t test. * p < 0.05 compared with the vehicle-treated control group. Data are shown as mean ± SEM (n = 3 per group).

Figure 3

The effects of hesperidin on neurogenesis in the hippocampus of 5xFAD mice. Mice were administered with vehicle or HSP (100 mg/kg) once daily for two months. (A) In vivo experiment scheme. (B) Representative images of BrdU⁺ cells in the hippocampus. (C) The quantification of BrdU⁺ cells. (D) Representative images of BrdU⁺(green)NeuN⁺(red) cells in the hippocampus. (E) The quantification of BrdU⁺(green)NeuN⁺(red) cells. Scale bar; 50 μm. Arrows; BrdU⁺ cells (B) or BrdU⁺NeuN⁺ cells (D). Statistical analysis included one-way analysis of variance and Tukey’s post hoc test. ^## p < 0.01 compared with the vehicle-treated WT mice. ** p < 0.01 compared with the vehicle-treated 5xFAD mice. Data are shown as mean ± SEM (n = 4 per group).

Figure 4

The effects of hesperidin on AMPK/BDNF/TrkB/CREB signaling in the hippocampus of 5xFAD mice. (A–E) Representative images of western blots (A) and quantification of AMPK (B), BDNF (C), TrkB (D), and CREB (E). Statistical analysis included one-way analysis of variance and Tukey’s post hoc test. ^# p < 0.05 and ^## p < 0.01 compared with the vehicle-treated WT mice. * p < 0.05 and *** p < 0.001 compared with the vehicle-treated 5xFAD mice. Data are shown as mean ± SEM (n = 4 per group).

Figure 5

The effects of hesperidin on spatial memory functions in 5xFAD mice. (A) Escape time (s) of mice over 7 days. (B) Representative swimming paths on day 8. (C–E) On day 8 of the probe trial, time in quadrant (C), crossing the platform number (D), and swimming speed (E) were recorded. Statistical analysis included one- or two-way analysis of variance and Tukey’s post hoc test. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with the vehicle-treated WT mice. * p < 0.05 and ** p < 0.01 compared with the vehicle-treated 5xFAD mice. Data are shown as mean ± SEM (WT mice, n = 12; WT+HSP mice, n = 12; 5xFAD mice, n = 12; 5xFAD+HSP mice, n = 13). n.s; not significant.