Liraglutide improves cognition function in streptozotocin-induced diabetic rats by downregulating β-secretase and γ-secretase and alleviating oxidative stress in HT-22 cells

Abstract

Diabetes has been regarded as an independent risk factor for Alzheimer’s disease (AD). Liraglutide could improve cognition in AD mouse models, but its precise mechanism remains unclear. In this study, we used STZ-induced diabetic rats and HT-22 cells to investigate the effects of liraglutide. The MWM test, MTT assay, ELISA, western blot, and immunofluorescence were used in this research. Diabetic rats induced by STZ displayed a longer escape latency and entered the target zone less frequently (p < 0.05) in the MWM test. Intraperitoneal injection of liraglutide improved the cognition of diabetic rats (p < 0.05) and reduced Aβ42 expression in the hippocampus (p < 0.05). In vivo experiments showed that HT-22 cell viability decreased in the HG group, but liraglutide (100 nmol/L and 1 μmol/L) enhanced HT-22 cell viability (p < 0.05). Oxidative stress markers were upregulated in HT-22 cells in the HG group, while liraglutide treatment significantly reduced these markers (p < 0.05). Western blot and immunofluorescence analyses demonstrated increased levels of Aβ, BACE1, and γ-secretase in HT-22 cells in the HG group (p < 0.05), whereas these levels were reduced in the liraglutide treatment group (p < 0.05). These effects were reversed by the nuclear factor kappa B (NF-κB) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors (p < 0.05). These findings suggest that liraglutide improved the cognition of diabetic rats and might exert its protective effects by reducing oxidative stress, downregulating BACE1 and γ-secretase expression, and decreasing Aβ deposition via the NF-κB and ERK1/2 pathways.

Introduction

Alzheimer’s disease (AD) is a common form of dementia, and its incidence continues to rise each year. The disease is characterized by the deposition of amyloid β (Aβ) peptides and tau protein aggregates [1, 2]. The progressive accumulation of Aβ, which plays a central role in neuronal death and memory impairment, is a key pathological feature of AD [3]. Aβ peptides are derived from amyloid precursor protein (APP) and are mainly processed by two enzymes: β-secretase (beta-site amyloid precursor protein cleaving enzyme, BACE1) and γ-secretase. Initially, BACE1 cleaves APP into an APP C-terminal fragment [4], and γ-secretase subsequently processes this fragment into Aβ40 and Aβ42 peptides [5]. Of these peptides, Aβ42 has been identified as the most hydrophobic, amyloidogenic, and neurotoxic.

Some studies have demonstrated a correlation between diabetes mellitus (DM) and AD, with DM considered an independent risk factor for AD. Aβ, an important biomarker of AD, has also been found in the brains of DM animal models [6]. Our primary study and other research have shown that Aβ deposits in the hippocampus and prefrontal cortex of streptozotocin (STZ)-induced diabetic rats, which exhibit cognitive dysfunction in the Morris water maze (MWM) test [7]. Research has shown that oxidative stress occurs before Aβ plaque formation and has suggested that oxidative stress may promote Aβ production [8, 9]. Other studies have shown that oxidative stress can upregulate BACE1 and γ-secretase expression, triggering APP processing and Aβ production [10]. Moreover, the γ-secretase complex includes presenilin (PS) proteins (PS1 and PS2), nicastrin, presenilin enhancer 2, and anterior pharynx defective 1. As the core catalytic subunit of γ-secretase, PS1 is the main active component of this enzyme and plays an independent role in γ-secretase activity. PS1 overexpression may be a risk factor for late-onset AD, while inhibition of PS1 expression has been shown to reduce Aβ production.

Given the relationship between DM and AD, drugs initially developed to treat DM have been repurposed for AD treatment. Liraglutide, a long-acting synthetic analog of glucagon-like peptide-1 (GLP-1), has been used as a therapeutic agent for DM. It is chemically similar to natural GLP-1 (97% homology) and shares several of its biological effects. Recent studies have shown that liraglutide can cross the blood-brain barrier [11] and has neuroprotective effects in conditions such as ischemic stroke, AD, and Parkinson’s disease (PD) [12]. Hansen et al. found that liraglutide prevents Aβ-induced impairments in spatial memory and hippocampal synaptic plasticity [13]. McClean et al. also demonstrated that liraglutide administration could reverse memory dysfunction, reduce Aβ deposition, and decrease microglial activation and insulin resistance in an AD mouse model [14]. However, the mechanism by which liraglutide downregulates Aβ deposition and mitigates DM-induced cognitive decline remains unclear. BACE1 is a key enzyme in Aβ production from APP. It has been demonstrated that STZ-induced diabetes exacerbates Aβ accumulation by upregulating BACE1 expression [15]. Additionally, γ-secretase is important for Aβ production. Whether liraglutide can influence the expression of BACE1 and γ-secretase (PS1) to reduce Aβ deposition remains to be determined.

This study aimed to elucidate a novel functional link between DM and AD by examining the effects of liraglutide on the cognition of STZ-induced diabetic rats. We also investigated the mechanism by which liraglutide protects HT-22 cells from high glucose (HG) treatment in vivo.

Materials and Methods

Animals and treatment

Eight-week-old male Sprague-Dawley rats, weighing approximately 180 ± 20 g, were obtained from the Animal Centre of Xi’an Jiaotong University. The animals were housed under standard conditions, adhering to the Guidelines for the Care and Use of Laboratory Animals. A DM model was established by intraperitoneally injecting 60 mg/kg STZ to 30 rats. The remaining 30 rats received an intraperitoneal injection of saline. Three days post-injection, blood samples were collected from the tail vein to monitor blood glucose levels. Rats with blood glucose levels above 16.7 mmol/L were considered to have successfully developed DM and were used for further experimentation. All rats were housed under a 12-hour light/dark cycle with controlled temperature (22 ± 0.5°C) and humidity (50–55%). By week 10, the 30 DM rats were randomly divided into two groups: DM group and DM + liraglutide group (25 nmol/kg intraperitoneal injection once daily for 14 days). Similarly, the 30 control rats were divided into normal control (NC) group and NC + liraglutide group (25 nmol/kg intraperitoneal injection once daily for 14 days). Throughout the 12-week study period, body weight and blood glucose levels were monitored weekly.

Open field test

An open field test was performed in week 11 to assess the locomotor activity of the rats. The testing apparatus was a rectangular box (100 × 100 × 50 cm) with black walls and bottom, equipped with a video tracking system mounted above the box. Each rat was placed in the box for 2 minutes, after which its locomotor activity was recorded for 5 minutes. The distance traveled and velocity were measured, and the computerized system automatically recorded horizontal locomotion (crossing) and vertical locomotion (rearing).

MWM test

The MWM test was carried out in week 12 to evaluate spatial learning and memory abilities after confirming the locomotion ability of the rats. The MWM device consisted of a circular water maze (120 cm diameter) divided into four quadrants, an escape platform (10 cm diameter), and a recording system. The test protocol and data recording followed the methodology described in our previous study [7].

Hippocampal HT-22 cell culture and treatment

The HT-22 immortalized hippocampal neuron cell line was purchased from Shanghai Zhongqiaoxinzhou Biotech (Shanghai, China). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco) in a humidified atmosphere of 95% air and 5% CO₂.

To assess glucose-induced neurotoxicity, HT-22 cells were treated with varying concentrations of glucose (25, 50, 100, 200 mmol/L) for 24 hours. Based on the results, 100 mmol/L was selected for subsequent experiments. To evaluate the neuroprotective effects of liraglutide, HT-22 cells were treated with liraglutide at different concentrations (10, 100, 1,000 nmol/L) for 24 hours, and 100 nmol/L liraglutide was used for further experiments. To investigate the underlying mechanisms, HT-22 cells were divided into five groups: normal glucose (NG, 5.5 mmol/L), HG (100 mmol/L), HG (100 mmol/L) + liraglutide (100 nmol/L) group, HG (100 mmol/L) + liraglutide (100 nmol/L) + SCH (SCH772984, ERK1/2 pathway inhibitor) group, and HG (100 mmol/L) + liraglutide (100 nmol/L) + BAY (BAY11-7082, NF-κB pathway inhibitor).

Cell viability assay

After 72 hours of treatment, cell viability was assessed using the MTT assay. HT-22 cells (1.0 × 10⁶ cells/mL) were seeded in 96-well plates (100 μL/well) and incubated for 24–48 hours at 37°C. MTT solution was added to each well at a final concentration of 0.5 mg/mL and incubated for 4 hours at 37°C. The absorbance of the resulting formazan crystals was measured at 595 nm using an Infinite 200 PRO microplate reader (Tecan, China).

Enzyme-linked immunosorbent assay (ELISA)

Hippocampal tissues from rats in each group were homogenized, and the concentrations of Aβ42 in the hippocampus were measured using Aβ42 ELISA Detection Kits (JianCheng Biotech Institute, Nanjing, China) following the manufacturer’s instructions. For HT-22 cells, both the cells and culture medium were collected after 24 hours of treatment. The levels of nitric oxide (NO), reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were determined using ELISA Detection Kits (JianCheng Biotech Institute, Nanjing, China), adhering to the manufacturer’s protocols. A BCA protein assay kit was utilized to quantify protein concentrations in the samples.

Western blot

Three rats from each group were euthanized, and their hippocampal tissues were harvested for western blot analysis. HT-22 cells were inoculated at a density of 10 × 10⁶ cells/mL and cultured in 5 mL of medium for 24 hours. Protein extraction was performed using nuclear and cytoplasmic extraction methods, and protein concentrations were quantified using a BCA Protein Assay Kit. For western blotting, the following primary antibodies were used: anti-BACE1 (sc-33711, 1:500), γ-secretase anti-PS1 (sc-365450, 1:500), and β-actin (sc-47778, 1:1,000) from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-Aβ (ab271968, 1:500) and secondary antibody (ab97035) from Abcam (Cambridge, MA, USA). The protein bands were visualized using a chemiluminescence (ELC) kit. The gray-scale values of the bands were analyzed using ImageJ software (version 1.50i, NIH, Bethesda, MD, USA).

Immunofluorescence

HT-22 cells (1.0 × 10⁶ cells/mL) were seeded in 6-well plates and cultured in 2 mL of medium for 24 hours. Cells were then fixed on coverslips with methanol at –20°C for 20 minutes, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 minutes at room temperature, blocked with 5% bovine serum albumin (BSA) in PBS for 30 minutes, and incubated with primary antibodies against Aβ, BACE1, or PS1 at 37°C for 2 hours. After washing, the cells were incubated with secondary antibodies in the dark for 1 hour at 37°C. The 4',6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei for 2 minutes at room temperature in the dark. BSA was used as a negative control. Images were captured using a Nikon fluorescent inverted microscope (Nikon Instruments Inc., Melville, NY, USA) and analyzed using ImageJ software (NIH).

Statistical analysis

All data were presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism 7 and SPSS statistics v23.0 software. One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used to determine statistical significance. A p-value of less than 0.05 was considered statistically significant.

Results

Open field test

The open field test was conducted to assess locomotion and rule out depression or reduced mobility as potential confounding factors in the MWM test. There were no statistically significant differences in any of the parameters among the four groups (p > 0.05) (Table 1).

Table 1 The results of Open-field Test among the 4 groups ($\stackrel{-}{\mathrm{x}}$ ± s)

Groups	n	The times of horizontal locomotion	The times of vertical locomotion
NC	15	100.64 ± 45.26	10.01 ± 1.09
NC + liraglutide	15	99.65 ± 46.03*	10.14 ± 1.26*
DM	15	98.89 ± 44.21*	10.27 ± 1.32*
DM + liraglutide	15	98.53 ± 46.28*,#	9.85 ± 1.13*,#

*p > 0.05 vs. control, ^#p > 0.05 vs. DM

NC: normal control group; DM: diabetes mellitus group

Liraglutide improved the cognition of STZ-induced DM rats

Liraglutide reduced fasting blood glucose levels in DM rats, but had no significant effect on body weight (Table 2). To explore the effect of the liraglutide on the cognition of diabetic rats, DM rats were administered with 25 nmol/kg liraglutide intraperitoneally once daily for 14 days from week 9. At week 11, open field testing was conducted, showing no significant differences in locomotion activity among the four groups (p > 0.05). A MWM test was then conducted across 4 groups of rats. Compared to the NC group, the NC + liraglutide group showed similar results. However, the DM group had a significantly longer escape latency compared to the NC group (48.04 ± 6.31 vs. 32.32 ± 7.1, p < 0.01) and a lower target entrance frequency (4.63 ± 1.86 vs. 9.19 ± 1.14, p < 0.01). Liraglutide treatment improved cognitive function in DM rats, as evidenced by a shorter escape latency (37.67 ± 7.25 vs. 48.04 ± 6.31, p < 0.05) and a higher target entrance frequency (8.53 ± 1.41 vs. 4.63 ± 1.86, p < 0.01) compared to the DM group (Table 3).

Table 2 The data of the 4 groups ($\stackrel{-}{\mathrm{x}}$ ± s)

Groups	n	Body weight (g) 12 weeks	Fasting blood glucose (mmol/L) 12weeks
NC	15	524.2 ± 22.30	8.84 ± 1.26
NC + liraglutide	15	507.3 ± 20.57	8.33 ± 1.53
DM	15	312.4 ± 23.54*	29.23 ± 3.89*
DM + liraglutide	15	322.2 ± 27.23	20.87 ± 2.77#

*p < 0.05 vs. NC, ^#p < 0.05 vs. DM.

NC: normal control group; DM: diabetes mellitus group

Table 3 Comparison of Morris Water Maze Test results among the 4 groups ($\stackrel{-}{\mathrm{x}}$ ± s)

Groups	n	Escape latencies	Frequency of entrance into the target zone
NC	15	32.32 ± 7.11	9.19 ± 1.14
NC + liraglutide	13	31.15 ± 7.03	9.36 ± 1.21
DM	13	48.04 ± 6.31**	4.63 ± 1.86**
DM + liraglutide	12	37.67 ± 7.25#	8.53 ± 1.41##

*p < 0.05 vs. NC, **p < 0.01 vs. NC, ^#p < 0.05 vs. DM, ^##p < 0.01 vs. DM.

NC: normal control group; DM: diabetes mellitus group

Liraglutide decreased Aβ42 expression in the hippocampus of DM rats

Hippocampal Aβ42 expression was measured using ELISA in the DM, DM + liraglutide, and NC groups. The results showed that Aβ42 expression was significantly higher in the DM group compared to the NC group (p < 0.01). However, liraglutide treatment significantly reduced Aβ42 levels in the hippocampus of DM rats (p < 0.01) (Fig. 1).

Fig. 1  Concentration of Aβ42 in the hippocampus across four groups, as determined by enzyme-linked immunosorbent assay

The Aβ42 expression in the DM group was significantly higher than that in the NC group (p < 0.01). In the DM + liraglutide group, Aβ42 levels were lower than those in the DM group (p < 0.01). **p < 0.01 vs. NC group; ^##p < 0.01 vs. DM group. Aβ42, amyloid beta 42; NC, normal control group; DM, diabetes mellitus group.

Liraglutide improved cell viability in HT-22 cells exposed to HG

MTT assays were performed to assess the viability of HT-22 cells exposed to HG. Compared to the NG group, HT-22 cell viability in the HG group was significantly reduced (100% vs. 60.65%, p < 0.01) (Fig. 2). Treatment with 10 nmol/L liraglutide did not significantly improve cell viability (60.65% vs. 64.45%, p > 0.05), but treatment with 100 nmol/L and 1 μmol/L liraglutide significantly enhanced HT-22 cell viability (60.65% vs. 70.41%, p < 0.05; 60.65% vs. 73.43%, p < 0.05, respectively) (Fig. 2).

Fig. 2  MTT assay of the viability of HT-22 cells across five groups

The viability of HT-22 cells in the high glucose (HG) group was significantly lower than that in the normal glucose (NG) group (p < 0.01). A concentration of 10 nmol/L liraglutide did not significantly increase the activity of hippocampal neurons (p > 0.05). However, concentrations of 100 nmol/L and 1 μmol/L liraglutide significantly enhanced the activity of HT-22 cells (p < 0.05). **p < 0.05 vs. NG group; ^#p < 0.05 vs. HG group. HG, high glucose; NG, normal glucose.

Liraglutide alleviated oxidative stress in HT-22 cells exposed to HG

Oxidative stress markers were measured using ELISA. The concentrations of NO, ROS, and MDA were significantly higher in the HG group compared to the NG group (p < 0.05, Fig. 3A, B, D). SOD activity was also elevated in the HG group compared to the NG group (p < 0.05, Fig. 3C). Liraglutide treatment reduced the levels of NO, ROS, and MDA compared to the HG group (p < 0.05, Fig. 3A, B, D) and also decreased SOD activity (p < 0.05, Fig. 3C).

Fig. 3  Oxidative stress markers in HT-22 cells from different groups, assessed by enzyme-linked immunosorbent assay

A: The concentration of nitric oxide (NO) in the HG group was higher than that in the NG group (p < 0.05), and liraglutide treatment reduced NO levels compared to the HG group (p < 0.05). B: The concentration of reactive oxygen species (ROS) in the HG group was significantly higher than in the NG group (p < 0.01), while liraglutide treatment decreased ROS concentration compared to the HG group (p < 0.01). C: Superoxide dismutase (SOD) activity in the HG group was higher than that in the NG group (p < 0.05), but liraglutide treatment resulted in decreased SOD activity compared to the HG group (p < 0.05). D: The concentration of malondialdehyde (MDA) in the HG group was higher than in the NG group (p < 0.01), and liraglutide treatment lowered MDA levels compared to the HG group (p < 0.01). *p < 0.05 vs. NG group; **p < 0.01 vs. NG group; ^#p < 0.05 vs. HG group; ^##p < 0.01 vs. HG group. NO, nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase; MDA, malondialdehyde; HG, high glucose; NG, normal glucose.

Liraglutide downregulated Aβ in HT-22 cells

The expression of Aβ in HT-22 cells was analyzed using western blot and immunocytochemistry. The western blot results indicated that Aβ levels were significantly higher in the HG group compared to the NG group (p < 0.01, Fig. 4A, B). Liraglutide treatment significantly reduced Aβ expression in the HG group (p < 0.01, Fig. 4A, B). Furthermore, treatment with extracellular signal-regulated kinase (ERK1/2) and nuclear factor-kappa B (NF-κB) inhibitors resulted in higher Aβ levels compared to the liraglutide treatment group (p < 0.05, Fig. 4A, B), suggesting that the downregulation of Aβ by liraglutide may involve these pathways.

Fig. 4  Western blot analysis of Aβ, BACE1, and γ-secretase expression in HT-22 cells from different groups

A: The expression levels of Aβ, BACE1, and γ-secretase were detected by western blot. B: The gray value ratio of Aβ/β-actin showed that Aβ expression was significantly higher in the HG group compared to the NG group (p < 0.01). The liraglutide treatment group exhibited lower Aβ levels than the HG group (p < 0.01). Treatment with NF-κB or ERK1/2 inhibitors resulted in higher levels of Aβ than the liraglutide-only group (p < 0.05). C: The gray value ratio of BACE1/β-actin demonstrated that BACE1 expression was higher in the HG group than in the NG group (p < 0.01), with liraglutide treatment leading to decreased BACE1 levels (p < 0.05). BACE1 expression in the HG + liraglutide + SCH and HG + liraglutide + BAY groups was lower than in the liraglutide treatment group (p < 0.05). D: The gray value ratio of γ-secretase/β-actin revealed that γ-secretase expression was higher in the HG group compared to the NG group (p < 0.01). In the HG + liraglutide group, γ-secretase expression was lower than in the HG group (p < 0.01). In the HG + liraglutide + SCH and HG + liraglutide + BAY groups, γ-secretase levels were higher than in the liraglutide-only group (p < 0.01 and p < 0.05, respectively). *p < 0.05 vs. NG group; **p < 0.01 vs. NG group; ^#p < 0.05 vs. HG group; ^##p < 0.01 vs. HG group; ^&p < 0.05 vs. HG + liraglutide group; ^&&p < 0.01 vs. HG + liraglutide group. Aβ, amyloid beta; BACE1, beta-site amyloid precursor protein cleaving enzyme 1; HG, high glucose; NG, normal glucose.

Liraglutide downregulated BACE1 and γ-secretase (PS1) expression in HT-22 cells via the ERK1/2 and NF-κB pathways

The expression of BACE1 and γ-secretase (PS1) in HT-22 cells were measured by immunofluorescence and western blot. BACE1 expression in the HG group was significantly higher than that in the NG group (p < 0.01, Fig. 4A, C), while liraglutide treatment significantly reduced BACE1 levels (p < 0.05, Fig. 4A, C). However, when ERK1/2 or NF-κB pathway inhibitors were applied, the effect of liraglutide on BACE1 was reversed, with BACE1 levels in the HG + liraglutide + SCH and HG + liraglutide + BAY groups being higher than those in the liraglutide treatment group (p < 0.05, Fig. 4A, C). Immunofluorescence analysis supported these findings, showing that the fluorescence intensity of BACE1 in the HG group was greater than that in the NG group (p < 0.01, Fig. 5). Liraglutide treatment decreased the fluorescence intensity of BACE1 in HT-22 cells (p < 0.01, Fig. 5), but this effect was attenuated in the ERK1/2 and NF-κB inhibitor treatment groups, where the fluorescence intensity was significantly higher than in the group treated with liraglutide alone (p < 0.05, Fig. 5).

Fig. 5  Immunofluorescence analysis of BACE1 expression in HT-22 cells from different groups

The fluorescence intensity of BACE1 in the HG group was significantly greater than that in the NG group (p < 0.01), and liraglutide treatment reduced BACE1 fluorescence intensity (p < 0.01). In the HG + liraglutide + SCH and HG + liraglutide + BAY groups, BACE1 fluorescence intensity was higher than in the HG + liraglutide group (p < 0.05). *p < 0.05 vs. NG group; **p < 0.01 vs. NG group; ^#p < 0.05 vs. HG group; ^##p < 0.01 vs. HG group; ^&p < 0.05 vs. HG + liraglutide group; ^&&p < 0.01 vs. HG + liraglutide group. BACE1, beta-site amyloid precursor protein cleaving enzyme 1; HG, high glucose; NG, normal glucose.

Similarly, western blot analysis demonstrated that γ-secretase subunit PS1 expression was elevated in the HG group compared to the NG group (p < 0.01, Fig. 4A, D). Liraglutide treatment significantly decreased PS1 expression in the HG group (p < 0.01, Fig. 4A, D). However, in the HG + liraglutide + SCH and HG + liraglutide + BAY groups, PS1 expression was higher than in the liraglutide treatment group (p < 0.01 and p < 0.05, respectively; Fig. 4A, D). Immunofluorescence also confirmed higher fluorescence intensity of PS1 in the HG group compared to the NG group (p < 0.01, Fig. 6). Liraglutide treatment reduced PS1 fluorescence intensity (p < 0.01, Fig. 6), while the addition of ERK1/2 or NF-κB inhibitors increased PS1 fluorescence intensity compared to liraglutide treatment alone (p < 0.05, Fig. 6).

Fig. 6  Immunofluorescence analysis of γ-secretase expression in HT-22 cells from different groups

The fluorescence intensity of γ-secretase in the HG group was significantly greater than that in the NG group (p < 0.01), and liraglutide treatment reduced γ-secretase fluorescence intensity (p < 0.01). The fluorescence intensity of γ-secretase in the HG + liraglutide + SCH and HG + liraglutide + BAY groups was higher than in the HG + liraglutide group (p < 0.05). *p < 0.05 vs. NG group; **p < 0.01 vs. NG group; ^#p < 0.05 vs. HG group; ^##p < 0.01 vs. HG group; ^&p < 0.05 vs. HG + liraglutide group; ^&&p < 0.01 vs. HG + liraglutide group. HG, high glucose; NG, normal glucose.

Discussion

This study demonstrated that liraglutide treatment improved cognitive function in STZ-induced DM rats, which exhibited cognitive decline at week 12. DM rats treated with liraglutide showed better cognitive performance compared to those in the DM group, and this was associated with reduced Aβ42 expression in the hippocampus. Liraglutide also attenuated Aβ42 deposition, suggesting its potential neuroprotective effects. Furthermore, the in vivo experiments revealed that liraglutide alleviated oxidative stress and downregulated the expression of BACE1, γ-secretase (PS1), and Aβ in HT-22 cells through the NF-κB and ERK1/2 pathways.

The STZ-induced DM rat model is well-established for studying diabetes-related cognitive dysfunction, which mirrors the cognitive deficits observed in DM humans. In this study, while the locomotor activity of DM rats remained normal in the open field test, spatial memory impairment was evident in the MWM test by week 12.

BACE1 is a key enzyme involved in the production of Aβ, which plays a crucial role in the pathogenesis of AD. Previous studies [16] have shown that BACE1 is upregulated in neurons under oxidative stress, chronic gliosis, traumatic brain injury, and hypoxic conditions [17]. In line with this, our findings showed that HT-22 cells exposed to HG had higher BACE1 expression and Aβ deposition compared to the control group. HG also upregulated the expression of oxidative stress markers, which likely contributed to the increased BACE1 expression. As Aβ deposition is known to induce oxidative stress, the upregulation of oxidative stress markers may have been driven by increased Aβ deposition.

γ-secretase is a complex enzyme that cleaves the APP fragment following β-secretase cleavage to produce Aβ [18, 19]. PS1 is the main active component of this enzyme and has an independent role in γ-secretase activity. In this study, we found that HG treatment increased the expression of PS1, which coincided with elevated Aβ expression. GLP-1 is an incretin hormone that induces insulin secretion to regulate glucose levels. Currently, GLP-1 agonists are used as a therapeutic strategy for type 2 diabetes mellitus (T2DM). Additionally, GLP-1 is expressed in neurons and acts as a neurotransmitter, functioning as a brain-gut peptide. GLP-1 receptors are widely distributed throughout the body and are abundantly expressed in the central nervous system [20]. The GLP-1 receptor regulates the activity of various central nerves and helps prevent neuronal injury [21, 22]. The HT-22 cell line is derived from mouse hippocampal neurons and expresses the GLP-1 receptor [23]. As a GLP-1 receptor agonist, liraglutide can be recognized by and act on this receptor [24]. Hunter et al. demonstrated that liraglutide, a long-acting analog of GLP-1, can cross the blood-brain barrier and bind to GLP-1 receptors in the brain, exerting a neuroprotective effect [25]. GLP-1 plays a significant role in the brain; some researchers have shown that it can protect neurons from neurotoxic influences, reduce apoptosis of hippocampal neurons, and improve spatial cognition [13, 26, 27]. In our study, we found that liraglutide improved cognition in STZ-induced DM rats, consistent with previous research findings [28, 29]. Further experiments demonstrated that liraglutide treatment downregulated Aβ expression. In vivo, liraglutide downregulated the expressions of BACE1 and γ-secretase (PS1) in HT-22 cells via the NF-κB and ERK1/2 pathways, potentially providing a mechanism by which liraglutide reduces Aβ expression.

Oxidative stress is a common factor in DM and many neurodegenerative disorders [30, 31]. Some studies have indicated that oxidative stress may be a shared feature between DM and AD. Thus, oxidative stress-induced inflammation may be one of the mechanisms through which DM affects AD [32]. When oxidative stress occurs, tissues generate ROS, leading to tissue damage [33, 34]. In the brain, the hippocampus and cerebral cortex are particularly vulnerable to oxidative stress; the ability to remove ROS is impaired in DM, AD, and other diseases [35]. A decrease in antioxidants and an increase in oxidative damage may contribute to the pathophysiology and cognitive decline observed in DM rats. In conclusion, this study found that liraglutide improved cognitive function in STZ-induced DM rats, possibly by alleviating oxidative stress and protecting HT-22 cells from HG-induced damage. Additionally, liraglutide downregulated the expression of BACE1 and PS1, components of γ-secretase, reducing Aβ deposition via the NF-κB and ERK1/2 pathways. However, further research is needed to better understand these mechanisms and to determine whether these findings can be extended to other animal models or clinical settings.

Declarations of Interest

None.

Funding

This work was supported in part by the National Natural Science Foundation of China (No. 81700736), Research project of Xi’an Municipal Health Commission (2022ms-12) and Xi’an Science and Technology Plan Project (24YXYJ0080).

Acknowledgments

Thanks for the language editing from the Elsevier Webshop.

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